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University Miaonlms International 300 N. Zeeb Road AnnAfbor, MI48106
8403574
Smoot, Jeffrey W.
PORCINE THYROID GDP-MANNOSE PYROPHOSPHORYLASE
The Ohio State University PH.D. 1983
University Microfilms International300 N. Zeeb Road, Ann Arbor, Ml 48106
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University Microfilms International
PORCINE THYROID GDP-MANNOSE PYROPHOSPHQRYLASE
Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University
By Jeffrey W. Smoot, B.A., B.S.
The Ohio State University 1983
Reading Committee* Approved by Dr. George A. Barber Dr. Edward J . Behrman Dr. George S. Serif a d v iso r Department of Biochemistry ACKNOWLEDGMENTS
X would like to acknowledge Dr. George A. Barber and Dr. Edward J. Behrman for their professional criticism , helpful insight, and tactful prodding.
I would especially like to acknowledge my advisor, Dr. George S* Serif, Whose guidance and support kept my enthusiasm and this project alive.
i l VITA
May 25, 1952 ...... Born in Los Angeles, C a lifo rn ia
1975 ...... • • ...... B. A .; B* S< University of California Irvine, California 1977-1983...... Graduate Research or Teaching Associate, The Ohio State University, Columbus, Ohio
PUBLICATIONS
"The Reduction of Aryl Disulfides with Triphenylphosphine and Water." Larry E. Overman, Jeffrey Smoot, and Joanne D. Overman, (1974) Synthesis 1_, 59-60
FIELDS OF STUDY Major Field: Biochemistry
Ui TABLE OP CONTENTS
Page ACKNOWLEDGMENTS...... i i VITA...... i i i LIST OF TABLES...... v ii LIST OF FIGURES...... v i i i
LIST OF PLATES...... X
ABBREVIATIONS...... x i INTRODUCTION...... 1 EXPERIMENTAL PROCEDURE A. M a te ria ls ...... 6 B. Methods Assays for Pyrophosphorylase A ctivity...... 1 Synthetic Assay...... 8 Degradative Assay...... 9 Detection of the Pyrophosphorylase in Polyacrylamide Gels...... 10 Pyrophosphorylase Activity in Crude Homogenates. 11 Protein Assay...... 12 Snake Venom (Phosphodiesterase) Assay...... 12 Chemical Synthesis of ( ^J-Mannose-l-phosphate... 13 Enzymatic Synthesis of ( c)-Mannose-l-phosphate.. 15 Preparation of Buffers...... 16 Chromatography Solvents...... 17 Paper Electrophoresis...... 18 Pipet Calibration ...... 19 Statistical Methods...... 19 Scintillation Cocktail • • • 19 pH Optimum Determination...... 19 Preparation of Polyacrylamide Gels Slab Gels Containing Sodium Dodecyl Sulfate 20 Non-denaturing Gels ...... 22 i) Agarose-acrylamide Composite Gels...... 23 ii) DATD Cross-linked Gels ...... 24 2-dimensional Electrophoresis...... 25 Electrophoretic Conditions...... 26 Visualization Procedures ...... 26 Iv page Sucrose Velocity Sedimentation Sedimentation Procedure ...... 27 Detection of Marker Enzymes ...... 28 Calculation of Relative M obility...... 29 Determination of the Pyrophosphorylase Specificity Specificity towards NDP-X substrates ...... 30 Specificity Towards Mannose-l-phosphate and Inorganic Pyrophosphate...... 30 Specificity Towards NTP Substrates ...... 32 Kinetic Constants Determinations Michaelis Constant for GDP-mannose...... 33 Michaelis Constant for Inorganic Pyrophosphate.. 34 Michaelis Constant for GTP...... 35 Michaelis Constant for Mannose-l-phosphate 36 Product Analysis ...... 36 Purification of GDP-mannose Pyrophosphorylase Homogenization...... 38 Centrifugation ...... 38 Synthesis of Blue Sepharose CL-6B Initial Coupling Procedure...... 39 Pinal Coupling Procedure...... 40 Blue Sepharose Chromatography...... 40 Preparation of DEAE-Sephacel...... 41 DEAE-Sephacel Chromatography...... 42 Preparation of Phenyl Sepharose...... 43 Phenyl SepharoBe Chromatography...... 43 P re p a ra tio n o f GTP-1 inked A garose...... 44 Agarose-GTP A ffinity Chromatography...... 44 S tability and Storage of GDP-mannose Pyrophosphorylase 44
RESULTS Purification of GDP-mannose Pyrophosphorylase Fresh vs. Frozen Tissue ...... 46 Blue Sepharose Chromatography...... 46 Chromatography on DEAE-Sephacel...... 47 Chromatography on Phenyl Sepharose...... 62 Agarose-GTP A ffinity Chromatography...... 65 Comments on Additional Separation Procedures.... 66 Polyacrylamide Gel Electrophoresis...... 72 pH Optimum...... • 79 Kinetic Constants Determination Michaelis Constant for GDP-mannose...... 82 Michaelis Constant for Inorganic Pyrophosphate.. 82 MichaeliB Constant for GTP...... 82 Michaelis Constant for Mannose-l-phosphate 89 GDP-mannose Pyrophosphorylase Specificity Specificity Towards NDP-X Substrates ...... 89 Specificity Towards Sugar-l-phosphates and Inorganic Pyrophosphate...... 89 Specificity Towards NTP Substrates ...... 94 v Page Product Analysis ...... 94 Sucrose Velocity Sedimentation ...... 100 DISCUSSION...... 103
BIBLIOGRAPHY...... I l l
vt LIST OF TABLES
Specificity of porcine thyroid pyrophosphorylase towards nucleoside diphosphate sugars
Specificity of porcine thyroid pyrophosphorylase towards sugar-l-phoaphates and inorganic pyrophosphate as determined by synthetic assays Specificity of porcine thyroid pyrophosphorylase towards NTP substrates as determined by synthesis assays Paper chromatography o f p u ta tiv e GDP-mannose Seven minute hydrolysis of putative mannose-l- phosphate
Paper chromatography of mannose released by alkaline phosphatase treatment of putative mannose-l-phosphate
Purification scheme for porcine thyroid GDP-mannose pyrophosphorylase LIST OF FIGURES
Figure Page 1 Core oligosaccharide of asparagine-linked 2 glycoproteins
2 Partial structure of the type A oligosaccharide 2 unit of porcine thyroglobulin 3. Structural heterogeneity of the type B oligo- 3 saccharide unit of porcine thyroglobulin 4 Elution profile of a crude homogenate of porcine 49 thyroid from Blue Sepharose 5 Elution behavior of a crude thyroid extract on 52 C ellex D
6 Elution profile of a Blue Sepharose dialyBate 54 from DEAE-Sephacel 7 Chromatofocusing of a Cellex D (peak III) 57 d ia ly s a te
8 Elution behavior of a Cellex D dialysate on 59 DEAE-Biogel A columns of fixed pH 9 The pH profile of a linearly applied pH gradient on 61 a 1.5 X 4.5 cm DEAE-Sephacel column 10 Elution profile of a DEAE-Sephacel concentrate 64 applied to Phenyl Sepharose 11 Elution profile of a DEAE-Sephacel concentrate 67 applied to Sephacryl S-300 12 Elution behavior of a DEAE-Sephacel concentrate 70 applied to hydroxyl apatite 13 E ffe c t o f pH on GDP-mannose pyrophosphorylase 81 enzymatic activity
14 Lineweaver-Burke plot for determining the apparent 83 Michaelis constant for GDP-mannose
vlll F igure Page
15 L in ewe ave r-B u rk e plot for determining the apparent 86 Michaelis constant for inorganic pyrophosphate 16 Lineweaver-Burke plot for determining the apparent 88 Michaelis constant for GTP 17 Lineweaver-Burke plot for determining the apparent 91 Michaelis constant for mannose-l-phosphate
18 Calibration curve and native molecular weight of 102 GDP-mannose pyrophosphorylase as determined by sucrose velocity sedimentation
Ix
r LIST OF PLATES
P la te Page
SOS polyacrylamide gel (10%) of representative 76 column e lu a te s in th e p u r if ic a tio n o f GDP-mannose pyrophosphorylase stained with Coomassie R-250 SDS pay 1 aerylamide gel of Plate 1 re-stained 78 by silver staining
x LIST OF ABBREVIATIONS
MOPS N-(morpholino)-propane sulfonic acid
MES N-(morpholino) -ethane sulfonic acid CHES cyclohexylaminoethane sulfonic acid CAPS 3-(cyclohexylamino)-propane sulfonic acid
TAPS trihydroxymethylaminopropane sulfonic acid (H)EPPS N-2-hydroxyethylpiperazine-N' -3-propane sulfonic acid Tris trihydroxymethylaminomethane
DEAE- diethylaminoethyl- SDS sodium dodecyl sulfate
TEMED N, N, N *, N1 -tetramethylethylenediamine Flurara 4-phenylspiro(furan-2(3H), 1 '-phthalan)-3#,3 '- dione w/v weight/ volume v /v volume/ volume w/w weight/ weight D.D. H20 deionized distilled water PPO 2-5-diphenyloxazole POPOP 1,4-bis-2-(5-phenyloxazolyl) -benzene
xl INTRODUCTION
Thyroglobulin is the predominant macromolecular product of the thyroid gland (1). In its native form, thyroglobulin is an iodinated glycoprotein dimer of approximately 670,000 daltons in molecular weight (2) and functions as the latent form of the hormone, thyroxin (T^ and T^). The carbohydrate portion of thyroglobulin, in the nomenclature of Spiro (3), is composed of about twenty-three asparagine-linked oligosaccharide moieties comprising two distinct classes i the type A unit (high mannose content. Fig. 2) and the type B unit (complex monosaccharide content, Fig. 3). Both oligosaccharide units are derived from What has come to be termed the "core oligosaccharide" con^osed of
(Man^tGlcNAc^ linked to an asparagine residue as shown in Fig. 1. This core oligosaccharide originates as a lipid-linked
(dolichol) precursor on which an antennary array of carbohydrate residues is assembled and then processed (4,5) in a specific sequential manner, the mechanism of which may be common to a variety of mammalian tissues (for a comprehensive review, see r e f . 6 ). The principle Bource of the mannose residues in the oligo saccharide moieties of glycoproteins is derived from GDP-mannose
(guanosine-5' - Of-D-mannopyranosy 1-1-pyrophosphate). However, the 1 2
M a n l ^ 6 j £ Man 1— 6GlcNAc 1 — 4GlcN Ac'-'-Asn
Man1v 3
Figure 1. Core oligosaccharide of asparaglne-llnked glycoproteins
Man1 — 6Man1 w .3 ^ 6 ^ t Man Man 1 — 4GlcN Ac 1 — 4GlcN Ac— Asn 3 Man*r
Figure 2. Partial structure of the type A oligosaccharide unit of porcine thyroglobulin (NeuAc)2— 3Gal 1 — 4GlcN Ac F iC J _Man1N!, (3) l*< M t 2 "Ng g (Neu Ac)2— 6Gal 1 — 4GlcNAc 1S Man1— 4G!cNAc1— 4GlcNAc ^ 3 (NeuAc)2-^-6Gal 1 — 4GlcNAc 1 —2Man 1 (6)
Fuc * * 1 (NeuAc)2— 6Gal1— 4GlcNAc1— 2Man1 ^ I « X 6 6 Man 1 — 4GlcNAc 1 —4GlcN Ac (NeuAc)2— 6Gal 1 —4GlcNAc 1 —2Man 1 ^
Figure 3. Structural heterogeneity of the type B oligosaccharide unit of porcine thyroglobulin final donor of the mannose residue may be GDP-mannose directly or indirectly after the transfer of the mannose portion of GDP-mannose to d o lic h o l phosp hate. In an e le g a n t s e r ie s o f experiments utilizing a GDP-mannose r dolichol phosphate transferase deficient cell line of a murine lymphoma, Kornfeld and co-workers were able to delineate the sequence and origin of eight of nine mannose residues in the major lipid-linked oligosaccharide produced by this cell line (7). In the particular case of bovine thyroglobulin, Spiro (8,9) and others (10,11) have demonstrated the participation of GDP-mannose, via the dolichol phosphate pathway, in the addition of mannose residues to the type A oligosaccharide. However, the sequence of addition and positions within the array have not yet been elucidated although the origin of the branch point mannose of the core oligosaccharide may be inferred (12,13). The cellular levels of GDP-mannose are produced by the enzymatic condensation of GTP (guanosine-5'-triphosphate) and a-D-mannopyranosyl phosphate by the enzyme GTP : O-D-mannopyran- osyl-1- phosphate guanylyltransferase, more commonly called GDP-mannose pyrophosphorylase. This enzyme is one of a large class of enzymes (14) catalyzing the generalized reaction:
NTP + X -l-P <—► NDPX + PPi nucleoside sugar nucleotide inorganic triphosphate phosphate .sugar pyrophosphate 5 Partial purification and characterization of GDP-mannose pyrophosphorylases has been reported from a number of sources
(15-18)i However, a detailed characterization and complete purification from a mammalian tissue has not been reported although the analogous enzyme, UDP-glucose pyrophosphorylase which catalyzes the synthesis of UDP-glucose (uridine-5'-a-D-glucopyran- osyl-l-pyrophosphate) from UTP and a-D-glucopyranosyl-1- phosphate, has been examined in detail (19, and references therein). nie importance of a detailed examination of the GDP-mannose pyrophosphorylase from thyroid tissue is four-fold: (1) it. is currently postulated that regulation of the synthesis of the oligosaccharide moeities of glycoproteins is at the level of the synthesis of the nucleotide sugars (20-22); the regulation of the thyroid GDP-mannose pyrophosphorylase is not yet defined, (2) the carbohydrate content of thyroglobulin is about 8 to 10% of the protein molecular weight for the species examined (23) of whidi mannose comprises approximately 52%, (3) GDP-mannose is the precursor of GDP-fucose (guanosine-5'-/?-L-fucopyranosyl- 1-pyrophosphate) Which is the donor of fucose residues to the type B oligosaccharide unit of thyroglobulin and has been shown to be formed de novo by thyroid enzymes (24), (4) the synthesis of thyroglobulin is hormonally regulated, a consideration that should have importance in the level of expression of GDP-mannose pyrophosphorylase and in the overall synthesis and degradation of thyroglobulin itself. EXPERIMENTAL PROCEDURE
A. M a te ria ls The following reagents and proteins were purchased from Sigma Chemical Conpany: MOPS, TAPS, MES, CHES, (H)EPPS, T ris,
UDP-glucose, UDP-mannose, GDP-mannose, GDP-glucose, ADP-glucose, ADP-mannose, dTDP-glucose, UDP-galactose, UDP-N-acetylglucosamine, CDP-choline, myosin, bovine serum albumin, bovine liver catalase, yeast alcohol dehydrogenase, ovalbumin, phosphorylase b, yeast inorganic pyrophosphatase, and E. coli 0-galactosidase. Calf intestine phosphatase (Grade I) was purchased from
Boehringer Mannheim, Acrylamide, bis-acrylamide, diallyltartardiam ide, SDS, TEMED, ammonium persulfate, Coomassie R-250 and G-250, and dithiothreitol were purdhased from Bio-Rad Laboratories. Guanosine triphosphate and GTP-linked agarose were purchased from P-L Laboratories.
Mannose, CAPS, and o-nitrophenyl-/9-D-galactoside were purchased from Calbiochem. The column m aterials, DEAE-Sephacel, Sepharose C1-6B, Sephacryl S-300, Octyl and Phenyl Sepharose, and DEAE-Biogel A are products of Pharmacia.
6 (*^C)-mannose, (l-^C)-glucose-l-phosphate, and GDP-(U^C)-mannose radiolabelled compounds were purchased from
New England Nuclear.
Whatman 3MM and DE 81 were purchased from Whatman Inc. Orange Ribbon 589-C is a product of Schleicher and Schuell. SB-2 anion
exchange paper is a product of Reeve Angel. Triton X-100, PPO, POPOP, and toluene were purchased from Research Products Incorporated.
Fluram is a product of Roche Diagnostics. All other chemicals were reagent grade or better. Unless otherwise noted, all sugars are of the D pyranose configuration.
All nucleotide sugars (with the exception of GDP-0-L-fucose), mannose-l-phosphate, and glucose-l-phosphate are thea isom ers.
B. Methods Assays for Pyrophosphorylase Activity
The pyrophosphorylase catalyzes the following reversible r e a c tio n :
GTP + -D-mannose-l-phosphate ■«— ► GDP-mannose + pyrophosphate
Synthesis or degradation of guanosine diphosphomannose by the pyrophosphorylase may be determined using an appropriate enzymatically coupled assay in conjunction with an anion exchange paper (Whatman DE 81 or Reeve Angel SB-2). However, these papers non-s^ecifically bind negatively charged compounds wder
conditions o£ low ionic strength (DC81 < 50 mM? SB-2 < 250 mM). Thus, characterization of bound compounds requires additional
analytical procedures.
Synthetic Assay
A reaction mixture typically contains 20 rrM MOPS.NaOH, pH24 =7.8? 5 mM MgCl2.6 HgO (Buffer A), 2 mM Li^TP.2
H20 10 jiM (U-^c)-mannose-l-phosphate (10 /iCi/pmole), 0.5 units of yeast inorganic pyrophosphatase, and pyrophosphorylase in a total volume of 100 pi. In a 1.5 ml microcentrifuge tube, the reaction is initiated by rapid addition of the pyrophosphorylase followed by a brief vortex mixing. A 35 pi aliquot is immediately withdrawn using a micropipettor and spotted onto a 1.6 X 1.6 cm anion exchange paper "disc". This disc is washed for twenty minutes with distilled water by being held between mesh screens that may be submerged into a distilled water holding tank. The remaining mixture is placed into a 37° water bath for the length of the assay interval. At the desired time, 0.5 units of calf intestine alkaline phosphatase is added to fhe mixture and an additional incubation at 37° is carried out to hydrolyze residual mannose-l-phosphate substrate. Finally, a second 35 pi aliquot is withdrawn, spotted onto an anion exchange paper, disc, and washed as previously described. Washed discs are placed into scintillation vials with one ml of 9 0.2 M KC1 in 0.1 N HC1 and shaken for twenty minutes to elute the bound radioactivity. Ten ml of scintillation cocktail is added to
the sample and the sample counted in a scintillation counter. Presence of the pyrophosphorylase in a protein sample is inferred by an increased retention of radioactivity over time to the anion
exchange paper.
Degradation Assay
A typical reaction mixture contains 20 nM MOPS.NaOH, P^24 » 7.8; 5 mM MgCl2.6 HjO (Buffer A), 10 M GDP-(U14C)- mannose (10 pCi/pmole) 2.5 mM Na4P207.10 HjO, 0.5 units
of calf intestine alkaline phosphatase* and pyrophosphorylase
in a total volume of 60 pi. In a 1.5 ml microcentrifuge tube, the reaction is initiated by rapid addition of the
pyrophosphorylase followed by a brief vortex mixing. A 25 pi
aliquot is immediately withdrawn using a micropipettor, spotted onto an anion exchange paper disc, and washed as described in
the synthetic assay procedure. The remaining mixture is placed into a 37° water bath for the length of the assay interval. At the desired time, a second 25 pi aliquot is withdrawn,
spotted onto an anion exchange paper disc, and washed as previously described. Washed discs are placed into, scintillation vials with one ml of 0.2 M KC1 in 0.1 N HC1 and shaken for twenty minutes to elute the bound radioactivity. Ten ml of scintillation cocktail is added and the sample 10 counted in a scintillation counter. Presence of the pyrophosphorylase in a protein sample is inferred by a loss of bound radioactivity over time from the anion exchange paper disc.
Detection of the Pyrophosphorylase in Polyacrylamide Gels
After electrophoretic separation in non-denaturing gels and removal from the gel tube, detection of the pyrophosphorylase activity
is typically performed in one of two methodst (1) cross-sectional slicing and assay (described below) of a single gel with concommittant staining of a second gel, or ( 2) longitudinal slicing of a single gel followed by cross-sectional slicing of one half of the gel and subsequent assay (described below) with protein staining of the intact second half of the gel. The first method is advantageous in that twice as much protein is available for staining and enzymatic assay compared to the second method. However, the first method suffers from uncertainties in assigning enzymatic activity to a given stained protein band due to differences in electrophoretic conditions between two separate gels and the expansion in length of a gel under the conditions required for staining. The second method avoids the electrophoretic disadvantage but it also suffers from expansion of the half-gel under the conditions required for staining. Described below is an assay procedure for use with the second (preferred) method. After electrophoresis, the gel is removed from the glass tube by rimming the gel with D.D. H^O using a 20 cm 23 gauge needle attached to a 50 ml disposable syringe followed by gentle expulsion of the gel with a pipet bulb placed over one end of the gel tube. The naked gel is placed into the trough of a lucite block vrtiich is designed to hold the gel straight for slicing. The gel is then carefully sliced longitudinally with a Stadie microtome blade. The halves of the gel are separated; one half is placed into a protein staining solution and the other half is cross-sectioned using a home-made slicer constructed from double-edged razor blades separated by plastic spacers. The gel slices have a nominal thickness of 2.2 nm. The individual pieces are placed sequentially into separate microcentrifuge tubes -and covered with 90 fil of Buffer B. The presence of the pyrophosphorylase may be detected in situ within the gel by addition of 8 fil of an assay mixture composed of 2.5 mM sodium pyrophosphate, 100 /IM GJP-fU^Cj-mannose (10 fiCL/fimole), and 15.5 units of calf intestine alkaline phosphatase. The degradative assay employing DEAE anion exchange paper is then used.
Pyrophosphorylase A ctivity in Crude Homogenates
Detection of pyrophosphorylase activity in crude thyroid extracts requires two modifications of the degradative assay described above. F irst, adenosine triphosphate (ATP) must be included in the assay mixture to a final concentration of five 12
nM. This prevents pyrophosphate-independent hydrolysis of the guanosine diphosphomannose substrate within the time course of the assay* Second, the pyrophosphate concentration in the assay mixture must be increased to 7.5 mM to minimize this co substrate's depletion and the resulting premature termination of the pyrophosphorolysis of GDP-mannose.
Protein Assay The protein assay is a modification of a procedure described by Udenfriend (25) to measure the relative florescent intensity of discrete protein samples. In a typical measurement, a 10-25 pi aliquot of a sample of interest is mixed with 1.5 ml of 0.2 M sodium borate, PH 24 ■ 9.2. One half ml of Fluram, dissolved in dry acetone to a concentration of 3 mg/ 15 ml, is added rapidly to the above mixture and immediately mixed. The relative florescence of the Bample is measured at 475 nm on excitation at 390 nm. The protein concentration is derived by comparison to a standard curve constructed in an analogous fashion using bovine serum albumin.
Snake Venom (Phosphodiesterase) Assay Lyophilized preparations of eastern diamondback rattlesnake venom (Crotalus adamanteus) contain an enzymatic activity that, upon reconstitution in a suitable buffer, is capable of hydrolyzing the anhydride bond of pyrophosphoric acid diesters 13
under conditions of alkaline pH. This activity can be conveniently
utilized to prepare (^C)-mannoso-l-phosphate from commercially available GDP-t^Cj-mannose (Hew England Nuclear) or from 14 GDP-( c)-mannose derived enzymatically by either porcine thyroid
GDP-mannooe pyrophosphorylase or an extract of baker's yeast
(described belo^). A typical assay contains 1 pCi of GDP-
(^C)-mannose (without cold carrier), 5 pi of 10 mg/ml
snake venom reconstituted in TAPS buffer (131 mM? pH ** 8 ),
in a total volume of 100 pi. The reaction is initiated by the addition of the venom and a thirty minute incubation at 37° is
carried out. Following incubation, the mixture is subjected to paper electrophoresis in Solvent I, the radioactive region of the electropherogram is detected by liquid scintillation counting, and
the mannose-l-phosphate recovered by elution with 95% ethanol followed by D.D. HjO. Conversion of GDP-mannose to GMP and
mannose-l-phosphate is quantitative.
14 Chemical synthesis of (U C)- a -D-mannose-l-phosphate Synthesis of 1,2,3,4,6-O-pentaacetyl-D-mannose
This procedure is a modification of the method of MacDonald (26). In a typical synthesis, 0.5 pCi of (U^C)-mannose
(200 p C i/ Mmole) is combined with 50 pi of 1 mM aqueous mannose in
a 5 ml pear-shaped 14/20 round-bottom flask and taken to dryness overnight over phosphorus pentoxide. 0.5 ml of acetic anhydride containing 20 pi of conc. sulfuric acid is added to the flask, 14 fitted with a calcium sulfate drying tube, and the flask placed in an ice bath overnight. The reaction is quenched by the addition of * * the mixture to 5 gm of ice. After the ice has melted, the aqueous solution is extracted three times with 10 ml of chloroform. The chloroform extracts are combined and dried overnight at 4° over
anhydrous sodium sulfate. The yield is approximately 90%.
Fusion and deacetyl at ion of the peracetate
The peracetate in dry chloroform is taken to dryness on a rotary evaporator in a pear-shaped 14/20 5 ml round-bottom flask.
Solid phosphoric acid (ca. 30 mg) is added to the flask and the
mixture is fused in vacuo for 45 minutes at 58°. After fusion,
the reaction flask is chilled in an ice bath 'followed by . the addition of ice cold 2 N LiOH in a 3.5 molar excess with respect
to the phosphoric acid. Deacetylation is allowed to proceed
overnight at 4°, The reaction mixture is filtered to remove the insoluble trilithium phosphate. The filtered solid is washed with a minimum amount of D. D. H^O, the filtrates pooled, and a volume of 2 N formic acid is added equivalent to the 0.5 molar excess o f 2 N LiOH.
Isolation of ot-P-mannoBe-l-phosphate
The aqueous material obtained is streaked onto the origin of
Whatman 3 MM chrom atography p ap er w etted w ith 0 .2 M ammonium form ate (pH 53 2.7). The material is electrophoresed at 1500 V for 15 a period of time required for a picric acid marker to migrate 15 cm from the origin. The position of a-D-mannose-l-phosphate (Rp^c “ 0.9) is detected by liquid scintillation counting after sectioning a portion of the electropherogram in the direction of
migration. The area of the electropherogram containing the product is cut out and is eluted from the paper with D. D. H^o following a wash with 95% ethanol to remove the formate buffer. The best
overall yield was approximately 40%. However, Because the overall
yield was quite variable (often less than 2%) the chemical synthesis was replaced by the enzymatic method described below.
Enzymatic synthesis of radiolabelled mannose-l-phosphate While mannose-l-phosphate may be prepared by the
pyrophosphorylase reaction catalyzed by porcine thyroid GDP-mannose pyrophosphorylase, the tedious nature of preparing a suitable extract, the lack of long-term stability of the extract,
and the expense of commercially prepared radiolabelled GDP-mannose preclude its practical utility. However, crude extracts of baker's yeast have been shown (27) to be capable of converting relatively inexpensive radiolabelled mannose to radiolabelled GDP-mannose in respectable yields (ca. 60%). The yeast-derived GDP-mannose is isolated following paper chromatography in Solvent I, subjected to hydrolysis by snake venom phosphodiesterase, and recovered by the method described for chemically synthesized mannose-l-phosphate. 16
Preparation of Buffers
Measurements of pH are made using an Orion Research model 701A pH m eter. Buffers containing MOPS are prepared by dissolving the components in D.D. H^O to 90% of the final volume, titration with 10 N NaOH to the desired pH, and dilution with D.D. HjO
to achieve the final concentration* Buffers containng imidazole are prepared by dissolving the appropriate amount of imidazole in D.D. H^O to 90% of the final volume, titratio n with 10% (v/v)
aqueous acetic acid to the desired pH, and dilution to the final
volume with D.D. H20. Phosphate buffer is used as supplied with the addition of 30% hydrogen peroxide to achieve the final peroxide concentration.
B u ffer Ai 20 mM MOPS.NaOH PH24 “ 7 *8 5 mM M gCl 2
B u ffer Bt 20 mM MOPS.NaOH P«24 “ 7 ,8 5 mM M gCl 2 50 nM KC1
B u ffer Ct 10 mM Im idazole.Acetate PH24 *“ 6 «5
B u ffer Dt 100 mM Imidazole.Acetate PH24 " 17 B uffer E: 100 mM HOPS.NaOH pH24 = 7 .9
25 mM MgClj 250 mM KC1
B u ffer F: 20 mM MOPS.NaOH pH24 = 7 .8
5 mM M gCl 2
50 mM KC1
10 rtM 2-mercaptoethanol
B u ffer G: 70 mM Phosphate PH24 **
0.06% H2 0 2
B u ffer Ht 20, mM MOPS.NaOH pH24 = 7.8
Chromatography S o lv e n ts
S o lv en t I : 1-BuOH : EtOAc : HOAc :P yr : w ater {5 : 5 : 4 t 4 t 3)
Solvent II * 1-propanol: ethyl acetate: water (7:1:2)
Solvent III: ethyl acetate: pyridine: water (60 : 25 : 20)
Solvent IV: 95% e th a n o l: 1M NH4OAc pH = 5.0 (7 : 3) 18
S olven t V: 95% e th a n o l : 1 M NH^OAc pH ** 3 .8 (5 I 2)
Solvent VI: isobutyric acidt ammonium hydroxide: water
(66 x 1 x 33)
Chromatography was carried out in 30 X 30 X 61 cm rectangular glass chromatography tanks at room temperature under chamber-saturated (no curtains) conditions. Twenty fil
samples were applied with a glass capillary at discrete points along an origin drawn 15 cm from the proximal end of the chromatogram.
Paper Electrophoresis
Electrophoresis was carried out at 1500 volts for a period of time required for the movement of a picric acid marker to migrate a distance of 15 cm from the origin. Paper for electrophoresis was prepared by dipping in the solvent, blotting with tissue paper to remove excess solvent, and application of the samples. Samples
are applied (10 to 20 /il/cm) with an adjustable pipettor by
streaking along a predetermined distance along the origin.
S o lv e n t I : 0 .2 M NH4HC02 pH24 = 2 .7 Pipette Calibration Variable volume pipettors were calibrated using the Phenol Red oolorimetric method suggested by Bio-Rad Laboratories.
S tatistical Methods
Where data interpretation was amenable to linear analyses (sucrose velocity sedimentation* assay rates* gel electrophoresis* kinetic constants)* a linear least squares program available as a preprogrammed function of a Texas Instruments TI-55 calculator was used.
Scintillation Counting Determinations of the amounts of radiolabel utilized in 14 3 analytical and preparative procedures* both C and H* were routinely measured using a scintillation cocktail described by Patterson and Greene (28). Samples adsorbed to paper were first eluted with 1 ml of D.D. H^O (chromatography paper) or 1 ml of 0.2 M KC1 in 0.1 N HC1 (DEAE or SB-2 paper) followed by the addition of 10 ml of cocktail. Quantitation of the samples was was measured using a Beckman LS-230 scintillation counter.
pH Optimum Determ ination The enzymatic activity of the pyrophosphorylase (degradative assay) was examined over a range of hydrogen ion concentrations from pH ® 4 to pH ® 10. The hydrogen ion concentrations were fixed using a series of sulfonic acid-based buffers originally described by Good (29). However, because these buffers are
• # zwitterionic, the setting of the ionic strength of these buffers at a constant value (an important parameter) cannot be achieved in
the normal straight-forward manner. Since at all pH values > 0 these buffers exist as either the sulfonic acid anion or zwitterion, it was arbitrarily- decided to hold the sulfonic acid anion component concentrations constant and consider the contribution of the zwitterion form as being that of an • electrically neutral component.
Preparation of Polyacrylamide Gels Slab Gels Containing Sodium Dodecyl Sulfate (SDS)
The apparatus used is one that was first described by Reid and Bieleski (30) with the subsequent modifications suggested by Studier (31). The buffer system used is a modification of the method of Laemmli and Favre (32). The cassette is assembled using 0.8 X 19 cm spacers of a vinyl-based plastic having a nominal thickness of 1.5 nm and is
sealed against leakage using white petrolatum. The separating gel
solution is introduced into the cassette using a 20 ml disposable syringe fitted with an 1.5 inch 18 gauge needle. This solution
( 30 mis) is composed of 9.48 % acrylamide (w/v), 0.52 % bisacrylamide (w/v), 0.1 % SDS (w/v), 0.375 M Tris.HCl, “
8 . 8, 6 .6 X 10"4% (v/v), and 0.02% ammonium persulfate (w/v). 21
Using a nodification of the method of Ames (33), the separating
gel solution is gently overlayed with D.D. H^O to a depth of 2 nvn Which results in a sharp interface on polymerization of the gel solution. After polymerization, the water layer is poured off and several ml of Tris buffer containing SDS at the same
concentraton as in the gel is introduced. The gel may be stored overnight at room temperature at this stage or the stacking gel
may be added im m ediately. The stacking gel solution (10 ml) is composed of 3.88 % acrylamide (w/v), 0.12 % bisacrylamide (w/v), 0.1 % SDS (w/v),
0.125 M Tris.MCI, pH24 - 6 . 8 , 3.3 X 10“4% TEMED (v/v), and 0.02% ammonium persulfate (w/v). A portion of the stacking gel solution, after removal of the overlay solution, is introduced into the cassette in an identical fashion as described above. The sample comb is inserted into the cassette and an additional amount of stacking gel solution is added to fill the cassette to the level of the notch in the one glass plate. Water is again gently overlayered onto the gel solution and removed following polymerization. The comb is then removed resulting in wells formed within the gel into which samples of interest may be introduced. * • Protein samples for electrophoresis are placed into microcentrifuge tubes and an aliquot of a denaturing solution, modified from Hames (34), is added, mixed, and incubated at 100° for three minutes. This denaturing solution is composed of 22 501 sucrose (w/v), 10% SDS (w/v), and 2.5% dithiothreitol dissolved in 0.5 M Tris.HCl, P ^ 4 “ € .8. Preparation is consisted by the addition of an aliquot of 0. 1% aqueous Bromophenol Blue commonly used as a tra c k in g dye. However, protein samples containing KC1 must be dialyzed against 0.125 M
Tris.HCl, PH24 “ 6 * 8 * prior to denaturation because the solubility of potassium dodecyl sulfate results in an adequate SDS/protein ratio with artifactual protein bands being visualized after staining.
Non-denaturing Gels
Highly porous gels for the separation of native proteins can be constructed using a modification of the method of Peacock and Dingman (35). These agarose/acrylamide-based gels use the discontinuous buffer system of Laemmli and Favre (32) with the omission of SDS, or they may be run in a continuous fashion by omitting the stacking gel and SDS corribined with the upper and lower reservoir buffers at a concentration the same as in the separating gel. Alternatively, highly porous gels may be constructed by using high relative concentrations of the cross-linking reagent diallyltartardiamide (DATD) as a substitute for the bisacrylamide as reported (36). These gels possess good mechanical strength and adhesiveness to glass while retaining the ability to sieve native proteins of high molecular weight. 23 (i) Agarose-acrylamide Composite Gels
Cylindrical gels are formed in 12 cm lengths of silylated Pyrex tubing of 7 nm O.D. and 5 nm I.D .. Gel tubes are prepared first by silylation for 30 minutes in 2% dichlorodimethylsilane in carbon tetrachloride. Then, the gel tubes are washed, rinsed with acetone, and oven-dried. Finally, the distal end of the gel tubes are double-wrapped with Parafilm, inserted into the end of a serum stopper, and submerged upright to a depth of 10 cm in a 4° water bath. The final composite gel solution (15 ml) is conposed o f 0.5% SeaKem agarose (w /v), 2.85% acrylam ide (w /v), 0.15% bisacrylamide (w/v), 0.125 M Tris.HCl, pH,. ■ 8 . 8, 6.7 X 10"4% TEMED (v/v), and 0.013% ammonium persulfate. This solution is prepared by the addition of 0. 8% aqueous agarose (w/v) to the acrylamide solution equilibrated at 42° without TEMED or ammonium persulfate. When the composite solution is at thermal equilibrium, the accelerator and the catalyst are added, mixed, and the solution introduced into the gel tubes. The tubes are filled to a depth of 10 cm using a pre-warmed 10 ml g la s s sy rin g e fitted with a four inch metal cannula. The gel solution is carefully overlayered with D.D. HjO until polymerisation is complete. The tubes are removed from the bath, and the agarose allowed to gel at room temperature. Gels may be stored for up to one month at 4°. Stacking gels, when used, are conveniently prepared from .a warm solution composed of 0.5% Isogel agarose
(w/v) and 0.125 M T ris .H C l pH,. ** 6 .8 which is added to the 24
previously prepared gels to a depth of1 cm and allowed to harden at room temperature. Completed gels are equilibrated at 4° for 30 minutes in the electrophoresis apparatus (Hoefer Scientific)
prior to application of the samples. Samples are prepared by addition of glycerol to a final
concentration of 10% and an aliquot of 0. 1% aqueous Bromophenol
Blue for use as a tracking dye* (ii)Acrylamide Gels Employing DATD as a Cross-linker Cylindrical gel tubes are prepared as described above. The
separating gel solution is a modification of the method of
Baumann and Chratnbach (3 6 ). In th e s e g e ls , th e p o ro sity , and hence restrictiveness and resolution, is controlled by the percentage of the total acrylamide in the completed gel solution. Therefore, a procedure for constructing gels containing 11% T; 15% C ("T" is the total acrylamide concentration;
"C" is the percentage of bisacrylamide in the total acrylamide concentration) is described (K^ » 1 for proteins \rfiose
M < 100, 000) which may be altered in restrictiveness as dictated by the individual need. The gel solution (10 ml) is composed of 9.35% acrylamide
(w/v), 0.65% DATD (w/v), 0.375 M Tris.HCl, pH 24 « 6 . 8, *•3 1 X 10 % TEMED (v/v), and 0.02% ammonium persulfate (w/v). At room temperature, the separating gel solution is introduced into the gel tubes using a 10 ml disposable syringe fitted with a 1.5 inch 18 guage needle inserted into an 8 cm piece of 1.2 nm (ID), 25
1*7 nm (OD) polyethylene tubing (Intramedic). The gel tutbes are filled to a depth of 10 cm and overlayered with D.D. as
previously described. After polymerization, the water layer is removed, replaced with Tris buffer at a concentration equal to that of the gel, and the gels stored overnight at room temperature prior to their use. Stacking gels, When used, may be conveniently formed using the method described under section (i) of this heading, or as follows.
A stacking gel solution (10 ml) is composed of 3.4% acrylamide
(w/v)? 0.6% DATD (w/v )t 0.125 M Tris.HCl, pH - 6 . 8; —3 1 X 10 % TEMED, and 0.02% ammonium persulfate (w/v). The stacking gel solution is introduced into the gel tubes to a depth of 1 cm, overlayered with D.D. HjO, and polymerization allowed to proceed. Equilibration of the completed gels and preparation of
protein samples for electrophoresis is as described in section
( i ) .
2-Dimensional Electrophoresis
This technique employs a modification of a procedure originally described by O'Farrell (37). Cross-sectional slices
from either composite gels or gels utilizing DATD are first equilibrated in a three fold excess of 0.5 M Tris.HCl, pHg^ “
6 . 8, and an aliquot of denaturing solution (section I) at 37° for thirty to one hundred twenty minutes in microcentrifuge tubes. Denaturation is completed by incubation at 100° for three 26 minutes. The gel slices and their respective solutions are placed into individual wells In the stacking gel of an SDS slab gel and electrophoresis carried out as described below.
Electrophoretic Conditions (i)Slab Gels
After introduction of samples into the wells, slab gels are electrophoresed at 15 mA (constant) for a period of time required for the tracking dye to completely enter the stacking gel. -The amperage is then increased to 25 mA (constant) for the duration of
the electrophoretic run. In addition, it is imperative that the slab gel be cooled using a forced-air fan to prevent distorting
curvature of the separated protein bands due to differential heat dissipation within the gel. (il)Cylindrical Tube Gels
Tube gels are electrophoresed at 1 mA (constant)/tube until the tracking dye has entered the stacking gel. The amperage is then increased to 3 nA (constant)/tube for the duration of the electrophoretic run. Cooling of the gels is achieved by magnetic stirring of the lower reservoir buffer Which is in contact with a cooling jacket through which cold water is circulated.
Visualization of proteins separated by PAGE
Three methods o f detection were commonly employed. 27 (i) A method described by Diezel (38) for use with isoelectric focusing gels may be employed for either slab or tube gels. This method, based on Coomassie Blue G-250, is rapid but the sensitivity is lew when used for tube gels* (ii) The method of Fairbanks (39) is time consuming but
provides the highest sensitivity of detection for stains employing Coomassie Blue R-250. Tube gels may be stained directly following electrophoresis, but for slab gels, this procedure was modified to include a one to three hour pre-fixation step with 12.5% trichloroacetic acid to prevent the loss of acetic acid soluble proteins.
(iii) The silver-based staining method of Sammons (40) cannot be used on tube gels. Slab gels were first fixed with 12.5% trichloroacetic acid (w/v) followed by the recently modified silver staining procedure (41).
Sucrose Velocity Sedimentation
Sedimentation procedure Linear density gradients of 5% to 20% sucrose (w/w) in Buffer B were formed in 9/16" X 3.5" polyallomer centrifuge tubes (Beckman Instruments, Palo Alto, California). Gradients were delivered into the centrifuge tubes using a Buchler Auto Densi-Flow in conjunction with a Buchler peristaltic pump joined to the outlet of a linear gradient former. Completed gradients can be stored for 4 to 12 hours at 4° prior to their use. 28
Samples containing marker enzymes and the pyrophosphorylase were carefully layered onto the top of the sucrose gradients.
Centrifugation was carried out for 10 hours at 4° in a SW-41 Ti rotor at 37,000 rpm using a Beckman L2-65B ultracentrifuge. Upon completion of centrifugation, the tubes were carefully removed from the rotor and fractionated into testubes using the Auto Densi-Flow/ peristaltic pump combination joined to a Buchler LC
100 fraction collector.
Detection of Marker Enzymes l)Yeast Alcohol Dehydrogenase
Detection of the distribution of the enzyme within the
fractionated gradient is achieved by measurement of the change in optical density at 340 nm produced by the reduction of NAD* to
NADH concommitant with the oxidation of ethanol to ethanal. The
enzymatic reaction is initiated at room temperature by the addition and rapid mixing of an aliquot to an assay mixture composed of 2.8% ethanol, 0.024% NAD* in Buffer B in a total volume of 2.05 ml. 2)Bovine Liver Catalase
Detection of the distribution of the enzyme within the fractionated gradient is achieved by measurement of the change in optical density at 240 nm produced by the conversion of hydrogen peroxide to water and molecular oxygen. The enzymatic reaction is initiated at room temperature by the addition of an aliquot to an 29 assay mixture composed of 0.06% hydrogen peroxide in Buffer G in a total volume of 2.50 ml. 3)E. coll ^-qalactosidase Detection of the distribution of the enzyme within the
fractionated gradient is achieved by measurement of the change in optical density at 420 nm produced by the hydrolysis of o-nitrophenyl- -D-galactopyranoside to D-galactose and the o-nitrophenylate anion. The enzymatic reaction is initiated by the addition of an aliquot to an assay mixture composed of 3 nM o-nitrophenyl-/?-D-galactopyranoside in Buffer P.
4)Porclne thyroid pyrophosphorylase The pyrophosphorylase distribution is determined using the degradative assay previously described.
Calculation of Relative Mobility
The relative mobility of the marker enzyme b and th e pyrophosphorylase is calculated as that volume removed from the centrifuge tube required to reach the midpoint of the fraction exhibiting peak enzymatic activity divided by the total volume of the gradient. The peak of enzymatic activity for the marker enzymes is taken as that fraction whose aliquot exhibits the maximum relative change in optical density per unit time. For the pyrophosphorylase, peak activity is taken as that fraction whose aliquot exhibits maximum release of radiolabelled mannose from guanosine diphospho-(U-^C)-mannose. 30
Determination of the Pyrophosphorylase Specificity Measurement of the S pecificity Towards NDP-X Substrates
The specificity of the pyrophosphorylase towards an array of nucleoside diphosphate sugars was assessed by a series of assays
vriierein an equimolar (10 jiM) concentration (with respect to GDP-mannose) of a given nucleotide sugar was added to a reaction mixture. A decrease in the rate of pyrophosphorolysis (degradation assay) would therefore reflect the extent of
specificity. Assay mixtures were composed of 0.6 nanomoles of NDP-X, 0.6 nanomoles of GDP-(U^C)-mannose (10 fjCi/jimole), 150 nanomoles of sodium pyrophosphate, 0.37 units of alkaline phosphatase, and pyrophosphorylase in a total volume of 60 fxl of Buffer A. The reaction was initiated by the addition of the pyrophosphorylase. Hie mixture was incubated at 37° for 15 minutes. Twenty-five fil aliquots were withdrawn at zero time and following the 15 minute incubation for quantitation as described earlier for the degradative assay.
Measurement of the Specificity Towards Mannose-l-phosphate and Inorganic Pyrophosphate
The pyrophosphorylase was examined for sugar-l-phosphate specificity by assessing the extent of synthesis when glucose-l-phosphate is substituted for mannose-l-phosphate in the synthesis assay. The reaction mixture was composed of 0.12 14 nanomoles of (U- C)-glucose-l-phosphate (or mannose-l- phosphate, both 10 pCi/ ^mole), 60 nanomoles of GTP, 15 units of inorganic pyrophosphatase, and pyrophosphorylase in a total volume of 60 pi of Buffer A. The reaction was initiated by the addition of the pyrophosphorylase followed by a brief vortex mixing. A 25 pi aliquot is withrawn and Bpotted onto
an anion exchange disc. This disc is washed, eluted, and the amount of bound radiolabel quantitated as described in the synthetic assay procedure. The remainder of the reaction
mixture was incubated for 10 minutes at room temperature. Following this incubation, the reaction was quenched by the addition of 1.8 units (7 ft 1) of alkaline phosphatase (15 minutes at room temperature). Finally, a 30 pi aliquot is withdrawn, spotted onto an anion exchange disc, and processed as above.
The specificity for pyrophosphate (degradation assay) was determined by assessing the extent of pyrophosphorolysis both when pyrophosphate is replaced by inorganic phosphate and When
inorganic phosphate is included with pyrophosphate as a potential in h ib ito r . Where inorganic phosphate was examined as a potential co-substrate, the reaction mixture contained 0.15 micromoles of potassium phosphate (monobasic), 0.91 nanomoles of 14 GDP-(U C)-mannose (10 pCi/ pmole), 0.5 units of alkaline phosphatase, and pyrophosphorylase in a total volume of 80 pi 32 of Buffer A. Where inorganic phosphate was examined as a potential inhibitor, the reaction mixture contained 0.15 to
0.60 micromoles of potassium phosphate (monobasic), 0.91 14 nanomoles o f GDP-(U C )-m annose (10 p C i/ pm ole), 0.5 units of alkaline phosphatase, 0.15 moles of sodium pyrophosphate, and pyrophosphorylase in a total volume of 80 pi Buffer A. The reactions were initiated by the addition of the pyrophosphorylase and incubated for 15 minutes at 37°.
Specificity Towards NTP Substrates The specificity of the pyrophosphorylase towards nucleotide triphosphate (NTP) substrates was determined by examining their ability to serve as nucleotidyl donors in the synthetic assay procedure described earlier.
Reaction mixtures are composed of 12 nanomoles of nucleotide triphosphate, 0.12 nanomoles of mannose-l-phosphate (25 pCi/p mole), 15 units of inorganic pyrophosphatase, and pyrophosphorylase in a total volume of 60 pi of Buffer A. The reactions are initiated by the addition of the pyrophosphorylase followed by a brief vortex mixing. Immediately after mixing, a 25 pi aliquot is withdrawn and spotted onto an anion exchange disc. This disc is washed, eluted, and the amount of bound radiolabel determined using the procedure described for the synthetic assay.
The remainder of the reaction mixture is incubated for ten minutes at room temperature. Following this incubation interval, 33
1.8 units (7 ill) of alkaline phosphatase is added to quench the reaction (IS minutes at room temperature). Finally, a 30
Hi aliquot is withdrawn, spotted on an anion exchange disc, and processed as above*
The ability of various nucleotide triphosphates to serve as nucleotidyl donors is expressed as a percentage of the amount of radiolabel bound to an anion exchange disc, following incubation and treatment with alkaline phosphatase, relative to a control assay containing guanosine triphosphate.
Kinetic Constants Determinations Michael is Constant for GDP-mannose
The K m for GDP-mannose was determined by a modification of the degradative assay already described. This modification does not datermine the amount of GDP-mannose substrate remaining at the conclusion of the assay (i.e. as described for detection of the pyrophosphorylase in various column eluates) but rather, it measures the amount of radiolabelled mannose liberated from the pyrophosphorolysis of GDP-mannose to mannose-l-phosphate by the coupling enzyme alkaline phosphatase as shown belowt
pyrophosphorylase GDP-mannose + PPi ^ GTP + mannose-l-phosphate 34
alkaline phosphatase mannose-l-phospihate______^ mannose + phosphate
Reaction mixtures were composed of 0.15 micromoles of sodium
pyrophosphate, 0.15 - 1.2 nanomoles of GDP-(U14c)-mannose (10 pCi/ pmole), 0.37 units of alkaline phosphatase, and sufficient pyrophosphorylase in a total volume of 60 pi of Buffer A to
result'in 5% conversion of the substrate in 3 - 24 minutes at 37°. At the pre-determined time point, 25 pi aliquots are withdrawn, spotted onto anion exchange discs, and the discs
placed into scintillation vials. One ml of 1 nW aqueous Dmannose is added to each vial and the vials gently shaken for twenty minutes at room temperature. After shaking, 0.6 ml aliquots are
withdrawn and placed into a second set of scintillation vials with 0.4 ml of deionized distilled water. Ten ml of scintillation cocktail is added to each vial, the contents briefly mixed, and the amount of radioactivity determined by liquid scintillation
co u n tin g .
Michaelis Constant for Inorganic Pyrophosphate
The for inorganic pyrophosphate was determined by using the modified degradative assay described above for the Michaelis
constant for GDP-mannose. However, in this instance the GDP-mannose concentration was fixed at 1.2 nanomoles per assay (20 pM) while the concentration of inorganic pyrophosphate 35
was allcared to vary over the range of 30 - 180 nanomoles per assay (0.5 - 3.0 mM). In addition, the reaction time was fixed at 10 minutes and the incubation carried out at room temperature.
Michaelis Constant for Guanosine Triphosphate The apparent Michaelis constant for GTP was established by using a modification of the synthetic assay. The reaction mixtures were composed of 0.3 - 3.0 nanomoles of GTP, 0.12 nanomoles of D-mannoso-l-phosphate (25 pCi/ pmole), 15 units of
inorganic pyrophosphatase, and pyrophosphorylase in a total volume of 60 pi of Buffer A. The reactions were initiated by the addition of the pyrophosphorylase followed by a brief vortex mixing. Twenty-five pi aliquots were immediately withdrawn and spotted onto anion exchange discs. These discs were washed, eluted, and the amount
of bound radiolabel determined as described for the synthetic assay. The remainder of the reaction mixtures were incubated for ten minutes at room temperature. Following this incubation, 1.8 units of alkaline phosphatase (7 /il) was added to each reaction and an additional fifteen minute incubation carried out at room temperature. Finally, 30 pi aliquots were withdrawn from each reaction mixture, spotted onto individual anion exchange discs, and processed .as above. 36
Michaelis Constant for a-D-Mannose-l-phosphate The for mannose-1 -phosphate was determined by the method used to establish the apparent Michaelis constant for GTP with the following modifications. The GTP concentration was fixed at three nanomoles per assay (50 pM) While the mannose-l-phosphate concentration (100 pCi/ pmole) was allowed to vary from 0.03 nanomoles to 1.0S nanomoles per assay (0.5 pM to 1.8 pM.)
Product Analysis
The synthesis of putative GDP-mannose was achieved by incubating 20 nanomoles of GTP, 0.14 nanomoles of
(14C)-mannose-1-phosphate (100 pCi/pmole), and 150 units of inorganic pyrophosphatase with an excess of the pyrophosphorylase in a total volume of 20 pi. The assay mixture was incubated at room temperature for thirty minutes, 0.15 micromoles of authentic GDP-mannose added, and the reaction quenched by incubation at 100° for one minute. The mixture was spotted onto 18 X 58 cm sheets of Schleicher and Schuell 589-C Orange Ribbon paper in 20 pi aliquots at discrete
points along an origin drawn 15 cm from the proximal end. Authentic standards of GMP, GDP, GTP, GDP-mannose, and mannose-l-phosphate were also spotted. Descending chromatography
in solvent-saturated chambers (no curtains) was carried out at roam temperature for 20-24 hours for each solvent (IV, V, VI). After development, the chromatograms were air-dried, the 37
UV-adsorbing standards located by using a UVS-12 Mineralight (U ltra-violet Products), and the mannose-l-phosphate and putative GDP-mannose located by liquid scintillation counting.
In the direction of pyrophosphorolysis of GDP-mannose, the authenticity of the putative mannose-l-phosphate produced was established by (1), 7-minute hydrolysis of the radioactive eluate isolated by paper electrophoresis (Solvent I) Which was derived by the pyrophosphorolysis of authentic GDP-(U14c)-mannoser (2), treatment of the isolate from (1) with alkaline phosphatase followed by paper chromatography in Solvents I, II, and III. The reaction mixture analyzed was composed of 0.2 pCi of GDP-mannose (269 pCi/pmole), 20 nanomoles of sodium pyrophosphate and
excess pyrophosphorylase in a total volume of 20 pi. The mixture was incubated for thirty minutes at 37°, stre a k e d o n to Whatman 3MM paper (10 X 57 cm) and electrophoreB ed as described in Methods. The radioactive region of the electropherogram was sectioned out, washed by descending elution with 95% ethanol, and the radioactive material collected in a minimal volume by elution with D.D. HjO. An aliquot of the eluate was divided into two parts and subjected to 7-minute hydrolysis (42). The remainder of the eluate was subjected to hydrolysis at 37° for 20 minutes using 0.5 units of alkaline phosphatase. Twenty pi aliquots were spotted, along with standards, onto Whatman 3MM paper (15 X 41 cm) at discrete points along an origin drawn 10 cm from the proximal end. The 38
chromatograms were run by descending chromatography in
solvent-saturated chambers (no curtains) overnight at room
temperature. Following development, the chromatograms were air-dried and the migration of the standards revealed by the
alkaline silver nitrate method (43). The position of putative
mannose was determined by liquid scintillation counting after
sectioning the chromatogram along the direction of migration.
Purification of GDP-mannose Pyrophosphorylase
Homogenization Thyroid glands from recently killed pigs are obtained from
a local abattoir and held on ice for transport to the laboratory.
Unless otherwise stated, the following procedures are performed at 4 ° .
For small scale extractions using one to four porcine thyroid
glands, homogenization is conveniently carried out using a Potter-
Elvehjem ground-glass tissue homogenizer with a close-fitting
Teflon-tipped pestle driven by an overhead mixer. Extractions,
with Buffer A (1»2, w/v), are performed in an ice bath with minced tissue that has been prepared by the removal of extraneous fat and
connective tissue. For extractions on a preparative scale (10-15
thyroid glands), homogenization is carried out as above in a Sorvall Omni-mixer stainless steel canister. Extraction is performed by four, one minute extractions at the top speed of the mixer interspersed with one minute waiting periods to allow any 39 heat generated by the mixer to dissipate.
Centrifugation The crude homogenate obtained above is centrifuged for twenty minutes at 27,000 x g in a Sorvall RCB-2 refrigerated centrifuge using a Sorvall SS-34 rotor. The clarified supernatant is then ready for fractionation by column chromatography.
Synthesis of Blue Sepharose CL-6B Initial coupling procedure In a modification of the method of Bohme (44), 150 ml of packed Sepharose CL-6B slurry is washed extensively with D.D.
H^O to remove the Thimerosal preservative. The washed gel is taken i£> in three volumes of D.D. H20 in a two L flask and equilibrated, with shaking, at 60° in a water bath. One gm of Cibacron F-3GA blue dye (60% by weight) is dissolved in 250 ml of D.D. HjO and added dropwise to the gel mixture. After two hours at 60°, 250 ml of a 20% aqueous NaCl solution is added and the shaking continued for an additional three hours. Following this, the mixture is divided equally into two 1 L flasks and re-equilibrated to 80°. After equilibration, 50 ml of an 8.8% aqueous Na^CO^ solution was added to each flask with shaking continued for an additional hour. Finally, the mixture is cooled to room temperature, suction filtered, and washed extensively with D.D. HjO until the filtrate becomes c o lo r le s s . 40 Final coupling procedure
The partially coupled gel is taken up in 273 ml of D.D.
HjO in a two lite r flask and equilibrated to 60° with shaking.
After equilibration, an equal volume of a 1% aqueous solution of
Cibacron F-33A was added dropwise. Shaking is continued for an
additional hour followed by equilibration at 80°. After two
hours at 80°, 7.2 gm of sodium carbonate in 82 ml of D.D. H^O is added. Shaking is continued for 1.5 hours. Following this, the
solution is cooled to room temperature, suction filtered, and
washed extensively with D.D. HgO until the filtrate becomes colorless. Finally, the coupled gel is washed extensively and
alternately with 0.02 M sodium acetate, pH ■ 4.5, and 0.02 M
sodium bicarbonate, pH ■ 9.0 prior to equilibration with a
desired starting buffer.
Blue Sepharose Chromatography
One hundred ml (packed bed volume) of Blue Sepharose
resin is equilibrated with 1.5 volumes of Buffer A in a 500 ml
erlenmeyer flask. The clarified supernatant (ca. 250 ml containing
approximately 4000 mg of protein) from the above centrifugation
step was added to the resin and gently swirled at five minute intervals over the course of an hour during which time adsorption of the enzyme to the resin occurs. Following adsorption, a 4 X 7 cm column is poured, the resin allowed to pack into a stable bed, and the unretained protein eluted from the column by extensive 41 washing with Buffer A. When the washing of the column was completed, as evidenced by an eluate with an optical density at
280 ran of zero, the column outlet was connected to a Buchler LC 100 fraction collector and ten ml fractions were collected. The
enzyme was eluted by switching to Buffer A containing 0.5 M KC1. Fractions exhibiting at least a 1% per minute loss of radiolabel (degradative assay) were pooled and dialyzed overnight against
3.8 L of Buffer B. This "batchwise" procedure was found to have a significant advantage over the more commonly used method of adsorption to a pre-formed column with subsequent elution. It was experimentally observed that with an increasing interval of time between the initial adsorption and ensuing elution there was a dramatic decrease in the recovery of the in itially bound enzyme. The batchwise procedure minimizes the column contact time thereby affording significantly greater yields of the eluted enzyme.
Preparation of DEAE-Sephacel As supplied, the material is in the chloride ion form. To prepare for use, the chloride ion is exchanged for an acetate counterion. This is achieved in two steps. First, the anion exchanger is extensively washed (> 20 volumes) with 1 N sodium hydroxide to convert it to the more easily displaced hydroxide form. Second, the hydroxide form of the exdhanger is washed (5 volumes) with 1 M sodium acetate to convert it directly to the 42 acetate form. Finally, the material is washed with Buffer C until the pH of the filtrate is that of the buffer.
DEAE-Sephacel Chromatography
The Blue Sepharose dialysate (ca. 300 ml) is titrated to the approximate starting pH (6.5) of the Buffer C equilibrated
DEAE-Sephacel column with 10% aqueous acetic acid. The solution is applied to the column (2.5 X 11 cm) at an approximate flow rate of
60 ml/hr. Four ml fractions are collected during the initial loading of the sample solution and modified later as described
below. After the protein solution has completely entered the
column, the column is washed with Buffer C until the optical
density of the eluate at 280 nm is zero. At this point, a 200 ml linear gradient is initiated and is composed of 100 ml of Buffer C (mixing chantoer) and 100 ml of Buffer D (reservoir charriber). The eluting fractions are collected into teat tubes containing
one ml of Buffer E to facilitate an immediate re-adjustment of
the eluant pH to a more alkaline value and thereby minimize loss due to denaturation at the acidic pH of the eluting buffer. Fractions exhibiting a loss of radiolabel of at least 1% per minute (degradative assay) are pooled and concentrated to approximately twenty ml in an Amicon model 52 stirred-cell concentrator using a virgin YM 10 Diaflo membrane under 50 psi o f N2 . 43
Preparation of Phenyl Sepharose Phenyl Sepharose is supplied as an aqueous suspension in 0.01 M sodium phosphate buffer (pH = 6.8) containing 1 M ammonium sulfate. Preparation for use requires only equilibration in the starting buffer.
Phenyl Sepharose Chromatography
A 2.0 X 1.5 cm column of Phenyl Sepharose (5.5 ml packed bed volume) was formed in a ten ml glass syringe and equilibrated with Buffer A. The DEAE-Sephacel concentrate was applied at a flow rate of approximately 10 ml/ hr. After the protein had been
loaded onto the column, the column bed was washed extensively
with Buffer A until the optical density of the eluate was zero. Following the wash, the buffer is changed to 50% ethylene glycol in Buffer A (w/w). IWo ml fractions are collected using a Buchler LC 100 fraction collector and the column development is monitored
spectrophotometrically at 280 nm. Fractions exhibiting an absorbance of greater than 0.01 units are pooled and concentrated
to approximately six ml in an Amicon model 8010 stirred-cell concentrator using a virgin YM 10 Diaflo membrane under 50 psi of N,,. The concentrate is then placed into dialysis tubing and dialyzed overnight against 1 L of Buffer B. Preparation of the GTP-llnked Agarose
This material Is commercially available from P-L Biochemicals as an aqueous suspension containing 50% glycerol. Preparation for
use only requires equilibration in starting buffer.
Aqarose-GTP A ffinity Column
A 0.5 to 1.0 ml aliquot of the Phenyl Sepharose concentrate is diluted with two volumes of Buffer A and applied at a nominal flow rate of 0.9 ml/hr to a one ml column of
GTP-linked agarose equilibrated in Buffer B in a 2 ml disposable syringe. After the sample has been applied, the column is washed with 12 volumes of Buffer B followed by 12 volumes of Buffer A without MgClg. The enzyme is eluted with four volumes of Buffer
A (without MgClj) containing 1 mM sodium pyrophosphate and 50 fiM GDP-mannose. The eluate is collected directly into the sample
chamber of an Amicon model 3 stirred-cell concentrator and concentrated to a nominal volume of 100 jxl using a virgin YM 10 Diaflo membrane under 50 psi of N^.
Stability and Storage of the Pyrophosphorylase Glycerol and sucrose at concentrations of 50% (w/w) were without effect. Frozen solutions of the enzyme with or without the above exhibited an in itial 50% loss of activity. The Blue Sepharose and DEAE-Sephacel preparations have a half-life of approximately Bixty days at 4°. The Phenyl Sepharose preparation has a half-life of about six weeks at 4° affinity column preparation is stable for only a few days at 4°, Dithiothreitol and 2-mercaptoethanol do not preserve or re-activate enzyme a c t iv i t y . RESULTS
Purification off GDP-Mannose Pyrophosphorylase
F rozen v b Freah Tissue
Where small quantities off the pyrophosphorylase are required for investigative purposes, frozen porcine thyroid tissue may be used provided that the full extent of purification is not the intended objective. Porcine thyroid pyrophosphorylase suffers an initial (24 hours) 50% loss in potentially recoverable activity as compared to that of fresh tissue extracts. For small scale extractions (one to four glands; ca. 30 grams), a
Potter-Elverihjem homogenizer may be used. Where highly purified samples are required, the initial fresh tissue extraction is easily carried out using a Sorvall Omnimixer and the homogenization regimen described in the Methods section.
Blue Sepharose Chromatography
While normally considered a psuedoaffinity resin for the isolation of NAD(P)-requiring dehyrogenases, Blue Sepharose has been shown to exhibit variable binding affinity for a number of proteins and enzymes utilizing nucleotides as substrates or co-factors (45). In the course of establishing a scheme for purification, it was discovered that the pyrophosphorylase also 46 47
exhibited a strong association for Blue Sepharose - an
association that, with increasing column contact time, lead to poor recoveries (ca. 10%). Attempts were made to elute the enzyme specifically using the substrate analog guanosine monophosphate (GMP) but, although large purification factors were achieved (ca. 0.1%), recoveries were often much less than one percent of the total activity applied. Thus, the elution regimen chosen represents a compromise that allows rapid and near conplete recovery at the expense of potentially greater specific activity. Therefore, Blue Sepharose was selected as the initial
separation strategem because, of the alternative procedures (discussed below), chromatography on Blue Sepharose afforded the beBt combination of yield and increase in specific activity. As described in the Methods section, the bulk of the enzymatic
activity is recovered in the Buffer B eluate while little activity is not bound or retained by the resin at KC1 concentrations greater than 0.5 M (Fig. 4).
Chromatography on DEAE-Sephacel
Ion exchangers are often employed as the initial fractionation step in separations of mixtures of proteins because of their large capacity to bind soluble or solubilized proteins. Commonly, separation procedures include a linear (or otherwise regularly dianging) gradient of an increasing concentration of a neutral salt dissolved in a buffer. This type of procedure was originally Figure 4. Elution behavior of a crude thyroid extract on Blue
Sepharose. Pyrophosphorylase activity is eluted by
applying an 0.5 M KC1 in Buffer A wash (at arrow). The activity is pooled and dialyzed against Buffer B
for chromatography on DEAE-Sephacel.
48 O.D. 280 nm 5.00 3.00 4.00 2.00 1.00 20 40 le Sepharose Blue rcin Number Fraction ° Pyrophosphorylase Pyrophosphorylase ° 280nm O.D. • Activity 60 80 100 oo 0 2 1 100 80 60 40 20
Relative Activity *0 50 explored for the initial fractionation of thyroid extracts, however, it was later abandoned in favor of the Blue Sepharose step (above) for reasons to be described below. First of all, the pyrophosphorylase (or enzymatic activities exhibiting a positive response in the assay) was distributed on Cellex D (DEAE-cellulose, Bio-Rad Laboratories) into three clearly resolvable elution volumes (see Fig. 5 for nomenclature). The majority of the relative activity appeared in the void volume (termed "peak I"). Two other activities ("peak III" and "peak IV" respectively) were eluted under a linear gradient of 0-0.5 M KC1 in Buffer A; the peak III activity eluted at approximately 50 rrM and the peak IV activity eluted at approximately 150 mM.
These two components (III and IV) were quite variable in their relative activity with respect to the peak I activity. Also, the latter two activities were unstable to dialysis against Buffer A
while the peak I activity was stable without further treatment. Although it was determined that the peak I activity represented the bulk of the activity recovered, it was found that the yield
was only fifty percent of the applied activity and the gain in specific activity was about two fold. While some additional purification was achieved using the more stable peak I material,
subsequent losses in the later stages of purification lead to the conclusion that the ion exchange step, in its present form, was unacceptable. Figure 5. Elution behavior of a crude thyroid extract on Cellex D. Peaks I to IV refers to the sequential order of eluting pyrophosphorylase activity as determined by either the degradation or synthesis assay*
51 O.D. 280nm 4.00 3.00 2.00 1.00 20 EK EK IVPEAK PEAK HI I PEAK EK II PEAK CKCI3 PrpiibrU* Activity PyroptioipboryUc* O • OJJ. 2S0nm OJJ. • rcin Number Fraction
0 10 4 10 8 20080 180 160 140 120 100 0.3 0.4 .t o 100 50 90 60 80 70 10 20 30
Relative Activity Ui 53 In the final purification scheme, DEAE-Sepahcel (a beaded form of cellulose) was used to initially adsorb the pyrophosphorylase at a pH at Which the enzyme appears to carry a net negative charge (pH,. “ 6.5), Elution is effected by employing a linear gradient of pH to a terminal value at Which the enzyme appears to carry a net positive charge (pH,. « 5.5; see Fig. 6). This procedure was selected on the basis of experiments on chromatofocusing on columns of DEAE-Biogel A
(Bio-Rad Laboratories) using the method of Sluyterman and Elgersma (46). As shown in Fig. 7, the buffering capacity was not constant across the range of pH chosen nor was it possible to reach the enzyme's apparent pi by chromatofocusing under the conditions employed. Subsequent elution of the enzyme usin g 10 mM im idazole a t pH25 “ 4.9, although separating the enzyme from the bulk of the adsorbed protein, resulted in poor yields and low specific activity. However, from this data it was posssible to define a range of pH, using small columns of DEAE-Biogel A at fixed pH values (Fig. 8), over which the pyrophosphorylase could be rapidly bound and released. A preliminary trial, in the absence of protein, establishing the linearity of the applied gradient is shown in Fig. 9). The final procedure described in the Methods section provides the same yield as in the case of a linearly applied salt gradient but achieves a significantly higher purification of about sixteen fo ld . Figure 6. Elution profile of a Blue Sepharose dialysate from DEAE-Sephacel (DEpH). The activity eluting under the pH gradient is pooled and concentrated for
chromatography on Phenyl Sepharose*
54 O.D. 280 nm 0.800 0.500 0.600 0.700 0.400 0.200 0.300 0.100 - - ~9 I - - i 0- -0 0 t I • / *
*
20 r aX) 'a ° * =r° 1 •- •Soi —i —ooo * o o o— i— i— i— ‘•wSJo-i— -• -• • — • — 1— 1 0 0 0 100 80 60 40 \ i ' -L o -1 o /- 120 r • a 140 o6.0 6 •o 160 1 5.0 5.5 6.5 X a 1 0 J 40 60 100 80 20 Relative Activity Ui IT Figure 7. Chromatography of a Cellex D (peak III) dialysate on DEAE-Biogel A. At "A", 10 mM imidazole acetate buffer, pH " 4.8, was applied. At "B", 0.5 M KC1 in Buffer A was applied to release the bulk of the bound protein. 56 O.D. 280nm 0.80 0.60 0.20 0.40 1.40 1.00 1.20 \ 0 0 0 0 0 0 0 80 70 60 50 40 30 20 10 \ pH — Prpopoyae Activity Pyrophosphorylase o 280nm O.D. • \ rcin Number Fraction \ \ \ \ J L u J \ \ i 0 .0 4 * - • ^ ♦ ^ - 0 100 90 1.0 1 n 10.0 8.0 5.0 6.0 7.0 . n 9.0 I . Q 100 50 60 0 10 30 80 90 20 70 40 Relative Activity in Figure 8. Elution behavior o£ a Cellex D dialysate on DEAE-Biogel A columns of fixed pH. At fraction 5 (arrow), 0.5 M KC1 in Buffer A was applied to release the bound protein. 58 59 1 0 0 0.20 8 0 pH-6.6 - O O.D. 280nm 6 0 O Pyrophosphorylnso Activity CM 0 .1 0 4 0 20 0.00 10 100 0 .4 0 8 0 pH-6.1 C 0 .3 0 - • O.D. 280nm 6 0 O Pyrophosphorylnso Activity CM 0 .2 0 4 0 0.10 20 0.00 o — 10 100 pH-5.5 8 0 • O.D. 280nm O Pyrophosphorylnso Activity 6 0 4 0 20 Relative Relative Activity Rejative Activity Relative Activity 1 23456 789 10 Fraction Number Figure 9. The pH profile of a linearly applied pH gradient on a 1.5 X 4.5 cm DEAE-Sephacel column. This was a model study for the method reported herein for the purification of the pyrophosphorylase. 60 61 6.7 6.5 6.3 £6.1 5.9 5.7 5.5 10 15 20 25 30 Fraction Number 62 Chromatography on Phenyl Sepharose In the course of several experiments involving discontinuous gel electrophoresis under non-denaturing conditions, it was noted that the pyrophosphorylase exhibited a marked tendency to precipitate at the stacking gel-resolving gel interface. This tendency to precipitate was observed even under conditions of low restrictiveness to migration provided by high porosity gels. An in itial experiment was performed to determine the elution behavior of the pyrophosphorylase on exposure to a hyrophobic resin, Octyl Sepharose (Pharmacia). A protein mixture (in this instance, an eluate from Blue Sepharose) and the column material were equilibrated to conditions of high dielectric (1 M KCl) as is normal for this type of chromatography. 'After application of the sample and subsequent washing at the same ionic strength, a linear gradient of increasing ethylene glycol (0-50% w/w) and decreasing KCl concentration (1.0-0.0 M) was applied. Analysis of the total column eluate indicated that the enzyme was neither eluted in the void volume (or subsequent wash) nor released under the elution conditions employed. It was therefore decided to utilize a lesB hydrophobic resin, Phenyl Sepharose (Pharmacia), and examine the results obtained using the same elution regimen employed for Octyl Sepharose. From this experiment it was observed that the pyrophosphorylase was sufficiently hydrophobic to bind to Phenyl Sepharose under the minimal ionic strength conditions of Buffer A (plus 25 nM KCl, fig. 10). However, a Figure 10. Elution profile of a DEAE-Sephacel concentrate applied to Phenyl Sepharose equilibrated in Buffer A. Enzyme activity is eluted (arrow) by applying a 50% ethylene glycol (w/w) in Buffer A wash. The activity is pooled, concentrated, and dialyzed for chromatography on Agarose-GTP affinity columns. 63 O.D. 280 nm 0.300 .0 fU -180 flU h 0.200 100h ^ 40 - ^ h 0 0 .1 0 0-0 _ o , / hnl phaose aro h ep S Phenyl rcin Number Fraction Prpopoyae ciiy 00 i0 n Activity Pyrophosphorylase O .. 280nm O.D. 0 0 40 30 20 —•—< — • *— ° 0 > ? % I? H - 60 20 Relative Activity ' O 65 concentration of ehtylene glycol of at least 50% (w/w) was still required for elution (this experiment was performed using a peak I eluate of Cellex D). At this stage, the purification approximated only 1.6 fold with yields in the range of twenty to forty percent. However, trtien this step is incorporated at its present place in the purification scheme, the purification approaches a value of approximately six fold with yields of about fifty percent. Chromatography on Agarose-linked GTP Affinity chromatography on the GTP-linked resin comprises the final column procedure. Ho column fractions are taken during development. The ensyme is adsorbed to the resin following a three-fold dilution with Buffer A, washed extensively with Buffer G, and s p e c if ic a lly e lu te d d ir e c tly in to an Amicon s t i r r e d - c e l l concentrator. This procedure is necessitated by the fact that the total yield of protein following elution is in the range of one to six micrograms and significant variations in the yield of enaymatic activity (40 to 80%) can be observed. This observation is mirrored in an inverse relationship (i.e. with respect to yields of activity) resulting in purification factors of thirty to forty-five fold respectively. 66 Conroents on Additional Separation Procedures 1 . Ammonium S u lf a te Fractionation within selected percentage ranges using saturated solutions of ammonium sulfate is a common crude separation procedure. However, such fractionation procedures cannot be employed in the purification of the pyrophosphorylase. Exposure of the enzyme to ammonium sulfate solutions results in the complete disappearance of detectable (crude extracts) activity both following in itial dilution for the assay procedures described and following extensive dialysis to remove trace amounts of residual ammonium sulfate. Thus, this step does not appear in the final purification scheme. 2. Gel Filtration Protein purification schemes employing separations based on molecular weight (Sephadex, Sepharose, Sephacryl) are usually employed in the later stages of any purification scheme due to their relatively low (eg. with respect to ion exchangers) capacity for protein loading. In regards to the final procedure described herein, this type of separation procedure would be most appropriate following the DEAE-Sephacel step. However, when attempts were made to exploit molecular weight differences of the proteins contained within the DEAE-Sephacel eluate using Sephacryl columns (Pharmacia), anomalous behaviors were observed (Fig. 11). Firstly, sucrose velocity sedimentation and SDS electrophoresis had shown that the native molecular weight of the Figure 11. Elution behavior of a DEAE-Sephacel dialysate applied to Sephacryl S-300. (A), elution volume for yeast /?-galactosidase; (B)« elution volume for bovine liver catalase? (C), elution volume for yeast alcohol dehydrogenase. 67 O.D. 280nm 4.00 r 4.00 2.00 3.00 1.00 yohshrls Activity Pyrophosphorylase o 280nm O.D. • 10 rcin Number Fraction 20 BC 30 40 50 100 80 90 70 60 50 10 20 30 40 Relative Activity oo O' 69 pyrophosphorylase was approximately 412,000 daltons and 407,000 daltons respectively. The apparent native molecular weight derived from gel filtration chromatography on Sephacryl S-300 Indicated that the molecular weight was lower than that of alcohol dehydrogenase (yeast, 150,000 daltons). As shown in Fig. 11, the enzymatic activity eluted within the bulk of a broad protein peak; concentration of the active fractions gave low yields and low purification values. Secondly, on several occasions, detectable enzymatic activity completely failed to elute. The reasons for such abberant behavior is as yet unclear, however in light of these results, further attempts at gel filtration of the pyrophosphorylase were abandoned. 3. Hydroxyl Apatite Hydroxyl apatite is most often included as one of the final steps of a purification scheme. Its intended purpose is to remove trace quantities of impurities from the then purified sample. The behavior of the pyrophosphorylase on adsorption and elution from hydroxyl apatite is shown in Fig. 12. The conclusion drawn from this figure is that the enzyme exhibits a non-specific interaction with the column material and is eluted in variable amounts relative to the phosphate concentration in the eluting buffer. Since no differential elution (i.e. separation of the enzymatic activity from extraneous protein at a fixed or limited range of phosphate concentration) could be achieved, this procedure was abandoned. Figure 12. Elution behavior of a DEAE-Sephacel dialyaate applied to hydroxyl apatite equilibrated in Buffer A. At "A", 20 rrM phosphate in Buffer A was applied; at "B", 40 nM phosphate in Buffer A was applied; at "C", 80 mM phosphate in Buffer A was applied. 70 O.D. 280nm 0.90 1.00 0.80 0.70 0.50 0.60 0.40 0.20 0.10 0.30 '-oo 10 rcin NumberFraction yohshrls Activity. Pyrophosphorylase o O.D.280nm• 20 30 40 50 100 90 80 70 60 50 10 30 40 20 Relative Activity 72 4. Concanavalln A-linked Sepharose The pyrophosphorylase, and the bulk of extraneous contaminating protein, derived from elution from DEAE-Sephacel failed to bind to this glycoprotein-binding (mannose specific) column m aterial. Therefore, no separation was achieved with this m a te ria l. 5. Procion Red Agarose The DEAE-Sephacel isolated material behaves in an identical fashion on elution from this column material as compared to its behavior on elution from Blue Sepharose. Therfore, there was no advantage in using this material. 6. Phospho-cellulose and Carboxymethyl-cellulose The pyrophosphorylase, and the bulk of the applied protein, showed no tendency to bind to these column m aterials. PAG Electrophoretic Behavior of GDP-Mannose Pyrophosphorylase Because of the variability of results achieved using gel filtration, it was suggested that perhaps an electrophoretic separation, in addition to providing an alternative method of native molecular weight determination, could be used as a method of purification. Initial attempts were made to fractionate a crude mixture containing the pyrophosphorylase on non-denaturing 5% acrylamide gels (2.6% cross-linking). A separation was achieved (as judged by Coomassie Blue staining), however, an analysis of sequential gel slices (degradative enzyme assay) showed that the 73 pyrophosphorylase activity was contained in the topmost (cathode) slice coincident with a stained band o£ protein. By an independent method (sucrose sedimentation; discussed below) the native molecular weight of the enzyme was approximated as 412,000 daltons. Since a 5% polyacrylamide gel of this cross-linking is too restrictive for proteins in this range of molecular weight, the use of agarose-acrylamide composite gels employing a continuous zone system (MOPS buffered) was adopted. As described by Hames (34), these gels are potentially capable of fractioning proteins approaching one million daltons in molecular weight. Using the continuous zone system, the enzyme entered the gel, however, the gel slice analysis revealed that the enzymatic activity was not confined to a discrete peak, but rather it was broadly dispersed over a two cm region of the gel. The diffuse nature of the enzymatic activity was coincident with a similarly diffuse protein band revealed by staining. Attempts were made to focuB the diffuse activity by utilizing a discontinuous zone method (32). The result of these experiments were analogous to the results obtained from the 5% acrylamide gels. That is, the enzymatic activity was confined to the topmost portion of the gel (i.e. the resolving gel). Since composite gels, in principle, should be non-restrictive to the enzyme, it was decided that the electrophoretic behavior was an artifact of the discontinuous system itself (i.e. under the conditions used to generate the protein "stacks", high local protein concentrations are achieved 74 - conditions which may lead to precipitation When the stack encounters the mere restrictive resolving gel). At a later date, the composite gels were superseded by the less arduous DATD cross-liked gels with identical results to those already described. In order to circumvent the problem of precipitation, the stability of the pyrophosphorylase in the presence of the mild, non-denaturing detergent Triton X-100 (Rohm and Haas) was assessed. The results of this experiment showed that there was no initial loss in enzymatic activity in the presence of the detergent nor any loss following exposure for twenty-four hours at concentrations of Triton X-100 up to 0.1% (w/v). Therefore, DATD-cross-linked gels with 0.1% Triton X-100 were constructed and the pyrophosphorylase subjected to electrophoresis using the discontinuous zone system. Following electrophoresis, the gel was sliced and stained in the manner already described. These results again showed a diffuse band of enzymatic activity with a conicident stained band of protein. The diffuseness of the pyrophosphorylase may, in part, be due to the detergent, however, contaminating proteins were observed to have been sharply focused. Plates 1 and 2 show the SDS electrophoretic protein banding patterns for representative eluates derived from the individual pooled column fractions in the course of purification. From left to right (lanes 1 to 8 respectively), the components arei (1) molecular weight standards (myosin, 212,000; phosphorylase b, 92,500; bovine serum albumin, 68,000; catalase, 57,500; ovalbumin, Plate 1. SDS polyacrylamide gel (10%) stained with Coomassle R-250 by the method of Fairbanks (39). From left to r i g h t i Lane 1: molecular weight markers; lane 2t crude thyroid extract; Lane 3t Blue Sepharose eluate; Lane 4t DEAE-Sephacel eluate; Lane 5: Phenyl Sepharose eluate; Lane 6t Agarose-GTP affinity column eluate; Lane 7t molecular weight markers# and Lane 8: buffer components. 75 Plate 2 SDS polyacrylamide gel of Plate 1 re-stained by silver s ta in in g . 77 P late 2 79 43,000); (2) crude thyroid extract; (3) Blue Sepharose eluate; (4) DEAE-Sephacel eluate; (5) Phenyl Sepharose eluate; (6) Agarose- OTP affinity column eluate; (7) molecular weight standards, and (8) buffer components for protein denaturation. Plate 1 shows the banding pattern revealed by Coomassie R-250 staining (39). Noticeably absent from lane 6 (the most purified fraction) is any evidence of protein banding. Plate 2 shews the banding pattern revealed by silver staining (40). This staining technique reveals the presence of two bands (lane 6) of approximately equal intensity and of identical coloration (an additional aspect of this particular technique, 40). Lane 8 (Plate 2) highlights an unfortunate disadvantage of silver staining - the production of artifacts that, following electro phoresis and subsequent staining, are expressed as bands that mimic electrophoretically separated proteins. In this regard, it should be noted that the two bands of lane 6 are not found in lane 9 and are therefore construed to be derived from the pyrophosphorylase itself. pH Optimum Figure 13 shows the profile of relative enzymatic activity aB a function of the hydrogen ion concentration at 37°. The pyrophosphorylase is active over a wide range of pH with maximal relative activity in the range of pH ** 6 to pH * 7.5. Relative activity was measured using the degradative assay procedure. Figure 13. Effect of pH on the GDP-mannose pyrophosphorylase enzymatic activity as determined by degradation assays. 80 Relative Activity 0 r 60 ro co A cn o o o o o ai O) * o ■ o X X CD CO 18 82 Kinetic Constants Determinations Michael is Constant for GDP-mannose Figure 14 shows the double reciprocal plot obtained from i n i t i a l v e lo c ity measurements Wherein th e GDP-mannose concentration was allowed to vary against a fixed saturating concentration of inorganic pyrophosphate. From the data obtained, and by using linear regression analysis, the apparent Michaelis constant for GDP-mannose is 1.9 jxM. Michaelis Constant for Inorganic Pyrophosphate Figure 15 shows the double reciprocal plot obtained from in itial velocity measurements wherein the inorganic pyrophosphate concentration was allowed to vary against a fixed saturating concentration of GDP-mannose (as determined above). From the data obtained, and by using linear regression analysis, the apparent Michaelis constant for inorganic pyrophosphate is 1.0 ntt. Michaelis Constant for GTP Figure 16 shows the double reciprocal plot obtained from in itial velocity measurements Wherein the guanosine triphosphate concentration was allowed to vary against a fixed saturating concentration of mannose-l-phospahte. From the data obtained, and by using linear regression analysis, the apparent Michaelis constant for guanosine-51 -triphosphate is 3.5 fxM. Figure 14. Lineweaver-Burke plot for determining the apparent M ichaelis constant for GDP-mannose. 83 2.0 X10 1.0 - 0.6 -0.5-0.4 -0.3 - 0.1 0 0.1 0.4 CGDP-Mannose] 00 Figure 15. Lineweaver-Durke plot for determining the apparent Michaelis constant for inorganic pyrophosphate. 85 9.0 8.0 7.0 6.0 - 5.0- 4.0- 3.0 y S 1.0 -1.0 -0.5 0 0.5 1.0 1.5 2.0 [PPi]»mM’’ 00O'* Figure 16. Lineweaver-Burke plot for determining the apparent Michaelis constant for guanosine triphosphate. 87 1 .2 5 r 1 V i - I 1 « I t » i i I ____ i I_____ L -0 .3 - 0.2 - 0.1 -1 1 ;j4M [GTP] 00 00 89 Michaelis Constant for Mannose-l-phosphate Figure 17 shows the double reciprocal plot obtained front in itia l velocity measurements Wherein the mannose-l-phosphate concentration was allowed to vary against a fixed saturating concentration of GTP (as determined above)* From the data obtained, and by using linear regression analysis, the apparent Michaelis constant for mannose-l-phosphate is 0.4 fiM. GDP-mannose Pyrophosphorylase Specificty Specificity towards NDP-X substrates Table 1 shows the results of a series of degradative assays Wherein an array of nucleoside diphosphate sugars were examined for their ability to influence the pyrophosphorolysis of GDP-mannose. At saturating concentrations of GDP-mannose (10 #iM) and at an equimolar concentration of NDP-X, there is no significant decrease in the rate of pyrophosphorolysis of GDP-mannose as measured by the anion exchange disc assay. Thus, the pyrophosphorylase is assumed to have a strict specificity towards th e n u c le o tid e su g ar, GDP-mannoBe. Specificity towards sugar-l-phosphates and pyrophosphate Table 2 shows that glucose-1-phosphate (2-epimer of mannose-l-phosphate) is not a substrate for the pyrophosphorylase (other radiolabelled sugar-l-phosphates were not available for Btudy). This table also shows that there is a strict specificity for inorganic pyrophosphate over inorganic phosphate. In the Figure 17. Linewoaver-Durke plot for determining the apparent Michaelis constant for mannose-l-phosphate. 90 3 .0 CMANN0SE-1-P043 ’ 91 92 Table 1. Specificity of porcine thyroid pyrophosphorylase towards nucleoside diphosphate sugars. Additions Relative Activity None 100 ADP-glucose 105 ADP-mannose 100 CDP-choline 93 CDP-glucose 100 GDP-fucose 97 GDP-glucose 100 dTDP-glucose 105 U D P-galactose 95 UDP-glucose 95 UDP-N-acetylglucoBamine 93 UDP-mannose 95 All sugar moieties have the D configuration with the exception of GDP-/?-L-fucose. 93 Table 2. Specificity of porcine thyroid pyrophosphorylase towards sugar-l-phosphates and inorganic pyrophosphate as determined by synthetic assays. Compound______Relative Activity at -D-mannose-l-phosphate 100 a-D-glucose-l-phosphate 0 Na4P2°7 (none) 0 Na4P20 ? ( 2 .5 mM) 100 KH2P04 (2.5 mM) 0 KH-PO. (1 0 .0 mM) 0 2 4 2.5 mM Na4P20? + KH2P04 (2.5 mM) 100 (10.0 mM) 100 94 absence of pyrophosphate, there is no appreciable pyrophosphorolysis of GDP-mannose while, in the presence of pyrophosphate, there is no measurable inhibition of the pyrophosphorolysis of GDP-mannose in the presence of inorganic phosphate at concentrations up to 10 nM. Speclficty towards NTP substrates Various nucleotide triphosphates were examined for their ability to serve as nucleotidyl donors to mannose-l-phosphate under synthetic assay conditions. Table 3 shews that pyrimidine nucleotides, and the purine nucleotide ATP, are not substrates for the pyrophosphorylase. Additionally, While the extent of synthesis is greatest When GTP is utilized as the nucleotidyl donor, there is an appreciable amount of synthesis in the presence of either dGTP or ITP; an observation that perhaps reflects the bulk tolerance specificity of the pyrophosphorylase towards these two substrates. Product Analysis While the results of the specificity studies described above would infer that the substrates and products of the reaction catalyzed by the pyrophosphorylase are GTP, mannose-l-phosphate, GDP-mannose, and inorganic pyrophosphate respectively, it is the non-specific nature of the anion exchange disc assay which requires that the products of the reaction (in both a synthetic and degradative sense) be characterized by additional methods. 95 / Table 3. Specificity of porcine thyroid GDP-mannose pyrophosphorylase towards NTP substrates as determined by synthesis assays. NTP Relative Activity GTP 100 dGTP 34 ITP 44 ATP 0 CTP 0 TTP 0 UTP 0 96 That is, the disc assays merely distinguish between radiolabelled compounds that bind or do not bind under the particular experimental conditions employed. Thus, it becomes necessary to identify these products by further chemical or chromatographic means. Table 4 shows the results of chromatographic separations of reactions run in the synthetic direction under conditions vdierein the conversion of GTP and mannose-l-phosphate to GDP-mannose and inorganic pyrophosphate is virtually quantitative (see Methods). In three separate chromatography solvent systems, the major radioactive product formed is shown to co-migrate with authentic standards of GDP-mannose. A trace quantity of radiolabel co-migrated with authentic mannose-l-phosphatej no free mannose was detected. Because the absolute amount of product synthesized was small, it was not possible to detect the co-production of inorganic pyrophosphate. Table 5 shows the results of a seven minute hydrolysis of a radiolabelled compound, isolated by electrophorectlc methods, produced in a degradative assay containing GDP-mannose and inorganic pyrophosphate. While this analysis distinguishes anomeric phosphates from other possible mannose-phosphate isomers, it cannot distinguish between the anomeric isomers themselves. However, these results establish that the position of attachment of the phosphate on the mannose moiety is the anomeric carbon. Table 6 shows that when the above isolate is treated with Table 4. Paper chromatography of putative GDP-mannose Solvent IV Solvent V Solvent VI % r g m p * Rf Compound GMP 0. 27 1.00 0. 24 GDP 0. 14 0 .4 4 0. 16 GTP 0. 08 0.21 0. 09 GDP-mannose 0. 21 0. 37 0. 10 GDP-mannose 0. 21 0 .3 7 0. 10 ( putative) m annose-1 -phosphate 0 .4 4 1.74 0. 18 * Migration relative to GMP Table 5. Seven minute hydrolysis o£ putative mannose-l-phosphate Compound______Percent Loss at seven minutes mannose-1 -pho sphate 0 ( putative, unboiled control) mannose-l-phosphate 97 ( treated) Percent loss was determined as the amount o£ radiolabel bound to an anion exchange disc following hydrolysis relative to the unboiled control. Discs were processed as described in Methods. Table 6. Paper chromatography of mannose released by alkaline phosphatase treatment of putative mannose-l-phosphate Rglc Compound Solvent I Solvent II Solvent 111 D-glucose 1.00 1.00 1.00 L-fucose 1.78 1.36 1.53 D -rham nose 2. 13 - 1.77 D-mannose 1. 21 1. 10 1. 20 D -m annose 1. 28 1. 11 1.20 ( putative) Migration was measured relative to glucose ( Rglc) 100 alkaline phosphatase to remove the phosphate aglycone (thus eliminating the possibility that the isolate is GDP-mannose itself), the radiolabelled compound w ill co-migrate with authentic mannose standards in each of three separate chromatography systems. Additionally, as in the case of inorganic pyrophosphate above, the absolute amount of labelled compound is small and therefore the co-production of GTP was not detected. Sucrose Velocity Sedimentation As described earlier, the peculiar chromatographic and electrophoretic behavior of this enzyme precluded the use of gel filtration and non-denaturing polyacrylamide gels as means of establishing the native molecular weight. As a result of these studies, it was decided that sedimentation in linear sucrose gradients would offer a plausible alternative to obtain the desired information. Figure 18 shows the sedimentation behavior of the pyrophosphorylase in the presence of three internal molecular weight standards. Under the conditions employed, the pyrophosphorylase exhibits a sedimentation behavior equivalent to a globular protein of approximately 412,000 in molecular weight (ca. 15 Svedberg units). 101 Figure IB. Calibration curve and native molecular weight of GDP-mannose pyrophosphorylase as determined by sucrose velocity sedimentation. Sucrose Density 5 .7 r 0 -Galactosidase • 5.6 GDPM Pyrophosphorylase 5.5 5 5.4 O) • Catalase o 5.3 5.2 • Alcohol Dehydrogenase 5.1 0.1 0.2 0.3 0.4 0.5 0.6 Rm 102 DISCUSSION The Impetus for the research reported herein is derived primarily from this laboratory's own research objectives (i.e. metabolism of L-fucose in thyroid tissue) but also from the earlier work of Hansen and co-workers (17, discussed below) whose observations on the calf liver pyrophosphorylase were thought to be inconsistent with this laboratory's findings regarding the spcificity requirements of other fucose-metabolizing enzymes of thyroid tissue. To date, the purification scheme shown in Table 7 represents the most extensive purification of GDP-mannose pyrophosphorylase from any source. In 1964, Preiss and Wood (18) published their findings for the enzyme (purification of 90 fold) derived from extracts of the soil bacterium Arthrobacter vlscoBiB. In 1965, Kornfeld and Ginsburg reported on the regulatory properties of the pyrophosphorylase in crude bacterial extracts of strains of Salmonella and Aerobacter. In plant systems, the activities of the UDP-glucose, GDP-glucose, and ADP-glucose pyrophosphorylasea are dominant and attention has been focused on the properties of these enzymes (48) with the notable exception of the participation of Q3P-mannose in cell wall polysaccharide biosynthesis (49). In mammalian systems exhibiting GDP-mannose pyrophosphorylase 103 Table 7. Purification scheme for porcine thyroid GDP-mannose pyropho spho rylase A ctivity P rotein Total Specific O verall Step m g fimoles/min Mmole s/m in/m k Yield Purification Crude 4522 1.46 3. 23 x 10‘ 4 100 1 Blue Sepharose 134 1. 19 9 .0 x 10"3 81 28 DEA E-Sephacel 5. 1 0. 59 1. 16 x 10_1 40 364 Phenyl Sepharose 0. 38 0. 30 0. 76 20 2184 GTP-agarose 0. 005 0. 034 24. 1 8 69. 900 105 activity, there exists solely the work of ’Jansen and co-workers who achieved a purification of 530 fold for a GDP-hexose pyrophosphorylase from calf liver. In contrast, the study of nammalian UDP-glucose pyrophosphorylases has been extensive (19). Therefore, this discussion w ill deal mainly with the properties of the calf liver enzyme in comparison to those properties of the thyroid enzyme except where information is available only on the UDP-glucose enzyme. Ihe native molecular weight of the thyroid pyrophosphorylase is somewhat smaller than its UDP-glucose counterpart (480,000 is the reported value versus ca. 412,000 for the thyroid enzyme). There is no available information in the literature for any mammalian GDP-mannose pyrophosphorylase. Additionally, i t is known that the UDP-glucose enzyme exhibits a monomer-to-oligomer interconversion (50) and, While no sim ilar information was reported for the GDP-hexose enzyme, there is suggestive evidence that this phenomenon may exist for the thyroid enzyme as well. This suggestive evidence is encompassed in the following observationst (1) there are three clearly resolvable peaks of pyrophosphorylase activity obtained by chromatography of crude thyroid extracts on columns of Cellex D (Fig. 5) developed with linear salt gradients; re-chromatography of the Cellex D peak I activity results in the same three peak elution profile, (2) gel filtration chromatography on columns of Sepharose 6B exhibits a peak of pyrophosphorylase-assay responding activity eluting just subsequent to the void volume followed by a later eluting broad peak of pyrophosphorylase-assay responding activity, (3) the marked tendency of the pyrophosphorylase to form an insoluble aggregate under the stacking conditions of discontinuous e le c tro p h o re sis* In t h i s re g a rd , While i t i s known th a t th e UDP-glucose enzyme exists predominantly as an octamer of identical subunits, an equivalent statement for the thyroid GDP-mannose enzyme cannot be made at the present time. Taken individually, the relative mobilities of the two protein bands (lane 6 of Plate 2) allow a range of native molecular wieghts of 300,000 to 340,000 daltons (assuming an octamer in analogy to the UDP-glucose enzyme). These values fall far below the 412,000 dalton value obtained by sucrose velocity sedimentation. To bring either SDS electrophoretic value into agreement with the sedimentation value, it is necessary to consider that the oligomeric structure of the purified pyrophosphorylase may be a decamer. Alternatively, the pyrophosphorylase may be a decamer composed of two non-identical subunits for Which a native molecular weight of 407,000 daltons may be calculated. Supportive evidence for this alternative hypothesis can be derived from the following two observations: (1) in the event that the pyrophosphorylase is composed of identical subunits, it would be unlikely that the two bands visualized, one of Which is a contaminant, would stain with equal intensity; (2) because proteins take on a distinctive coloration When visualized by the silver stain method (40), it is 107 unlikely that the pyrophosphorylase and the single possible contaminant would develop the same coloration. Finally, the disparity between the sedimentation and electrophoretic values can also be attributed to inherent errors in measuring native molecular weights by sedimentation techniques (e.g. partial specific volumes, axial ratios, and degree of hydration). The calf liver GDP-hexose enzyme was reported to be able to catalyze the synthesis of several different nucleotide sugars. In contrast to this observation, both the UDP-glucose pyrophosphorylase and the thyroid GDP-mannose pyrophosphorylase have strict specificities towards their respective substrates. With regards to the partial activities of the thyroid enzyme towards the dGTP and ITP nucleotide triphosphates, this is most likely due to the bulk tolerance of the enzyme rather than representing any in vivo biological significance (i.e. dGTP concentrations are normally very low except during the S phase of the cell cycler ITP is normally only a transient entity in the synthesis of purine nucleotides). Additionally, the Michaelis c o n sta n ts fo r GDP-manmnose fo r both th e c a lf l i v e r and th e thyroid enzyme are consistent with a reported value for the s te a d y -s ta te c e l lu l a r c o n c e n tra tio n o f GDP-mannose (one micromolar, ref. 20) in cultured thyroid cells (no known value for liver cells). Since the steady-state concentrations of GTP, mannose-l-phosphate, and inorganic pyrophosphate are not presently known for thyroid tissue, no significance can be 108 attached to the Michaelis constants for these metabolites at this tim e. There is preliminary evidence that the regulation of oligosaccharide synthesis occurs at the nucleoside diphosphate sugar le v e l. Kean (22) has shown th a t GDP-mannose (and not dolichol phosphomannose ) stimulates the incorporation of the second residue of N-acetylglucosamine into dolichol diphosphate chitobiose in chicken embyro retina. Additionally, Ronin and co-workers (20) have shown that GDP-mannose (at the in vivo concentration of one micromolar) severely inhibits the incorporation of glucose from UDP-glucose into dolichol phosphate in cultured porcine thyroid cells. Thus, the importance of the regulation of the cellular GDP-mannoBe concentration, produced by the pyrophosphorylase, has been established but not yet quantified. Interestingly, as determined by the present research reported herein, the Michaelis constant for GDP-mannose (1.5 ^M) suggests that the enzyme is operating under sub-saturating conditions (steady-state is ca. one micromolar, see ref. 20). Since the NDP-X : dolichol phosphate glycosyltransferases are normally present in vanishing small quantities and, since the Michaelis constants for these enzymes are similar to the pyrophosphorylase itself, this would suggest that the production of QlP-mannose must also be under some form of regulation. More explicitly, the sim ilarity in Michaelis constants would imply that the subsaturating level of GDP-mannose is not due to 109 depletion of the pool levels by the glycosyltransferases. Instead, the regulatory control may be at the level of activity of the pyrophosphorylase itself or perhaps through regulation of the availability of substrates for the pyrophosphorylase. The regulation of the GDP-mannose pyrophosphorylase in strains Salmonella was examined by Kornfeld and Ginsburg (47). They determined that, in strains producing mannose-containing cell wall polysaccharides, GDP-mannose (and GDP-fucose) acted as feedback inhibitors of GDP-mannose synthesis. With the calf liver enzyme, Hansen and co-workers determined that the pyrophosphorylase was inhibited by nucleotide sugars and sugar-l-phosphates. However, since no criteria of purity for the enzyme was given, and in contrast to the strict specificity of the thyroid enzyme, this conclusion must be considered controversial. On the other hand, in defense of Hansen's observations, it was noted that in the course of the purification of the thyroid GDP-mannose enzyme it was considerably more difficult to quantify enzymatic activity when synthesis assays were employed. Indeed, the determination of the Michaelis constant for mannose-1-phosphate required a ten-fold higher specific activity of radiolabel, with respect to GDP-mannose, when measurements at sim ilar concentrations were attempted. Unfortunately, no comparative measurements of the relative forward and reverse reaction rates were made on the same isolated preparation. Finally, it is known that the UDP-glucose enzyme of human erythrocytes shows a highly selective inhibition no by UDP-glucose (i.e* a constant degradative reaction rate is observed versus a rapid inhibition of the synthetic reaction rate; see ref. 19). At the level of hormonal regulation, there are no reports as yet published on the effect of either TSH (thyroid stimulating hormone) or GH (growth hormone) on the synthesis or activity of GDP-mannose pyrophosphorylase. However, in chicken oviduct tissue, Lucas (51) has determined that the effect of estrogen, diethylstilbesterol, and progesterone is to regulate the availability of dolichol phosphate for oligosaccharide-lipid biosynthesis. Unfortunately, there is no comparable information for thyroid tissue. The catalytic mechanism of the GDP-mannose pyrophosphorylases has not been examined. However, in E. coli ADP-glucose m pyrophosphorylase, Preiss (52) has demonstrated the involvement of an arginine residue Which, through chemical modification by phenylglyoxal, results in a 2 to 3 fold greater inhibition of the synthetic reaction rate relative to the degradation reaction rate. Additionally, Frey and co-workers (53) have detailed the stereochemical reaction pathway of the yeast UDP-glucose pyrophosphorylase by the use of the phosphothioate substrate analog UTP(otS). In this study it was shown that the normally pro-bhiral alpha phosphate (pro-R) of UTP is the sole diastereomer utilized in an ordered binding mechanism. Ill With regards to future studies on the pyrophosphorylase, of immediate interest would be an investigation into the possible effect of GDP-mannose as a feedback inhibitor in analogy to the UDP-glucose pyrophosphorylase. Alternatively, studies may be directed towards potential cellular metabolite regulators or regulation by some form of protein-directed covalent modification. In conjunction with these studies, hormonal stimulation studies on cultured thyroid cells, perhaps through development of a radioimmunoglobin precipitation assay, may provide useful information on the regulatory aspects at the DNA transcriptional and RNA translational levels. 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