Vol. 69 METABOLISM OF TETRACHLOROBENZENES 189 3. 1:2:3:5-Tetrachlorobenzene is very slowly REFERENCES metabolized. Only about 5 is oxidized to and % Azouz, W. M., Parke, D. V. & Williams, R. T. (1952). excreted as 2:3:4:6-tetrachlorophenol in 6 days. Biochem. J. 50, 702. Some 14 % is eliminated unchanged in the faeces, Azouz, W. M., Parke, D. V. & Williams, R. T. (1953). 12 % in the breath and 23 % remains in the tissues Biochem. J. 55, 146. after 6 days. There is evidence that some 9 % of the Azouz, W. M., Parke, D. V. & Williams, R. T. (1955). dose is dechlorinated and eliminated in the ex- Biochem. J. 59, 410. pired air as less chlorinated benzenes. Some 5 % of Cameron, G. R., Thomas, J. C., Ashmore, S. A., Buchan, the dose may also be excreted in the urine as di- J. L., Warren, E. H. & Hughes, A. W. M. (1937). and tri-chlorophenols. Injected 1:2:3:5-tetra- J. Path. Bact. 44, 281. chlorobenzene is partly excreted as such in the Conrad-Billroth, H. (1932). Z. phy8. Chem. 19, 76. El Masri, A. M., Smith, J. N. & Williams, R. T. (1958). faeces, probably via the bile. Biochem. J. 68, 587. 4. 1:2:4:5-Tetrachlorobenzene appears to be the Fewster, M. E. & Hall, D. A. (1951). Nature, Lond., 168,78. least readily metabolized of the three isomers. Holleman, A. F. (1920). Ree. Trav. chim. Pays-Bas, 39, 736. Only about 2 % is converted into 2:3:5:6-tetra- Huntress, E. H. (1948). Organic Chlorine Compound3. chlorophenol in 6 days; 48 % of the dose was found London: Chapman and Hall. in the tissues after 6 days, and 16 % was in the Jondorf, W. R., Parke, D. V. & Williams, R. T. (1955). faeces and 2 % in the expired air. Dechlorination Biochem. J. 61, 512. products could account for 15 % of the dose, about Mead, J. A. R., Smith, J. N. & Williams, R. T. (1958). 10 % of the dose appearing in the expired air as less Biochem. J. 68, 61. Parke, D. V. & Williams, R. T. (1955). Biochem. J. 59,415. chlorinated benzenes and 5 % in the urine as di- and Paul, J. (1951). Ph.D. Thesis, University of Glasgow. tri-chlorophenols. Dechlorination of the tetra- Smith, J. N., Spencer, B. & Williams, R. T. (1950). chlorobenzenes is believed to occur in the gut, Biochem. J. 47, 284. probably under the influence of bacteria. Spencer, B. & Williams, R. T. (1950). Biochem. J. 47, 279. Sperber, I. (1948). J. biol. Chem. 172, 441. The work was supported by a grant from the Agri- Stekol, J. A. (1936). J. biol. Chem. 113, 279. cultural Research Council. Tiessens, G. J. (1931). Rec. Trav. chim. Pays-Bas, 50, 112.

Glutamic-Alanine and Glutamic-Aspartic Transaminases of Wheat Germ

BY D. H. CRUICKSHANK* Botany School, Univer8ity of Cambridge AND F. A. ISHERWOOD Low Temperature Research Station for Research in Biochemistry and Biophysics, University of Cambridge, and Department of Scientific and Industrial Research (Received 18 December 1957) Since the discovery of the transamination reaction In the present investigation a study has been by Braunstein & Kritzmann (1937), transaminase made of some of the properties of two partially systems have been studied inextracts from anumber purified transaminase enzymes from wheat germ. ofplantandanimaltissuesandmicro-organisms.This Wheat germ was chosen as the plant material work has been reviewed by Braunstein (1947) and because Leonard & Burris (1947) had previously Cohen (1951, 1954). Results of many of the early demonstrated that it contained active tranam.inase investigations were conflicting, as crude enzyme systems. The two transaminase systems were as preparations and non-specific quantitative methods follows: were used. Recently much information on the pro- perties of animal and microbial transaninases has (1) L-Glutamic acid+pyruvic acid beenobtainedwithpurifiedenzymepreparations and oc-oxoglutaric acid+ L-alanine specific quantitative methods. There is, however, (catalysed by glutamic-alanine transaminase) littleprecise information on theplanttransaminases. (2) L-Glutamic acid + oxaloacetic acid = * Present address: Division of Food Preservation and c-oxoglutaric acid + L-aspartic acid Transport C.S.I.R.O., Botany School, University of Sydney, Australia. (catalysed by glutamic-aspartic transaminase) 190 D. H. CRUICKSHANK AND F. A. ISHERWOOD I958 These reactions were followed by the specific At specified times samples were removed for analysis. chromatographic methods recently developed by For the estimation of a-keto acids, 0X1 ml. of the enzyme Isherwood & Cruickshank (1954a, b respectively). digest was added to 0.5 ml. of 0011m-2:4-dinitrophenyl- hydrazine in 0-2N-HC1 in ethanol contained in a stoppered centrifuge tube. The cx-keto acids were then estimated by MATERIALS AND METHODS the method of Isherwood & Cruickshank (1954a). For the quantitative determination of the cx-amino acids, a sample Enzyme preparation (0-1 ml.) of the enzyme reaction mixture was added to Commercial wheat germ (kindly supplied by Dr J. Pace of 0.05 ml. of 0-3N-acetic acid. The mixture was centrifuged the Research Association of British Flour Millers, Cereal and samples of the supernatant were used for the estima- Research Station, St Albans) was extracted three times tion of the x-amino acids by the method of Isherwood & with 1-5 vol. of ether and dried at room temperature. Cruickshank (1954b). Removal of the fat caused the original flaky material to Results have been expressed as micromoles of reactant disintegrate into a powder. This defatted powder (15 g.) present in 1 ml. of enzyme digest. The percentage trans- was suspended in 60 ml. of water in a stoppered bottle and amination (% T), used to express transaminase activity, is the mixture agitated gently for 3 hr. by slowly rotating the defined as the percentage of the initial substrate trans- bottle between rollers. The mixture was then centrifuged; aminated during a specified time. This has been determined the milky supernatant was adjusted to pH 5*7 with from the amount of substrate utilized or the amount of 2N-acetic acid and the liquid again centrifuged. The clear, reaction product formed: the results should be identical brown supernatant was brought to pH 7 with 2x-NaOH whichever method is used. and the liquid treated with saturated (NH4)2SO4 (pH 7) at 50. The fraction precipitated between 33 and 66 % satura- RESULTS tion was suspended in a few millilitres ofwater, dialysed for 15 hr. at 5° against 1% (w/v) KCI and finally adjusted to The purified enzyme preparation contained only pH 7-5 or 8-0 with N-NaOH. No oc-keto acids or amino acids glutamic-alanine and glutamic-aspartic trans- could be detected in this preparation by the chromato- aminases. A preliminary examination of the crude graphic methods referred to above. aqueous extract of wheat germ showed that other transaminases were present, for transamination Substrates occurred to a small extent between a-oxoglutaric x-Keto acids. Commercial samples of ac-oxoglutaric acid, acid and valine, leucine, phenylalanine, tyrosine and oxaloacetic acid and pyruvic acid (as sodium pyruvate) tryptophan. These other transaminases were lost were used. Chromatographic examination of their 2:4- during the purification procedure described above. dinitrophenylhydrazones showed that they were free from other keto acids. Immediately before use neutral 0-2M- Measurements in the glutamic-alanine system solutions were prepared (with the addition of dil. NaOH if necessary) and these were diluted to O-1M with 0-2M- Preliminary experiments showed that there was phosphate buffer (KH2PO4-NaOH), pH 7-5 or 8-0. no significant change in any of the reactants cx-Amino acids. Commercial preparations were used. (reaction 1) when incubated singly with the enzyme These were found by paper chromatography to be free preparation and that the value obtained for rate of from other amino acids. For enzyme studies the amino transamination was the same whichever com- acids were prepared as 0*2M neutral solutions and diluted to ponent was determined. Some results are given in 01m immediately before use with 0-2m-phosphate buffer Table 1. (KH2PO4-NaOH), pH 7-5 or 8-0. Pyridoxal phosphate. A sample of the calcium salt of The results indicated that the glutamic-alanine pyridoxal phosphate was kindly provided by Dr E. F. transamination system was not being influenced by Gale, F.R.S., Department of Biochemistry, University of side reactions involving the formation or destruc- Cambridge. Immediately before use, 40Icg. was dissolved tion of any ofthe reactants, and that the estimation in 1 ml. of 0 2M-phosphate buffer, pH 7-5 or 8-0. of any one of the reactants would serve as a measure of the progress of the reaction. Procedure Progress curves for the forward and reverse To small tubes were added 1 vol. (0.2-0.4 ml.) of 0.2m- reactions are shown in Fig. 1. The points on the phosphate buffer (KH2PO4-NaOH), 1 vol. of 01M-ac- curves were obtained by estimating oc-oxoglutaric amino acid solution and 1 vol. oftransaminase preparation, acid and pyruvic acid at each specified time. and the mixture was incubated at 250 for 10 min.; 1 vol. of The initial rates for the forward and reverse 01M-oc-keto acid solution was then added. To test the reactions were almost equal: at 7-5 min. 27-4 %of effect of inhibitors or coenzymes this procedure was the L-glutamic acid had been converted into a- slightly modified. Enzyme solution (2 vol.) was incubated oxoglutaric acid and 26X6 % of the L-alanine into for 15 min. at 250 with 2 vol. of inhibitor or coenzyme and in 2 vol. of phosphate buffer and then 1 vol. of 0-2M-cx-amino pyruvic acid. Equilibrium was reached 30 min., acid and 1 vol. of 0-2m-a-keto acid were added. Control the system remaining unchanged after incubation experiments were carried out in which the enzyme pre- for a further 30 min. The equilibrium was slightly paration, phosphate buffer and water were incubated with in favour of the formation of alanine and oc-oxo- and without addition of each a-amino acid or a-keto acid. glutaric acid, the separate determination of pyruvic Vol. 69 TRANSAMINASES OF WHEAT GERM 191 and a-oxoglutaric acids giving practically the same figure for the equilibrium mixture (about 55 % of E o o the L-glutamic acid was changed). The equilibrium P4 P4 N constant for reaction (1),

K= [oc-oxoglutaric acid] [L-alanine] acid] acid]' 1-: 1-: [L-glutamic [pyruvic was after determining all four reactants, ' calculated 'e-u a I o. o. and was found to be 1-4 at pH 75. Ca1 6 1 Cao P4a - o , Measurements in the glutamic-apartic 8y8tem . op 0 St OD I eo were p4*^ I.., Preliminary experiments showed that there 0 ¢ no significant changes in the concentrations of L- *b .E ° glutamic, L-aspartic or a-oxoglutaric acids when 0 0 they were incubated singly with the enzyme pre- o _~ paration. However, oxaloacetic acid was slowly ii converted into pyruvic acid and carbon dioxide in 0 - eq "' aqueous solution (with KH2PO4-NaOH buffer, &d ~,<- 0 a-7i 19 o pH 8) and this instability meant that a perfect w I CB _e equilibrium as described in reaction (2) could not S °C be reached in practice. In the present experiments the loss in oxaloacetic acid due to non-enzymic decarboxylation to pyruvic acid was found to be S 2-4% in lOmin. and a similar figure was obtained 0Go cI cP o when oxaloacetic acid was incubated with the r S oG enzyme preparation. Some preliminary results for . O) the transamination reaction are described in Table 2. These have been corrected for the slight I ao *i 10#los due to non-enzymic decarboxrylation men- "0 tioned above but it is noticeable that even after

CB 0 -4 CB 0eq p 0) a ^ this correction the % T based on oxaloacetic acid

0 1(

0 0c 20 .20 tdm g a 4E Bo tP 0) T CP C O.i O I A co 0 014 0) t_0 01- 60 o8 a-A

80 0

p ~ 0 100 a5 C Time (min.) a aa Fig. 1. Rates of forward and reverse reactions catalysed 4- + by glutamin-alanine transaminase. Ordinate numbers on d CB E- "e3 0- + 0 the right show % T measured by pyruvic acid disap- cs d aD pearance (or a-oxoglutaric acid formation) in reaction (1), with the substrates glutamic acid +pyruvic acid. Ordinate numbers on the left show % T measured by r'P~k a-oxoglutaric acid disappearance (or pyruvic acid formation) in (1), with the substrates alanine +.-oxo- glutaric acid. Substrate concn. 0O025M; temp. 250; pH 7*5. E, oa-Oxoglutaric acid; *, pyruvic acid. 192 D. H. CRUICKSHANK AND F. A. ISHERWOOD I958 estimations was slightly higher for the forward re- reached for both reactions in 30 min., but the action and lower for the reverse reaction (reaction 2) observed position of equilibrium depended on than the % T from estimations of the other whether oxaloacetic acid or oc-oxoglutaric acid was reactants. This indicated that the rate of oxalo- determined. It was 75 % in favour of the forward acetic acid decomposition was greater in the com- reaction, if oxaloacetic acid formation and dis- plete reaction mixture than that observed in the appearance were measured, and 68 % if oc-oxo- presence of the enzyme alone. It appears that the glutaric acid was determined. Small changes presence of glutamic or aspartic acids and, to a detected in the period from 30 to 60 min. were lesser extent, a-oxoglutaric acid increases the non- probably caused by the decarboxylation of the enzymic breakdown (Nisonoff, Henry & Barnes, oxaloacetic acid: control experiments showed that 1952). Though an active glutamic-alanine enzyme the non-enzymic destruction of oxaloacetic acid was present in the extract used, the amount of increased from 7 to 13 % during the period 30- alanine formed from the pyruvic acid produced in 60 min. Calculation of the equilibrium constant this way was not detectable on a paper chromato- was made by directly estimating the concentration gram at 10 min. By comparison the % T of of each of the reactants after 30 and 60 min. The glutamic acid was of the order of 42%, which relatively slow change in the oxaloacetic acid meant that the rate of the side reaction was concentration was not sufficient to affect seriously relatively small under the experimental conditions. the equilibrium concentrations of the various The results in Table 2, based on initial reaction reactants. The equilibrium constant velocities, show that the forward reaction was [oc-oxoglutaric acid] [L-aspartic acid] about twice as fast as the reverse reaction. Complete progress curves for the forward and [oxaloacetic acid] [L-glutamic acid] reverse reactions are shown in Fig. 2. These were was 5-0, with initial substrate concentrations based on estimations of ac-oxoglutaric acid and 0-025M, pH 8 and temperature 250. oxaloacetic acid. The forward reaction was 5-10 % more rapid if Enzyme propertiem measured by disappearance of oxaloacetic acid Pyridoxal phosphate. Addition of pyridoxal than if measured by a-oxoglutaric al,cid formation. phosphate (10 ,ug. of the calcium salt to 1 ml. of the In the reverse reaction the rate of disgappearance of reaction mixture) to digests containing the purified o-oxoglutaric acid was higher thawn the rate of enzyme from wheat germ increased the glutamic- formation of oxaloacetic acid. Eq[uilibrium was aspartic enzyme activity by 50-80%, depending on the treatment to which the enzyme had been 0 100 subjected during purification, but did not affect the glutamic-alanine enzyme activity at all. The difference between the two enzymes was probably 20 o 80 wmm -- - due to the difficulty of resolving the apoenzyme c0 from coenzyme in the latter. Attempts to resolve E distilled 40 V 60 the enzyme complexes by dialysis against C water, aq. 0-002N-NH3 soln. or 1 % KCI for 72 hr. 4) were inconclusive, as the enzymes were largely 60 °° 40 irreversibly inactivated by the treatments. C Effect of piH. The effect of pH on transaminase activity (measured as % T in 10 min., under V standard conditions) is shown in Fig. 3. The optimum pH of the glutamic-alanine system 100 O, I I was approximately 7-5 but there was little change 0 15 30 45 60 between pH 7 and 8. The glutamic-aspartic Time (min.) transaminase showed maximum activity between Fig. 2. Rates of forward and reverse reawctions catalysed pH 8-0 and 8 5. by glutamic-aspartic transaminase. 0 rdinate numbers Effect of temperature. The effect of temperature on the right show % T measured by oxaloacetic acid on enzyme activity (measured as % T in 10 min. disappearance (or a-oxoglutaric acidI formation) in under standard conditions) is shown in Fig. 4. The reaction (2), with the substrates glutaimnic acid + oxalo- ,, optimum temperature for each reaction was acetic acid. Ordinate numbers on the left show the %T between 400 and 500. The fact that very little measured by oc-oxoglutaric acid disappearance (or oxaloacetic acid formation) in (2), witth the substrates difference in activity was found between 400 and aspartic acid + a-oxoglutaric acid. S ubstrate concn. 500, under the experimental conditions used, sug- 0.025ar; temp. 25°; pH 8. 0, a-Oxog lutaric acid; *, gested that inactivation of the enzymes was oxaloacetic acid. proceeding at 500. Vol. 69 TRANSAMINASES OF WHEAT GERM 193 Table 3. Effect of some inhibitors on glutamic-alanine and glutamic-aspartic transaminase activity Substrate conon. 0-025M; temp. 250; pH 7-5; incubation period 10 min. Substrate Substrate , ~~~A. Glutamic acid + pyruvic acid Glutamic acid + oxaloacetic acid oa-Oxoglutaric m-Oxoglutaric acid formed acid formed Final concn. (jumoles/ml. Inhibition (pmoles/ml. Inhibition Inhibitor (mM) of digest) (% of digest) (%) 7.5 _ 8-8 Iodoacetate 1-0 7*5 0 8-0 9 Malonate 10-0 7*0 7 8-8 0 KCN 1.0 4-5 40 7-0 20 AgNO3 0.1 5-0 33 8-8 0 AgNO3 1-0 0.0 100 5*3 40 Semicarbazide 1-0 6*2 18 7-1 19 Semicarbazide 2-0 2-3 70 3-5 60 Hydroxylamine 1-0 0-8 90 1-4 84 601 5or

0 4wco 45 4C .E

v 30 u c 4,o co40 *E 30 U L. ,

'A

I I 9 6 7 8 9 pH Fig. 3. Effect of pH on transaminase activity. % T measured by oa-oxoglutaric acid formed. Substrate 10 concn. 0-025mr; temp. 250; incubation period 10 min.; pH 6-8, 0-05M-phosphate buffer (KH2PO4-NaOH); pH 8-9, 0-05M-sodium borate-potassium chloride buffer I I I I Glutamic-alanine v (Na2B407-KCl). O, transaminase, 20 30 40 50 substrates glutamic acid+pyruvic acid; 0, glutamic- Temperature (0) aspartic transaminase, substrates glutamic acid + oxaloacetic acid. Fig. 4. Effect of temperature on transaminase activity % T was measured by a-oxoglutaric acid formed. Substrate concn. 0-25M; incubation period 10 miin. The temperature coefficient (Qlo) was calculated 0, Glutamic-alanine transaminase, substrates glutamic for each reaction from the ratio % T (a-oxoglutaric acid +pyruvic acid, pH 7-5; 0, glutamic-aspartic acid) at 350/% T (x-oxoglutaric acid) at 250. The transaminase, substrates glutamic acid + oxaloacetic Qlo of reactions (1) and (2) was 1-3 and 2-1 re- acid, pH 8. spectively. Inhibitor8. The effects of different inhibitors DISCUSSION on the activity of the two enzymes is listed in Table 3. The present study indicates that the glutamic- The glutamic-alanine transaminase was com- alanine enzyme from wheat germ has many pro- pletely inhibited by mM-AgNO3 but only 30 % perties similar to those of the corresponding inhibited by 0-1 mm-AgNO3. The glutamic-aspartic enzymes from animals and micro-organisms. The transaminase was less sensitive to the presence of equilibrium constant for the reaction, 1-4 at pH 7-5 Ag+ ions. Iodoacetate (mM) and malonate (10 mM) and 250, is close to the latest figure of 1-53 at did not affect the transaminase activity. pH 7-4 and 250 reported by Krebs (1953) for the 13 Bioch. 1958, 69 194 D. H. CRUICKSHANK AND F. A. ISHERWOOD I958 animal enzyme. Lenard & Straub (1942) reported the glutamic-alanine enzyme from wheat germ. 1*43 and Cohen (1940) 10 for this enzyme from Cohen (1940) reported a figure of 400 for the animal sources. The enzyme showed its maximum animal enzyme. The effect of pyridoxal phosphate activity at pH 7 5, but there was only a small on the purified enzyme from wheat germ suggests change in activity between pH 7 and 8. Kritzmann that a considerable resolution into apoenzyme and (1939) records 7-4 for plant and animal trans- coenzyme had occurred during the isolation pro- aminase systems and Rautanen (1946) 6-9 for the cedure. This contrasts sharply with our experience glutamic-alanine enzyme from many plant tissues. with the glutamic-alanine enzyme, which did not The effect of temperature on the rate of the trans- split at all readily, but is similar to the observations amination reaction is complicated by the inactiva- of O'Kane & Gunsalus (1947) on pig heart-muscle tion of the enzyme above 500, but if this is ignored transaminases. They found that the glutamic- an apparent optimum was found between 400 and aspartic enzyme was resolved by heating at 560 500 under the experimental conditions used. followed by ammonium sulphate precipitation, Rautanen (1946) reported a figure of 410, but a whereas the glutamic-alanine enzyme was not longer incubation period was used to measure the split. The sensitivity of the glutamic-aspartic initial rate of the reaction. The effect of pyridoxal enzyme from wheat germ to hydroxylamine and phosphate on the purified enzyme was small, though semicarbazide is similar to that of the glutamic- it seems clear that this compound is the coenzyme alanine enzyme and is much greater than with the for transaminase enzymes from animal tissues and corresponding animal enzymes (Braunstein, 1947). micro-organisms (Lichstein, Gunsalus & Umbreit, The configuration of the enzyme complex is 1945; Schlenk & Snell, 1945; Green, Leloir & probably different for the two groups of enzymes. Nocito, 1945). Attempts to resolve the purified The separation and purification of the glutamic- enzyme into apoenzyme and coenzyme by dialysis alanine and glutamic-aspartic transaminases of against various aqueous solutions were only heart muscle have been reported from several partially successful, and it seems that the plant laboratories (Meister, 1955), so that it is clear that enzyme must be much less easily split without reactions (1) and (2) are catalysed by two different denaturation ofthe protein moiety than some ofthe enzymes in the animal. In the present work on animal enzymes. The inhibition of the glutamic- wheat-germ extracts, we have not separated the alanine transaminase from wheat germ by silver individual enzymes but there seems little doubt nitrate and potassium cyanide, and the lack of that two separate enzymes are also responsible for inhibitory effect with iodoacetate and malonate, catalysing reactions (1) and (2) in the plant. are in general agreement with the results reported Treatment of the original extract with ammonium for animal transaminases (Braunstein, 1947). The sulphate gave a fraction in which the glutamic- main differences between the enzyme from wheat aspartic enzyme was largely in the apoenzyme germ and that from animal sources is the sensi- form whereas the glutamic-alanine enzyme was tivity of the former to hydroxylamine and semi- still unresolved. The effect of silver nitrate on this carbazide. This suggests that an aldehyde or keto fraction from wheat-germ extract also suggested group is present in the enzyme complex and that if that two different enzymes were responsible. The it is attached to pyridoxal phosphate, the link glutamic-alanine enzyme was inhibited at lower between the apoenzyme and the coenzyme must be concentrations of Ag+ ion than the glutamic- different in the plant and animal enzymes. aspartic enzyme. If a single protein had been The properties of the glutamic-aspartic enzyme responsible for both enzyme activities the effect of from wheat germ were similar to those of the Ag+ ions might be expected to be similar for both enzymes extracted from animal tissue and from enzymes. micro-organisms. The equilibrium constant at SUMMARY pH 8 and 25' was 5 0. Figures of 5-4 at pH 7.5 and 25°, 6-7 at pH 7-4 and 250 and 7-8 at pH 7-4 and 1. The transamination reactions 37.50, have been reported by Darling (1945), Krebs L-glutamic acid + pyruvic acid = (1953) and Nisonoff, Barnes & Enns (1953) cx-oxoglutaric acid + L-alanine respectively for animal enzymes. The enzyme showed optimum activity between pH 8-0 and 8-5. (catalysed by glutamic-alanine transaminase) For the corresponding enzyme in oat embryo and (Albaum & Cohen, 1943), E8cherichia coli (Lichstein L-glutamic acid+ oxaloacetic acid = & Cohen, 1945) and Streptococcus faecaUts (Lichstein o-oxoglutaric acid + L-aspartic acid et al. 1945) optimum activity was shown at pH 8.6, 8-5 and somewhere between 5-7 and 9-5 respec- (catalysed by glutamic-aspartic transaminase) tively. The activity of the enzyme reached an have been studied with partially purified enzyme apparent maximum between 400 and 500, as with preparations from wheat germ. Vol. 69 TRANSAMINASES OF WHEAT GERM 195 2. The glutamic-alanine transaJninase has an Cohen, P. P. (1954). In Chemical Pathway8 of Metaboli8m, equilibrium constant of 1-4 at 250 and pH 7 5, and vol. 2, p. 29. Ed. by Greenberg, D. M. New York: an optimum pH of 7*5. Academic Press Inc. 3. enzyme has an Darling, S. (1945). Acta physiol. 8cand. 10, 150. The glutamic-aspartic Green, D. E., Leloir, L. R. & Nocito, V. (1945). J. biol. equilibrium constant of 5 0 at 250 and pH 8 and an Chem. 161, 559. optimum pH between pH 8-0 and 8-5. Isherwood, F. A. & Cruickshank, D. H. (1954a). Nature, 4. The effect of pyridoxal phosphate and some Lond., 173, 121. inhibitors on the transaminases suggests that the Isherwood, F. A. & Cruickshank, D. H. (1954b). Nature, two systems may be catalysed by two different Lond., 174, 123. enzymes, as are the animal glutamic-alanine and Krebs, H. A. (1953). Biochem. J. 54, 82. glutamic-aspartic transarninase systems. Kritzmann, M. G. (1939). Biokhimiya, 4, 691. L6nard, P. & Straub, F. B. (1942). Stud. Inst. med. Chem. This work forms part of the programme of the Food Univ. Szeged, 2, 59. Investigation Organization of the Department of Scientific Leonard, M. J. K. & Burris, R. H. (1947). J. biol. Chem. and Industrial Research. The authors wish to thank 170, 701. Professor C. S. Hanes, F.R.S., who initiated this study, and Lichstein, H. C. & Cohen, P. P. (1945). J. biol. Chem. 157, Professor G. E. Briggs, F.R.S., for his interest during the 85. course of the work. We are grateful to the University of Lichstein, H. C., Gunsalus, I. C. & Umbreit, W. W. (1945). Adelaide, University of Cambridge, and Agricultural J. biol. Chem. 161, 311. Research Council for financial assistance for one of us Meister, A. (1955). Amino Acid Metaboli8m Sympo8ium, (D.H.C.). p. 19. Ed. by McElroy, W. D. & Glass, H. B. Baltimore: REFERENCES Johns Hopkins Press. Nisonoff, A., Barnes, F. W. & Enns, T. (1953). J. biol. Albaum, H. G. & Cohen, P. P. (1943). J. biol. Chem. 149, 19. Chem. 204, 957. Braunstein, A. E. (1947). Advanc. Protein Chem. 3, 1. Nisonoff, A., Henry, S. S. & Barnes, F. W. (1952). J. biol. Braunstein, A. E. & Kritzmann, M. G. (1937). Nature, Chem. 199, 699. Lond., 140, 503. O'Kane, D. E. & Gunsalus, I. C. (1947). J. biol. Chem. 170, Cohen, P. P. (1940). J. biol. Chem. 138, 585. 436. Cohen, P. P. (1951). In The Enzymes, vol. 1, part 2, Rautanen, N. (1946). J. biol. Chem. 163, 687. p. 1040. Ed. by Sumner, J. B. & Myrbaick, K. NewYork: Schlenk, F. & Snell, E. E. (1945). J. biol. Chem. 157, Academic Press Inc. 425.

The Nature of the Link Between Protein and Carbohydrate of a Chondroitin Sulphate Complex from Hyaline Cartilage

BY HELEN MUIR Medical Unit, St Mary'8 Hospital, London, W. 2 and Postgraduate Medical School, Ducane Road, London, W. 12 (Received 24 September 1957) Chondroitin sulphate from hyaline cartilage has prolonged extraction with water (Shatton & been prepared by several procedures which give Schubert, 1954). The yield from alkaline extractions products with physical properties differing accord- is high, but extraction with neutral salt gives much ing to the method used for extraction. Successively smaller yields and the remaining polysaccharide larger quantities can be extracted from fresh can be extracted only with alkali (Blix & Snellman, cartilage by water (Partridge, 1948 b), neutral 1945; Einbinder & Schubert, 1950). By making a salts such as 30 % potassium chloride (Einbinder & solution of potassium chloride mildly alkaline with Schubert, 1950) or 10 % calcium chloride (Meyer & potassium carbonate Einbinder & Schubert (1950) Smyth, 1937; Blix & Snellnan, 1945) and by increased the yield of chondroitin sulphate con- alkali, which has been used extensively (Morner, siderably. Alkali is therefore particularly effective 1889; Levene & La Forge, 1913; Jorpes, 1928; in releasing soluble chondroitin sulphate from Fiirth & Bruno, 1937; Bray, Gregory & Stacey, cartilage, but the polysaccharide is obtained in a 1944; Mathews, Roseman & Dorfman, 1951). comparatively degraded form as judged by the Shrinkage of collagen in cartilage by heat-treat- relatively low molecular weight of such prepara- ment increases the yield of water-soluble chon- tions, although the values depend partly on the droitin sulphate (Partridge, 1948 b), as does very method of measurement. Values ranging from 975 13-2