Proc. Nati. Acad. Sci. USA Vol. 76, No. 12, pp. 6172-6176, December 1979 Biochemistry Uroporphyrinogen I synthase from human erythrocytes: Separation, purification, and properties of isoenzymes (heme synthesis/porphobilinogen deaminase/porphyria) K. MIYAGI*, M. KANESHIMA*, J. KAWAKAMI*, F. NAKADAt, Z. J. PETRYKAt, AND C. J. WATSONt Departments of *Clinical Pathology and Biochemistry, College of Health Sciences, University of the Ryukyus, Naha, Okinawa 902, Japan; and *University of Minnesota Medical Research Unit at Northwestern Hospital, Minneapolis, Minnesota 55407 Contributed by Cecil James Watson, September 10, 1979

ABSTRACT Uroporphyrinogen I synthase [porphobilinogen other reagents of analytical grade were obtained from Wako ammonia-lyase (polymerizing), EC 4.3.1.8] from human eryth- Purechemical Ind. Ltd. and Nakarai Chem. Ltd. Phosphate rocytes was separated into two active protein peaks (A and B) the present experiments were made on DEAE-cellulose, by ammonium sulfate fractionation, on buffers used throughout Sephadex G-100, and' on DEAE-Sephadex A-50 with a NaCl of K2HPO4 and KH2PO4. gradient. The final purification was 613 and 743 times for A and Enzyme Source. Human whole blood withdrawn for blood B. respectively. The corresponding yields were 2.2 and 3.4%. transfusion was kept at 4-6'C for 3-7 days after being with- Fraction A was separated further into two (Al and A2) active drawn. To 200 ml of whole blood were added 0.327 g of citric protein bands'and fraction B into three (B1, B2, and B3) on ana- acid, 2.63 g of sodium citrate, 0.251 g of sodium dihydrophos- lytical polyacrylamide disc gel electrophoresis. Bands Al and A2 were identical with B1 and B2; B3 represented a third isoen- phate, and 2.32 g of glucose. Whole blood (400 ml) obtained zyme. Molecular weights (mean ± SEM), measured by gel fil- from two persons was used as a starting material. tration and sodium dodecyl sulfate/polyacrylamide gel elec- Enzyme Assay. UPGI-S activity was assayed by measuring trophoresis, were 38,000 ± 1000 for B1 and 40,000 ± 1000 for B2 the formation of uroporphyrinogen as follows. The incubation and B3. Isoelectric focusing on 4% polyacrylarmide gel separated mixture contained 50 mM Tris-HCl at pH 8.2, 600 mM por- both fractions A and B into three active protein bands. Maximal phobilinogen, and the enzyme solution in a total volume of 1.5 activity of the enzyme was found in gel cuts (5-mm) at pH 5.6 ml. Incubation was at 370C for 3 hr. After the incubation, for both fractions A and B. protein was precipitated with 1.5 ml of 25% (wt/vol) trichlo- Two enzymes are involved in the cyclic polymerization of four roacetic acid. After 15 min in the dark, 0.1 ml of an ethanolic molecules of porphobilinogen into uroporphyrinogen III, solution of 0.2% I2 and 0.4% KI was added. The residual iodine uroporphyrinogen I synthase [UPGI-S; porphobilinogen am- was reduced by the addition of 0.1 ml of 0.4% K2S204, and the monia-lyase (polymerizing), EC 4.3.1.8] (1) and uroporphyri- formed porphyrin was measured fluorometrically; as a stan- nogen III cosynthase (2). UPGI-S acting alone on porphobili- dard, 10 jig of coproporphyrin in 12.5% trichloroacetic acid nogen forms uroporphyrinogen I. Uroporphyrinogen III co- solution was used. synthase by itself has no catalytic function on either porpho- An enzyme unit is defined as the amount of enzyme that bilinogen or uroporphyrinogen I but, in conjunction with catalyzes the formation of 1 ,ug of uroporphyrinogen per hour UPGI-S acting on porphobilinogen, it forms uroporphyrinogen under standard conditions. The specific enzyme activity is III, the natural precursor of heme and chlorophyll. expressed as number of enzyme units per mg of protein. UPGI-S has been isolated and purified from bacteria, higher Determination of Protein Concentration. Protein con- plants, and avian and mammalian erythrocytes (3-10). Al- centration was measured by the method of Lowry et al. (12). though the enzyme has been isolated from many different Protein concentration in samples containing 2-mercaptoethanol sources, the properties of purified UPGI-S are similar. The was determined after dialysis against distilled water. enzyme from mammalian tissue can be replaced in vitro by the Polyacrylamide Disc Gel Electrophoresis. The preparation enzyme from higher plants (6). This enzyme from many dif- of the gel and electrophoresis was according to the method of ferent sources also has similar molecular weights (3-5, 7, 11). Davis (13). The following modifications were introduced. The In all of the reports, the final purified enzyme preparation has sample solution was mixed with an equal volume of 40% been shown to be a single protein. However, little work has been (wt/vol) sucrose. The separation gel contained 5 mM phosphate done on the enzyme from human erythrocytes, and the prop- at pH 7.4, 0.1 mM 2-mercaptoethanol, and 0.3% glycerol. A erties of human UPGI-S have not yet been well defined. constant current of 2 mA per gel was applied until the protein The present paper describes the purification of UPGI-S from went into the separation gel; then the current was increased to human erythrocytes and its separation into three active pro- 3 mA per gel and the gel was maintained at 4VC for 2.5 hr. teins. The developed gel was cut into halves lengthwise. One half was stained for protein identification with 1% Amidoblack 1OB MATERIALS AND METHODS in 7% acetic acid for 30 min. Destaining was performed elec- Reagents. Porphobilinogen was purchased from Sigma. trophoretically to shorten the procedure. The other half was DEAE-cellulose (DE-23) was from Whatman Ltd. Sephadex kept in a small amount of 0.05 M phosphate buffer at pH 7.4 G-100, DEAE-Sephadex A-50, and marker proteins for the at room temperature. Comparison of the halves indicated the from Phar- desired protein band to be cut for isolation. molecular weight determinations were obtained Proteins were eluted electrophoretically by the method of macia. Reagents for polyacrylamide disc gel electrophoresis and Braatz and McIntire (14). The buffer used for protein elution was 0.134 M mM The publication costs of this article were defrayed in part by page phosphate, pH 7.4/1 2-mercaptoethanol. charge payment. This article must therefore be hereby marked "ad- Electrophoresis was carried out with constant current (2 mA vertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Abbreviation UPGI-S, uroporphyrinogen I synthase. 6172 Downloaded by guest on September 24, 2021 Biochemistry: Miyagi et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6173 per tube) at 40C for 4 hr. The sliced gels and the protein eluates equilibration of the Sephadex and the elution were carried out were passed through a nylon filter, the column was washed with the same buffer. The eluates containing enzyme activity several times with small amounts of eluting buffer, anfdthe were combined. eluates were combined. Heat Treatment. The enzyme preparation from the above Isoelectric Focusing on 4% Polyacrylamide Disc Gel. The step was heated at 60°C for 15 min, cooled in an ice bath, and separation gel solution was prepared by mixing 3.2 ml of solu- centrifuged at 10,000 X g for 10 min. The precipitate was dis- tion A, 3.0 ml of solution B, 1.0 ml of 0.5% ammonium persul- carded. fate solution, 1.2 ml of 40% Ampholine (pH 3.5-10), and 15.6 DEAE-Sephadex A-50. The supernate after heat treatment ml of 5 mM phosphate, pH 7.4/0.1 mM 2-mercaptoethanol/ was concentrated in a collodion bag to approximately 5 ml and 0.3% glycerol. Solutions: A, 30 g of acrylamide and 1.5 g of then applied to a column (1.5 X 60 cm) of DEAE-Sephadex methylenebisacrylamide dissolved in water (total volume, 100 A-50 preequilibrated with 0.05 M phosphate, pH 7.4/0.01 M ml); B, 4 mg of riboflavin and 0.45 ml of N,N,N',N'-tetra- 2-mercaptoethanol. Elution was carried out with the same methylenediamine dissolved in water (total volume, 100 ml). buffer containing 0.2 M NaCl. The sample solution consisted of 1 vol (300 ,ug) of enzyme so- Rechromatography on DEAE-Sephadex A-50 with Linear lution and 1 vol of the mixture of 20% glycerol and 4% Am- NaCI Gradient. The enzyme preparation from the above step pholine (pH 3.5-10). The sample-protecting solution contained was concentrated in a collodion bag to approximately 5 ml. 1.5 ml of glycerol and 40% Ampholine per 100 ml. The glass Without dialysis, the enzyme solution was applied to a column tube (10 X 0.5 cm inside diameter) was filled with 7 ml of of DEAE-Sephadex A-50 (1.5 X 60 cm) preequilibrated with separation gel solution. After 30-40 min, no more than 50 ul 0.05 M phosphate, pH 7.4/0.01 M 2-mercaptoethanol. The of sample solution (desalted through Sephadex G-25) was ap- concentration of NaCl was increased linearly from 0 to 0.2 plied on the top of the gel, and then protection solution was M. layered on. The electrophoresis was carried out at 200 V at 4VC Determination of Molecular Weight. The molecular for 3 hr. weights of enzyme preparations were determined by two One of the simultaneously run gels was stained for proteins methods. (i) The gel filtration method of Andrews (16). A and the other one was cut into 5-mm-long sections. Each gel Sephadex G-100 column (1.5 X 70 cm) was preequilibrated segment was put in 5 ml of distilled water in a capped test tube with 0.05 M phosphate, pH 7.4/1 mM 2-mercaptoethanol. The and kept overnight at room temperature; then its pH was void volume was determined with blue dextran and the elution measured. A third gel was cut into 5-mm-long sections, minced volumes of known molecular weight protein markers (3 mg of with 1.5 ml of 0.05 M Tris-HCI, pH 8.2/600 mM porphobili- blue dextran and 5 mg each of RNase A, chymotrypsinogen A, nogen, and incubated at 370C for 20 hr. Deproteinization and ovalbumin, and albumin) were determined by absorption at determination of uroporphyrin formed was the same as de- 280 nm. (ii) Sodium dodecyl sulfate/polyacrylamide disc gel scribed above. electrophoresis. The enzyme solution was incubated with porphobilinogen for 3 hr, irradiated with UV light for 10 min, PURIFICATION OF UPGI-S and then analyzed according to the method of Weber and Os- The whole procedure described below was carried out at 4- born (17). The protein markers were phosphorylase b, albumin, 80C. carbonic anhydrase, trypsin inhibitor, and a-lactalbumin. DEAE-Cellulose. Plasma was removed from anticoagulated human whole blood after centrifugation; the erythrocytes were washed three times with equal volumes of 1.15% KCI solution. RESULTS The packed cells (150 X g for 10 min) were hemolyzed by thorough mixing with an equal volume of distilled water and Purification of the enzyme continuous stirring for 1 hr in an ice bath. The hemolysate was The results of the purification procedure up to column chro- centrifuged at 27,000 X g for 90 min; the supernate was filtered matography on DEAE-Sephadex A-50 are summarized in Table through glass wool, and the filtrate was applied onto a column 1. The loss of enzyme during purification on DEAE-cellulose (3 X 40 cm) of DEAE-cellulose (15) (fibrous form) preequili- was rather large, the total enzyme activity being 30-50% of the brated with 0.03 M phosphate buffer at pH 7.3. The column original activity. Complete removal of hemoglobin resulted in was washed with the same buffer until the color of cellulose the greater loss of enzyme. Such removal of hemoglobin at this turned whitish pink, free of hemoglobin. The cellulose was stage was not necessary for large-scale preparation because the removed from the column and washed once with 70 ml of the column chromatography on DEAE-Sephadex A-50 removed same buffer by mechanical stirring. Then, the enzyme was hemoglobin contamination completely. eluted into 70 ml of 0.134 M phosphate buffer at pH 7.4, after Ammonium sulfate fractionation purified the enzyme about 30 min of magnetic stirring. The supernate was collected after 2-fold. The fraction precipitated between 0 and 30% saturation centrifugation at 23,000 X g for 10 min. This process was re- contained 13.9% of the total protein (0-90% fraction) and 10.1% peated three times and the supernates were combined. of the enzyme activity; the fraction between 70 and 90% satu- Ammonium Sulfate Fractionation. To the combined su- ration contained 7.2% of the protein and only 0.4% of the en- pernate from DEAE-cellulose purification, solid ammonium zyme activity. The ammonium sulfate fractionation did not sulfate was added, a small amount at a time, until 70% satura- remove hemoglobin contamination sufficiently. tion. During this procedure, the pH was carefully maintained Column chromatography of the enzyme preparation on at about 7.4 by the addition of aqueous ammonia. The solution Sephadex G-100 further eliminated impurities. A typical elution was stirred continuously for 1 hr and the precipitate was col- diagram is shown in Fig. 1. Maximal UPGI-S activity was ob- lected after centrifugation at 10,000 X g for 15 min. served between the third and fourth protein peaks. Sephadex G-100. The precipitate from the above step was Heat treatment of the enzyme at 60°C for 15 min did not dissolved in a small volume of 0.05 M phosphate, pH 7.4/0.01 decrease the enzyme activity appreciably, but 40% of the M 2-mercaptoethanol/3% glycerol. Insoluble material was protein was removed. removed by centrifugation. The enzyme solution was then Chromatography on DEAE-Sephadex A-50 with 0.2 M NaCl applied to a column (1.5 X 85 cm) of Sephadex G-100. Pre- removed hemoglobin contamination eluted in the first protein Downloaded by guest on September 24, 2021 6174 Biochemistry: Miyagi et al. Proc. Natl. Acad. Sci. USA 76 (1979)

Table 1. Purification of UPGI-S from human erythrocytes Specific Total Total Enzyme activity, Purifi- vol, protein, activity, units/g cation, Yield ml mg units protein -fold % Supernate (27,000 X g) 375 71,250 2826.562 39.67 1.0 100.0 DEAE-Cellulose 225 405 874.124 2,158.3 54.4 30.9 Ammonium sulfate (0-70% saturation) 146.9 666.858 4,540.5 114.5 23.6 Sephadex G-100 33 69.1 604.500 8,753.3 220.7 21.4 Heat treatment 36 40.5 594.000 14,666.7 369.7 21.0 DEAE-Sephadex A-50 (with 0.2 M NaCl) 37 15.9 310.800 19,534.9 492.4 11.0 DEAE Sephadex A-50 (with 0-0.2 M NaCl): Fraction A 20.0 2.6 62.52 24,326.8 613.2 2.2 Fraction B 21.8 3.3 96.50 29,510.7 743.9 3.4

peak, far in advance of the enzyme in the second protein peak resolved into 7 main protein bands and fraction B was resolved (Fig. 2A). This second peak was rechromatographed on into 15. The enzyme activity was found in fraction A in two DEAE-Sephadex A-50 with a linear gradient of NaCl from 0 separate protein bands designated A1 and A2; in fraction B it to 0.2 M. The enzyme activity was separated into two peaks: was in three bands designated B1, B2, and B3 (Fig. 3). According the first one was sharp and corresponded to the protein peak; to the mobility on polyacrylamide disc gel, A1 corresponded the second one was contained in a rather complex protein peak to B1, and A2 corresponded to B2; this was confirmed by the (Fig. 2B). These two protein peaks were collected separately molecular weight estimations described below. Thus, in total, and designated fractions A and B. The final purifications of three isoenzymes were purified into single protein bands. The fractions A and B were 613- and 744-fold with yields of 2.2 and activities of these isoenzymes decreased considerably after 3.4% (Table 1). polyacrylamide gel electrophoresis. Therefore, it is incorrect Fraction A seemed to be more labile than fraction B, or the to compare the activities of each isoenzyme in Table 2 directly impurities in fraction B may have stabilized the enzyme. In with activities of fractions A and B in Table 1. some instances, especially when long-stored erythrocytes were Isoelectric Focusing. UPGI-S was also separated into three used, the enzyme activity in fraction A was lost even after enzymatically active protein bands by isoelectric focusing on identical elutions of proteins. On occasion, the whole fraction 4% polyacrylamide disc gel of both fractions A and B (Fig. 4). A was not detectable. The maximal UPGI-S activity was observed in fraction A at pH When the enzyme preparation from the heat treatment was 5.66 (range, 5.24-5.96) and in fraction B at pH 5.64 (range, subjected directly to chromatography on DEAE-Sephadex A-50 5.64-6.14). These isoelectric points were slightly higher than with linear gradient of NaCl, the resolution of the enzyme ac- those of UPGI-S from wheat germ (4) and from Rhodopseu- tivity into two separate protein peaks was not observed. Thus, domonas spheroides (3). the preliminary purification on DEAE-Sephadex A-50 with 0.2 M NaCl was necessary. Properties of the enzymes Identification of Three Isoenzymes. Fraction B was further purified on DEAE-Sephadex A-50 with a NaCl gradient from 0 to 1.0 M. No difference in elution pattern was observed with E the change of NaCl gradient from 0 to 0.5 M; however, the S0 elution time was prolonged. cn Fraction A and purified fraction B were subjected to ana- C lytical polyacrylamide disc gel electrophoresis. Fraction A was 00 ._ eo ._l 0) E 0) N 0 c 0a wL Co .ea' 4-1 0 4-- co a) EcB *10 20 30 40 50 Fraction '10 15 20 225 30 33 FIG. 2. Elution diagram of UPGI-S (0) from human erythrocytes Fraction on DEAE-Sephadex A-50 with 0.2 M NaCl (A) and with a linear FIG. 1. Elution diagram of UPGI-S from human erythrocytes on gradient of NaCl from 0 to 0.2 M (B). (A) Enzyme solution (42 mg of Sephadex G-100. Enzyme solution (80 mg of protein in 5 ml) from protein in 5 ml) from Sephadex G-100 was applied to a column of 0-70% ammonium sulfate fractionation was applied to a column of DEAE-Sephadex A-50 preequilibrated with the same buffer as in Fig. Sephadex G-100. Preequilibration of the gel and elution were carried 1 and eluted with buffer containing 0.2 M NaCl. (B) Pooled enzyme out with 0.05 M phosphate, pH 7.4/0.01 M 2-mercaptoethanol. solution from the above step (41 mg of protein in 4 ml) was applied

Fractions (3 ml) were collected at a flow rate of 1 ml/5 min. , Ab- to a column as in A. The NaCl gradient was linear from 0 to 0.2 M. sorbance at 280 nm; 0 --- e, UPGI-S activity. Other conditions were as in Fig. 1. Downloaded by guest on September 24, 2021 Biochemistry: Miyagi et al. Proc. Natl. Acad. Sci. USA 76 (1979) 6175 3 4 5 *slim> z4.i. I-. *r;, I&.4.J. 1 2 ir 6 7 8 B1 - -A, B2- -A2 41 B3 B2 B, AA I J

, .... B2 B3

.k ..4 : A2 ff

,7.

FIG. 3. Separation of UPGI-S from human erythrocytes on analytical polyacrylamide disc gel electrophoresis. Lanes 6-8 represent prolonged electrophoresis. Lanes: 1, fraction B; 2, fraction A; 3, final purified B2 + B3; 4, final purified B2; 5, final purified B1 + B2; 6, fraction A; 7, final purified Al; 8, final purified A2-

Molecular Weight. The molecular weights of fractions A and as single protein band on starch gel electrophoresis (5), and the B were determined by gel filtration (16). Both fractions had a enzymes from R. spheroides (3) and spinach leaf (4) were also mean (+SEM) molecular weight of 36,000 i 500. B1, B2, and shown to be single protein bands on analytical and preparative B3 were isolated into single protein bands and subjected to so- polyacrylamide disc gel electrophoresis. The present experi- dium dodecyl sulfate/polyacrylamide disc gel electrophoresis ments reveal that UPGI-S from human erythrocytes can be (17). Molecular weights were 38,000 i 1000 for B1 and 39,500 separated into three active protein bands on analytical poly- + 1000 for B2 and B3. The RF values of these isoenzymes on the acrylamide disc gel electrophoresis. gel were in the order B1 < B3 < B2 (Fig. 5). A1 and A2 had the According to the mobility on disc gel electrophoresis, these same molecular weights as B1 and B2, respectively. three isoenzymes may have either different molecular weights Other Properties. The optimal pH for UPGI-S assay for or different electric charges, or both; we favor the last possi- fractions A and B was pH 7.9 for incubation in 0.05 M phos- bility. The molecular weight measured by sodium dodecyl phate buffer. The more purified enzyme became more labile. sulfate/polyacrylamide disc gel electrophoresis showed con- The enzyme was stable through ammonium sulfate fraction- sistently that B1 was smaller than B2 and B3. On the other hand, ation; however, the stability decreased rapidly after Sephadex on analytical polyacrylamide disc gel electrophoresis the mo- G-100 chromatography. The more purified enzyme lost its bility of B1 (expected to be the fastest according to molecular activity when it was frozen; this agrees with other reports (3). weight) was less than that of the other two; B3 was the fastest. The idea of the isoenzymes having different electric charges DISCUSSION is also supported by the facts that: (i) B1 (Al) from fraction A was eluted with lower NaCl concentration than fraction B UPGI-S has been believed to be a single enzyme species (11). containing B3; (ii) isoelectric focusing separated the enzyme The enzyme purified from avian erythrocytes was identified preparation into the three active protein bands. It is probable that the enzyme may be composed of a single basic unit that Table 2. Final purification of UPGI-S from human erythrocytes has a single active site and a few small subunits that do not have Specific active sites but have electric charges; these may play a part in Enzyme activity, the process of enzyme synthesis or maturation. Protein, activity, unit/g Ratio of The basic question remains: What roles do these isoenzymes Fraction mg units protein specific activities play in the porphyrin biosynthetic pathway? Enzyme synthesis or is one Al 0.208 0.288 1384.6 1:4.2 maturation of the possibilities. Another possibility with A2 0.176 1.024 5818.21 far-reaching implications is that isoenzymes of UPGI-S are separate entities-i.e., each isoenzyme is synthesized inde- B1 0.089 0.1760 1977.2 pendently. If so, each isoenzyme would have a specific role and B2 0.090 0.1467 1630.0 3:2.5:1 probably a separate regulatory mechanism (18). By changing B3 0.096 0.0624 650.0 the ratio of the amount of these isoenzymes, the total activity Al and A2 were eluted electrophoretically (14) and the enzyme ac- of UPGI-S may be regulated. Numerous examples can be found tivity was assayed. B1, B2, and B3 in the corresponding gel sections in the literature Structural isomerism of UPGI-S also has were minced in a test tube with 1 ml of 3 mM porphobilinogen in 0.05 (19). M Tris*HCl buffer (pH 7.4) and 2 ml of 0.134 M phosphate buffer (pH been postulated; however, we have seen no relevant published 7.4); the mixture was incubated at 370C for 3 hr. reports. Downloaded by guest on September 24, 2021 6176 Biochemistry: Miyagi et al. Proc. Natl. Acad. Sci. USA 76 (1979)

1.2 8.0 'A 0 \' o-Lactalbumin @00 a lk 0 0~~~~T0 @0 0.8F 6.0 0 *0.9 Trypsin inhibitor

0 0 T 0 m :t 0. 0 0 0.6 .0 4.01' '0.6 IA I E Carbonic N s,X II a .a anhydrase \ B3 u 0. ._)co 0.41- . a . . a . a . . a s-B2 (A,) ir Go Ovalbumin a (1) 0

. . 9 . 9 I . v :3. 0.21 Albumin *

8.0 B 1.2 ' Phosphorylase b aa a° a) 2 . a A a a .A .a a o E 2 4 6 8 10 N Molecular weight X 10-4 6.0 0.9 U aa~~~~~a FIG. 5. Determination of the molecular weight of final purified I a UPGI-S from human erythrocytes on sodium dodecyl sulfate/poly- I a@°@ *. 'a X acrylamide gel electrophoresis. I...... 4.0 .-- 0.6

4. Higuchi, M. & Bogorad, L. (1975) Ann. N.Y. Acad. Sci. 244, e 401-418. @3LLLIl 5. Llambias, E. B. C. & Batlle, A. M. Del C. (1971) Biochem. J. 121, 9 12 15 18 327-340. Gel slice 6. Levin, E. Y. & Coleman, D. L. (1967) J. Biol. Chem. 242, 4248-4235. FIG. 4. Isoelectric focusing of UPGI-S from human erythrocytes 7. Llambias, E.B. C. & Batlle, A. M. Del C. (1971) Biochim. Biophys. on 4% polyacrylamide disc gel. (A) Fraction A. (B) Fraction B. Par- Acta 227, 180-191. f A B were tially purified enzymes (fractions and of Fig. 2B) applied & to 4% polyacrylamide disc gels for isoelectric focusing. Protein dis- 8. Stevens, E., Frydman, R. B. Frydman, B. (1968) Bwochim. Acta tributions are shown at the bottom of each figure. Each gel was cut Biophys. 158,496-498. 9. Russell, C. R. & Pollack, S. E. (1978) J. Chromatogr. 166, into 5-mm-long sections. 0, 0, pH; bars, enzyme activity. 632-636. The great significance of the UPGI-S isoenzymes relates to 10. Frydman, R. B. & Feinstein, G. (1974) Biochim. Biophys. Acta 350,358-373. their role in abnormalities of porphyrin metabolic pathway 11. Tait, G. H. (1978) in Handbook of Experimental Pharmacology, expressed in pathological conditions-e.g., acute intermittent eds. DeMatteis, F. & Aldrige, W. N. (Springer, Berlin), Vol. 44, porphyria (20) and congenital erythropoietic porphyria (21). pp. 14-18. The deficiency of UPGI-S in the former has been repeatedly 12. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R.J. reported (20, 22, 23). The excess of UPGIS in the latter (21) still (1951) J. Biol. Chem. 193,265-275. remains controversial and puzzling. The UPGI-S isoenzymes 13. Davis, B. J. (1964) Ann. N.Y. Acad. Sci. 121,404-427. A. R. would provide an explanation for the observed decrease or in- 14. Braatz, J. & McIntire, K. (1977) Prep. Biochem. 7, 495- 509. crease activities. in UPGI-S 15. Cornford, P. (1964) Biochem. J.91, 64-73. The final hypothesis postulates organ specificity of UPGI-S 16. Andrews, P. (1965) Biochem. J. 96,595-606. isoenzymes (e.g., hepatic, nephritic, or erythropoietic); specific 17. Weber, K. & Osborn, M. (1969) J. Biol. Chem. 244, 4406- isoenzymes would be predominant in a particular organ as 4412. expressed by different ratios of UPGI-S isoenzymes. 18. Brodie, M. J., Moore, M. R., Thompson, G. G., Campbell, B. D. & Goldberg, A. (1977) Biochem. Soc. Trans. 5, 1466-1468. 19. Markert, C. L., ed. (1975) in Isoenzymes (Academic, New York), Note Added in Proof. Additional work on UPGI-S isoenzymes in Vols. 1-4. human erythrocytes has demonstrated six isoenzymes. Previously their 20. Miyagi, K., Cardinal, R., Bossenmaier, I. & Watson, C. J. (1971) occurrence was missed completely because they were not so apparent J. Lab. Clin. Med. 78,683-695. and were considered negligible. Their occurrence has been confirmed 21. Miyagi, K., Petryka, Z. J., Bossenmaier, I., Cardinal, R. & Watson, repeatedly. The intensity of protein stain on disc gel electrophoresis C. J. (1976) Am. J. Hematol. 1, 3-21. may vary (24). 22. Magnussen, C. R., Levine, J. B., Doherty, J. M., Cheesman, J. 0. & Tschudy, D. P. (1974) Blood 44, 857-868. 1. Bogorad, L. (1958) J. Biol. Chem. 233,501-509. 23. Heilmeyer, L. & Clotten, R. (1969) Klin. Wochenschr. 47, 2. Bogorad, L. (1958) J. Biol. Chem. 233, 510-515. 71-74. 3. Jordan, P. M. & Shemin, D. (1973) J. Biol. Chem. 248, 1019- 24. Miyagi, K., Petryka, J., Kaneshima, M., Kawakami, J. & 1024. Pierach, C. A. (1979) Intern. J. Biochem., in press. Downloaded by guest on September 24, 2021