Multiple forms of human phosphoglycerate kinase

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS Vol. 193, No. 2, April 1, pp. 415-421, 1979

Multiple

Forms of Human

Phosphoglycerate

WOLFGANG K. G. KRIETSCH,2 INGE U. FREIER, lnstitut

fiir Physiologische

AND

Physikalische Biochemie und Zellbiologie GoethestraJe 33, 8000 Miinchen 2, West Germany

Chemie,

Received

August 11, 1978; revised November

Kinasel STEFAN W. EBER der Universittit

Miinchen,

14, 1978

Phosphoglycerate kinase was isolated by affinity chromatography from human skeletal muscle and erythrocytes. As in the tissue extracts, the purified enzyme showed in Cellogel electrophoresis one major and two minor bands with phosphoglycerate kinase activity. The multiple forms were separated by chromatography on CM-Sepharose. From the three separated forms, A, B, and C, the latter was not detectable in electrophoresis of tissue extracts or in the purified unresolved phosphoglycerate kinase. The faintest, most anoditally migrating form observed in the tissue extracts could not be isolated in pure form by chromatography on CM-Sepharose. The electrophoretic mobility of the phosphoglycerate kinase forms depended strongly on the buffer systems used. The different forms had identical molecular weight, substrate affinity, and heat stability and were inhibited to the same extent by antibody. They could also not be separated by column affinity chromatography. Small differences were found in thiol group content and in the specific activity, the latter being a consequence of diminished free sulthydryl residues. Exposure to either reductive or oxidative conditions changed the specific activity, but did not result in interconversion among the pure forms. The multiple forms probably arise as a result of epigenetic factors occurring after the primary polypeptide chain has been synthesized.

Beutler (1) found on starch gel electrophoresis three bands of phosphoglycerate kinase (PGK)” activity in hemolysate from normal human erythrocytes. This pattern was observed in several laboratories (Z-4). The stability of the multiple forms was demonstrated by Chen et al. (2>, in a procedure in which frozen hemolysate was stored for as long as 5 years. Stored lysates revealed no apparent change in the pattern of the three bands. Yoshida and Watanabe (5) obtained in the last step of the purification of the human erythrocyte enzyme, a chromatography on CM-Sephadex, an elution profile which showed, besides a major enzyme peak, two minor peaks. These two peaks comprised less than 5% of the total 1 This work was supported by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 51. * To whom correspondence should be addressed. 3 Abbreviations used: PGK, phosphoglycerate kinase; DTE, dithiothreitol; TEA, triethanolamine; bicine, N,N-bis(2-hydroxyethyl)glycine; tricine, N-[2-hydroxy-l,l-bis(hydroxymethyl)ethyl]glycine; DTNB, 5,5’-dithiobis(2-nitrobenzoic acid). 415

activity. The separated enzyme peaks, however, each showed the same electrophoretic pattern in starch gel as the unfractionated enzyme or the crude extract. Yoshida and Watanabe therefore assumed that the two minor forms were artifacts resulting from the association of the enzyme with protein impurities of higher acidity. With the adsorbent ATP-rib-Sepharose, pure PGK can conveniently be isolated in sufficient amounts to separate minor forms. Through chromatography on CM-Sepharose, the purified enzyme could be segregated in three distinct, nonconvertible forms. Several properties of the multiple forms have been characterized. Since PGK is a monomeric enzyme (5, 6), of which only one cistron on the X chromosome for the enzyme of human somatic cells is known (7, S), epigenetic factors involving modifications of a primary chain and the phenotype product of a single cistron offer a broad spectrum of reasons accounting for multiplicity in enzymes. Included are polymerization, conjugation 0003-9861/79/040415-~$02.00/0 Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

416

KRIETSCH,

FREIER,

with other groups, enzymatic modification of the polypeptide chain, specific modification of residues, and conformational changes. When epigenetic factors are involved, ascertaining the reasons for the multiple forms becomesmuch more complex since only very slight differences may occur among the various forms.

AND EBER

benzophenoxazine). The stain was “positive.” By Beutler’s procedure, the stain was “negative.” Preparation of antibodies. Unresolved human skeletal muscle PGK and the pure main form were dialyzed against 20 mrd Tris, pH 7.2. Two to three milligrams of protein (in 0.5 ml) was mixed with 0.5 ml of Freund’s complete adjuvant and injected subcutaneously into rabbits in three different places. The injection was repeated once weekly for 2 weeks, after which the Freund’s adjuvant was exchanged against sodium alginate adjuvant for an additional 3 weeks of MATERIALS AND METHODS injection. Each rabbit received lo-15 mg of enzyme All procedures, unless otherwise specified, were protein throughout the immunization schedule. Blood was taken 1 week after the final injection by punccarried out at O-PC. Enzyme assay. Enzyme activities were measured ture of the marginal ear veins. y-Globulins were isolated by the standard techin the backward reaction at 365 nm and 25°C (6). The unit used was 1 pmol NADH consumed/mm, with niques of (NH,),SO, fractionation, and the fourfold a molar absorption coefficient for NADH of 3.4 x 103 concentrated globulin was stored at -30°C in 100 mM M-’ cm-* (10). To prevent inactivation, the enzyme Tris, pH 8.0. The presence of antibody against PGK was diluted in 100 mM triethanolamine buffer (pH was visualized by the double immunodiffusion (Ouch7.5), containing 0.1% (w/v) bovine serum albumin terlony) test. Immunological studies. Varying amounts of antiand 1 mM DTE. Protein determination. The protein concentration serum were added to 0.2 U of PGK, and the volume of the native enzyme was determined spectrophoto- was brought to 0.2 ml with 0.1 M TEA-HCl buffer, metrically at 280 nm with an E$$ ,,m= 6.8 (l-cm light pH 7.6, containing 150 mM NaCl. After 18 h of incupath). This absorption coefficient was evaluated from bation at 4”C, the mixture was assayed for PGK. Since the original unresolved enzyme and from the pure the antibody inhibits PGK, the activity in the supermain form by three independent weight measure- natant, after centrifugation at 30,OOOgfor 10 min, was the same as before centrifugation. It was found ments. that normal rabbit serum had no effect on enzyme Electrophwesis and activity staining. Polyacrylamide gel electrophoresis was carried out in 10% activity. Thiol groups. For the titration of the sulfhydryl acrylamide containing 3’70 mM Tris buffer at pH 8.8. A 25 mM Tris-100 mM glycine buffer, pH 8.3, with content, the three purified forms were incubated for 0.1% (w/v) sodium dodecyl sulfate was used as a running 20 h with 2 mM DTE in degased 200 mM Tris, pH 8.0, buffer (9,ll). Electrophoresis was performed at in a closed vessel. This was followed by exhaustive approximately 10°C for 8 h with a constant voltage dialysis against the same degased buffer without of 200 V. The gels were stained overnight with 0.025% DTE. Determination of thiol groups was carried out by spectrophotometrical titration with DTNB (13). (w/v) Coomassie brilliant blue dissolved in methanolIsolation of the multiple forms. The PGK was acetic acid-water (l&2:10, v/v/v) and destained with the same solvent. For the estimation of the molecular isolated from human skeletal muscle and erythrocytes weight, glyceraldehyde phosphate dehydrogenase, through affinity chromatography and gel filtration as triosephosphate isomerase, and catalase were used described previously (9). CM-Sepharose CL-6B was washed and equilibrated with 10 rnrd sodium phosfor calibration. For activity staining, samples of 2-5 ~1 PGK (5-10 phate buffer, pH 7.0, and poured into a 2 x 40-cm U/ml) were applied on Cellogel sheets 0.5 mm thick. column. The purified PGK, dialyzed against the same Electrophoresis was performed at 250 V for 200 min buffer, was added, and the column was eluted with a linear gradient from 10 to 40 mM of sodium phosphate at 4°C in 50 mM Tris buffer containing 2 mM EDTA, buffer (each vessel containing 350 ml). and adjusted to pH 7.0 or 8.1 with 1 M citric acid. Enzyme properties. The Michaelis-Menten conIn further tests, Tris buffer was used, but the citric acid was exchanged against phosphoric acid and boric stants (K,) were determined in 100 mhf TEA-HCl acid. In addition, electrophoretic runs were conducted buffer, pH 7.5, 8 mM MgS04, 0.3 mM NADH, glycwith TEA-HCl, bicine, tricine, and sodium phos- eraldehyde phosphate dehydrogenase (5 U/ml), and phate. To compare electrophoretic mobility, all buffers 80 mu/ml PGK at 25°C. The constants for ATP were were adjusted to pH 7.0 at nearly identical ionic determined with 10 mM 3-phosphoglycerate, and the strength. constants for 3-phosphoglycerate were determined The gels were stained using the technique of Oehl- with 2 mM ATP. For each kinetic constant, the enzyme schlegel and Brewer (12), but replacing phenoxy- activity was measured at nine substrate concentrations (ATP, 0.03-2 mrvf;3-phosphoglycerate, 0.08-10 methosulfate with Meldola blue (&dimethylamino-2,3-

MULTIPLE

FORMS OF PHOSPHOGLYCERATE

1

i'.

KINASE

417

C2ooa

FIG. 1. CM-Sepharose CL-6B chromatography of the human skeletal muscle PGK. The protein was eluted with a linear gradient from lo-40 mM sodium phosphate buffer. The flow rate was 40 ml/h. mM). The apparent K, values were obtained graphically from standard double reciprocal LineweaverBurk plots. The heat stability at 54°C was measured in 100 mM TEA-HCl buffer, pH 7.5. For the denaturation-renaturation studies, 2.5 mg of the pure main form of PGK (dried in vacuum at 60°C) was dissolved in a 6 M guanidinium chloride solution, pH 8.0. After incubation at 37°C for 1 h, the solution with the completely inactivated enzyme was diluted tenfold with 50 mM Tris-citrate buffer, pH 8.1, and exhaustively dialyzed against the same buffer. The reactivated enzyme was then examined by Cellogel electrophoresis. Materials. All chemicals were of the highest purity available. Enzymes, substrates, TEA-HCl, Tris, and Meldola blue were purchased from Boehringer (Mannheim, Germany). CM-Sepharose CL-6B was obtained from Deutsche Pharmacia (Freiburg, Germany), and Cellogel was from Celtec GmbH (Bad Homburg, Germany). DTE, DTNB, bovine serum albumin, aaylamide, N,N’-methylene bisacrylamide, sodium dodecyl sulfate, /3-mercaptoethanol, guanidin hydrochloride, bicine, tricine, nitroblue tetrazolium salt, and Coomassie brilliant blue were purchased from Serva (Heidelberg, Germany). All other chemicals were from E. Merck (Darmstadt, Germany).

was isolated from skeletal muscle of two female donors and one male donor (60, 71, and 55 years of age) and chromatographed on CM-Sepharose Cl-6B. With the shallow sodium phosphate gradient (lo-40 mM) employed, four protein peaks with corresponding enzyme activity were eluted (Fig. I). This elution pattern was obtained in all three cases and from one preparation (20 mg) of erythrocyte PGK. In all samples, the share of the four fractions on the total enzyme activity was 0.9-1.6% for fraction I, 0.9-1.0% for fraction II, 3.2-4.9% for fraction III, and 92.6-95% for fraction IV. The Cellogel electrophoresis in a Tris-citrate buffer of the four separated fractions from a single donor is shown in Fig. 2. The protein in fraction I, which was eluted from the

RESULTS

Isolation of the Multiple Forms Purified PGK from human skeletal muscle and erythrocytes (9) migrated as a single sharp band in the acrylamide gel electrophoresis in the presence of dodecyl sulfate. In three separate samples, 240 mg of PGK

FIG. 2. Cellogel electrophoresis in Tris-citrate, pH 8.1, with activity staining (12). a, unresolved skeletal muscle enzyme; b-e, the four fractions I(b)-IV(e) from the CM-Sepharose chromatography. The capital letters A-C group the multiple forms. 0 is the fastest band which could not be isolated in pure form.

418

KRIETSCH,

FREIER,

column with the void volume, did not bind to the cation exchange resin. This fraction had the most heterogeneous protein composition and contained mainly the form from fractions III and IV, in addition to a faint band with the fastest anodic mobility (Fig. 2). The fact that fraction I had a different banding pattern in Cellogel from the original extract proves that the column was not overloaded. It cannot yet be explained why fraction I is eluted with the void volume. Possibly, the PGK in this fraction associated to a greater extent with more acidic protein(s), as already suggested by Yoshida and Watanabe (5). According to this assumption, the specific activity of PGK (400 U/mg) in fraction I is distinctly lower than in the three other fractions. Fraction II contained also slight impurities from the other fractions (about 8%), but in fraction III, the portion is less than l%, and fraction IV is pure. Through rechromatography under the same conditions, the main forms of fractions II and III are obtainable free of impurifications by other forms. As the Cellogel electrophoresis exhibits (Fig. 2), the PGK had at least three different forms. According to their anodic mobility in T&-citrate, these forms were designated A (fraction III), B (fraction IV), and C (fraction II). For the forms A and B, the order of anodic mobility was in accordance with their binding strength to the cation exchanger. In contrast to its ability to bind to the exchanger, C had the slowest mobility. Electrophoretic Mobility

AND EBER

+

start

FIG. 3. Cellogel electrophoresis in bicine, pH 7.0, with activity staining. From left to right the three forms C, A, and B.

two minor forms, A and C, migrated with the same mobility to the cathode (Fig. 3). In the TEA-HCl and sodium phosphate buffers, PGK migrated to the cathode and did not separate into multiple forms. Wrobel and Stinson (14) found in yeast PGK a binding site for anions which is not at the active site. The dissociation constants for anions differ between 0.4 PM and 182mM. In part, this strong binding of anions to PGK could explain the observed dependence of the electrophoretic mobility on the anions. If ATP was added to the T&-citrate buffer, the anodic mobility of the three forms was further increased and became identical. This had been already observed by Beutler (1) on human erythrocyte hemolysate. It can be assumed that ATP binds to the active site and to the anion binding site. Thus, through the additional negative charges and maybe through changes which could be induced by the ATP binding, differences in the electrophoretic mobility between the three forms of PGK disappeared.

Since the electrophoretic mobility of PGK is also influenced by the buffer systems Properties of the Multiple Forms used, the degree of binding to the exchanger The three forms were not interconverticannot be directly compared to the mobility. The strong dependence on the anion type ble by repeated freezing (-30°C) and thawwas remarkable. If Tris was used as a cation ing (+2O”C). Furthermore, no alteration at pH 7.0, the mobility was the fastest with in the electrophoretic mobility was obtained citrate, very slow with phosphate, and with either by incubation with muscle extract borate PGK had not migrated even after free of PGK (unbound protein and NaCl 150 min. However, in Tris-citrate and peak of the affinity chromatography) (9), Tris-phosphate, the different forms had or with 2 InM DTE for 20 h. The PGK forms the same relative mobility. On the other renaturated from 6 M guanidinium chlohand, in bicine and tricine buffers the main ride have the same electrophoretic mobility form, B, moved toward the anode and the as before the denaturation.

MULTIPLE

FORMS OF PHOSPHOGLYCERATE

TABLE I

PROPERTIESOFTHE MULTIPLEFORMS

Properties

Number of experiments (n)

4 Ulmg protein 8-10 U/ml antiserum K, for ATP(m@ 3 K, for 3PG” (mM) 3 Half-life at 2 54°C (min) Molecular weight 5 Thiol groups/m01 3-6 1% enzyme 3 E 280“In 3 Ratio E 280mn,260 nm

Forms A

C B (mean values)

700 8.8 0.38 1.08

1050

690

8.6

8.8

0.38

0.38

1.10

1.12

38 43,000

35 43,000

33 43,000

6.4 -

7.0 6.8

6.5 -

1.58

1.59

1.42

R3PG, 3-phosphoglycerate.

With the sensitivity of the methods applied (gel chromatography and dodecyl sulfate-acrylamide electrophoresis), the multiple forms had the same molecular weight of 43,000. The apparent K, values for the substrates Mg-ATP and 3-phosphoglycerate were identical, but the specific activities of the three forms were distinctly different (Table I). Heat stability was studied at 54”C, and all three forms had nearly the identical half-life of 33-38 min. Thiol Groups

419

KINASE

thiol groups revealed minor differences between the three forms. The highest content of free thiol groups (7.0 groups/m01 enzyme) belonged to the B form, the two minor forms A and C having contents nearly 8% lower (Table I). The fact that the cystein residues of the two minor forms were not completely reduced by the incubation with DTE may have been due to the isolation procedure. In contrast to the main form, the A and C forms were eluted from the CM-Sepharose column in very diluted solution and had to be concentrated by dialysis against 30% polyethylene glycol. For that reason, it is possible that a small portion of the very reactive thiol groups was irreversibly oxidized. Antibody Titration Antibodies prepared against unresolved human PGK formed in Ouchterlony immunodiffusion a single line of precipitation with the unresolved PGK and the three separated forms. The precipitation lines fused completely showing the presence of identical determinants in the three forms. The same result was obtained with antiserum against the pure B form. Therefore, the

,

1000 -

l l ,* ,

Without effective protection of the two .; most reactive thiol groups, PGK very quickly lost its enzymatic activity. Thiol groups ;;L P and specific activity were determined from E" the three forms of each preparation before 3 500 and after reduction with DTE (Fig. 4). After the final step of the isolation procedure, the three forms had specific activities between 100 and ‘700U/mg and a thiol content of 4-6 groups/mol. Through the reduction 100, I with 2 mM DTE in degased buffer, the speol, cific activities increased to 700-1000 U/mg, 4 7 5 6 8 and the number of thiol groups increased Mol SH/Mol PGK to 6.5-7.0/mol. From these experiments, it was evident that the specific activity of FIG. 4. The dependence of specific activity on the PGK strongly correlates to the number of thiol group content in PGK. The thiol groups were thiol groups (Fig. 4). However, after the determined with DTNB (13), and the specific activity reduction with DTE, the titration of the is expressed in units per milligram of protein.

420

KRIETSCH,

FREIER,

AND EBER

total PGK activity was only l%, and because C migrated immediately behind the main form, B. Under these conditions, both forms fuse. Only if the purified B and C forms were applied in separate slots on the electrophoresis in Tris buffer, were the slight differences in mobility recognizable. The three forms were homogeneous in dodecyl sulfate-acrylamide electrophoresis and had the same molecular weight. They were not interconvertible. With the exception of specific activity (U/mg protein), the three forms were identical in all kinetic parameters investigated. The content of free thiol groups has no influence on the electrophoretic mobility. This means that the small differences between the three forms (6.5-7.0 thiol groups/ mol) cannot be the reason for multiformity. However, the three forms vary in their affinity to the buffer ions. In Tris buffer they migrate with different mobility to the anode. Yet in bicine or tricine buffer, B migrates to the anode and A and C migrate with the same mobility to the cathode. In TEA-HCl and sodium phosphate, all three forms have the same cathodic mobility. Such alteration of the effective charge caused by buffer ions was also observed in rat (15), rabbit (16), and yeast (16) PGK. Moreover, the binding of citrate ions has been proposed DISCUSSION to explain the lowering of the isoelectric PGK consists of three distinct compo- point of a human erythrocyte PGK variant nents as shown in Fig. 2 (see slot a). The (21). The differences in anion affinity in the band with the strongest staining intensity has the slowest mobility. The fastest band three forms of human PGK can be attributed is frequently very faint. This PGK pattern to changes in the anion binding sites. The was uniformally observed in the hemolysate importance of the anion binding site is not of normal blood donors (l-4, 20) and in all known, but it may possibly be that this site human organs with the exception of the is necessary to attach PGK to specific carrier proteins (e.g., to membranes) or to testes. The enzyme from human skeletal muscle modulate the enzymatic activity. Correand erythrocytes purified by affinity chro- sponding to this assumption then, the three matography (9) could be separated into forms should have different functions in three forms by column chromatography on the human somatic cells. For a small portion CM-Sepharose. Two of these forms were (l-5%) of the human erythrocyte enzyme, demonstrable in the Cellogel electrophore- binding to the erythrocyte membrane was sis of tissue extracts from all human somatic reported (17- 19). If only one cistron codes for somatic PGK organs. The third isolated form, C, was only discovered after chromatography on (7, 8), the multiple forms should be a reflection of nongenetic, posttranscriptional modCM-Sepharose. The C form was not detected in the origi- ification or partial conformational differnal tissue extract because its share of the ences. Beutler (1) assumed that the banding

preparation was considered to be free of impurities, and the three forms were deemed immunologically identical. PGK is inactivated by association with antibody. If the determinants are the same for all forms, they should be inactivated to the same degree by increasing amounts of antibody. For all three forms, the ratio of the antiserum required to inhibit a given amount of units was identical. One milliliter of antiserum inhibited 8.8 U of A, 8.6 U of B, and 8.8 U of C (n = 5). Therefore, the specific activity expressed in units per milliliter of antiserum of the three forms was identical. In contrast, the specific activity, determined in units per milligram of protein, was about 30% lower for A and C than for the B form. This difference may have been due to the specific interaction of the inhibitory antibody with only enzymatitally active molecules. In the Ouchterlony immunodiffusion, the A and C forms had definitely more intense precipitation lines than the B form if identical amounts of units of all three forms were applied (60 U/ml), indicating that the specific activity of both are lower than for the B form. In the assay for units per milligram of protein, the inactive molecules were also taken into account.

MULTIPLE

FORMS

OF PHOSPHOGLYCERATE

of the normal erythrocyte enzyme appeared to be due to either binding of its substrate, ATP, or to configurational changes which could be induced by ATP. However, the ratio of absorbance from 280 to 260 nm is identical for the A and B forms and is only insignificantly lower for the C form (Table I), indicating that the three forms have not bound ATP. Since the three forms have the same electrophoretic mobility in the presence of ATP, the binding of ATP cannot be the reason for the observed multiform&y. Furthermore, neither freezing-thawing nor the denaturation-renaturation studies changed the electrophoretic mobility of the forms. Therefore, conformational forms may be excluded. Limited postsynthetic modifications, such as acetylation, deamination, glycosylation, etc., which do not involve gross changes in charge and enzyme conformation seem to be most likely. In four rare variants of human PGK all having increased electrophoretic mobility, the relative mobility and intensity of the multiple forms remained the same (3). This unchanged pattern could be best explained as the result of the same epigenetic modification of one original polypeptide chain. In another rare variant, PGK “Miinthen” (20), only one form was detected. It is supposed that in this variant, the transformation process was not possible, perhaps because the amino acid or conformation at which the modification occurred was exchanged. ACKNOWLEDGMENTS The authors gratefully acknowledge the fruitful discussions with Dr. H. Krietsch. Thanks also to M. L. Everett for proofreading and typing the menuscript. REFERENCES 1. BEUTLER, E. (1968) Biochem. Genet. 3, 189-195. 2. CHEN, S.-H., MALCOLM, L. A., YOSHIDA, A., AND GIBLETT, E. R. (1971) Amer. J. Hum. Genet. 23, 87-91.

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3. CHEN, S.-H., AND GIBLETT, E. R. (1972) Amer. J. Hum. Genet. 24, 229-230. 4. OMOTO, K., AND BLAKE, N. M. (1972) Ann. Hum. Genet. (London) 36, 61-67. YOSHIDA, A., AND WATANABE, S. (1972) J. Biol. Chem. 247, 440-445. KRIETSCH, W. K. G., AND B~CHER, TH. (1970) Eur. J. Biochem. 17, 568-580. MEERA KHAN, P., WESTERVELD, A., GRZESCHIK, K. H., DEYS, B. F., GARSON, D. M., AND SINISCALCO, M. (1972) Amer. J. Hum. Genet. 23, 614-623. 8. GRZESCHIK, K. H., ALLERDICE, P. W., GRZESCHIK, A., OPITZ, I. M., MILLER, 0. J., AND SINISCALCO, M. (1972) Proc. Nut. Acad. Sci. USA 69, 69-73. 9. KUNTZ, G. W. K., EBER, S., KESSLER, W., KRIETSCH, H., AND KRIETSCH, W. K. G. (1978) Eur. J. Biochem. 85, 493-561. 10. BUCHER, TH., LUSCH, G., AND KRELL, H. (1976) Quality Control in Clinical Chemistry, pp. 301-310, Walter de Gruyter and Co., Berlin and New York. 227, 11. LAEMMLI, U. K. (1970) Nature (London) 680-685. 12. OEHLSCHLEGEL, F. J., AND BREWER, G. I. (1972) Ezperimentia 28, 116-117. 13. ELLMAN, G. L. (1959) Arch. Biochem. Biophys. 82, 70-72. 14. WROBEL, I. A., AND STINSON, R. A. (1978) Eur. J. Biochem. 85, 345-350. 15. FRITZ, P. I., AND WHITE, E. L. (1974)Biochemistry 13, 444-449. 16. HASS, L. F., CABAN, C. E., KAPPEL, W. K., OTTINGER, W. E., WHITE, F. L., KRANZ, K. R., ANDFRIT~, P. I. (1974)Comp. Biochem. Physiol. 47B, 889-893. 17. SCHRIER, S. L., BEN-BASSAT, I,, JUNGA, I., SEEGER, M., AND GRUMET, C. (1975) J. Lab. Clin. Med. 85, 797-810. 18. NILSSON, O., AND RONQUIST, G. (1969) Biochim. Biophys. Acta 183, 1-9. 19. DE, B. K., AND KIRTLEY, M. E. (1977) J. Biol. Chem. 252, 6715-6720. 20. KRIETSCH, W. K. G., KRIETSCH, H., KAISER, W., DONNWALD, M., KUNTZ, G. W. K., DUHM, J., AND BOCHER, TH. (1977) Eur. J. Clin. Invest. 7, 427-435. 21. YOSHIDA, A., AND WATANABE, S. (1972) J. Biol. Chem. 247. 446-449.