Enzymatic modifications of human fibroblast and leukocyte interferons

The sensitivity of highly purified human fibroblast interferon and partially purified human leukocyte interferon to several proteolytic and glycolytic enzymes was determined with respect to antiviral activity, isoelectric point, molecular weight, and thermal stability. Leucine aminopeptidase altered the distribution of isoelectric points for both interferons but produced little change in molecular weights; this enzyme somewhat reduced the activity of only leukocyte interferon. Treatment of fibroblast interferon with carboxypeptidases A and B did not greatly decrease antiviral activity, but it did slightly reduce the molecular weight of the interferon and substantially altered the distribution of isoelectric point values; similar treatment of leukocyte interferon caused some loss in activity, especially of the 17,000-molecular-weight species. Both interferons were inactivated rapidly by treatment with the endoproteases trypsin, pepsin, bromelain, and subtilisin. Chymotrypsin shifted the isoelectric points of both interferons, but only leukocyte interferon was significantly inactivated. Treatment with neuraminidase and beta-galactosidase changed the isoelectric point distribution but did not affect the activity or thermal stability of either interferon; such a treatment reduced the molecular weight of fibroblast interferon and the size heterogeneity of leukocyte interferon. Treatment with neuraminidase and then leucine aminopeptidase greatly reduced the activity of both interferons, especially leukocyte interferon. The data indicate that biologically active forms of fibroblast and leukocyte interferons can be distinguished by their relative sensitivity to certain proteases.     

Multiple molecular forms of mouse liver arginase

Hepatic arginase (L-arginine amidinohydrolase, EC 3.5.3.1) is an oligomer composed of three or four subunits. The present studies indicate heterogeneity in the size and charge of arginase subunits in mouse liver. Two types of arginase subunits with molecular weights of approximately 35,000 and 38,000 have been found. These two subunits are detected in liver cytosol or in purified preparations of arginase after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Two dimensional SDS-PAGE revealed multiple ionic forms of arginase for both the 35,000 and 38,000 subunits; the subunits contain basic proteins (pI range 7.8-9.1) and acidic proteins (pI range 5.8-6.4). Limited proteolysis by trypsin eliminated the molecular weight differences between the subunits without substantially affecting either their isoelectric points or activity. Comparative peptide maps and amino acid analyses of the 35,000- and 38,000-Da subunits showed that they were very similar. The data indicate that a neutral peptide (approx 3000 Da) is responsible for the differences in subunit molecular weight and that the multiple sized and charged forms are variants of the same protein.     

Cathepsin D of rat spleen. Affinity purification and properties of two types of cathepsin D.

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin--Sepharose 4B and concanavalin-A--Sepharose 4B, and chromatography on Sephadex G-100 and DEAE-Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE-Sephacel chromatography), termed cathepsin D-I, represented about a 1000-fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D-II, represented about a 900-fold purification and about a 3% recovery. Both enzymes showed four (pI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D-I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.     

Epstein-Barr virus induction by a serum factor. Purification of a high molecular weight protein that is responsible for induction

Serum contains a factor that induces Epstein-Barr virus antigens in latently infected human lymphoblastoid cell lines and that cooperates with chemical inducers. Here we report the purification of a protein that is responsible for the effect. Using ammonium sulphate precipitation, chromatography on DEAE-agarose and cellulose, molecular sieve chromatography on Bio-Gel A-5, and glycerol gradient centrifugation, the factor was enriched about 300,000-fold compared to calf serum. Under neutral conditions, the protein chromatographed as a high molecular weight protein of about 5.5 X 10(5) both in its active and inactive form. Both forms of the molecule sedimented at about 7 s. The factor is an acidic protein with a pI of 5. It seems to be composed of high molecular and low molecular weight subunits. Nanogram amounts of purified factor are sufficient for measurable inducing effects.     

Purification and characterization of acid beta-D-galactosidase from rabbit spleen

beta-D-Galactosidase has been purified to apparent homogeneity from rabbit spleen. The purification steps involved ammonium sulphate precipitation, DEAE-cellulose, concanavalin A-Sepharose, Sephadex G-200, and Sepharose 4B-(epsilon-aminocaproyl)-2-deoxy-beta-D-glucosylamine affinity chromatographies. In the DEAE-cellulose step, the beta-D-galactosidase was separated into two molecular forms, designated I and II, with similar pH optimum, Km, substrate specificity, and sensitivity to substrate analogues and other substances. Form I was purified 1,800-fold with a yield of about 2% of the total activity. This form is heat-labile, it has an acid optimal pH (4.0), an isoelectric point of 6.7 and a molecular weight of 75,000 daltons. Form II has an optimal pH of 3.6 and three different pI values (5.3, 5.7, and 6.7) whose relative proportions can be modified by treatment with neuraminidase. Form II appeared to be a multimeric form (IIA) of about 600,000 daltons at pH 4.0, which was reversibly dissociated to an oligomeric form (IIB) with an apparent molecular weight of 120,000 at neutral pH values. Both IIA and IIB were purified separately and showed an acid pH optimum and an heterogeneous pI (from 4.6 to 7.2). The dissociation of IIA into IIB can be generated spontaneously, but is increased by the presence of urea in the elution buffer, suggesting that both are aggregates of a common subunit.     

Purification to homogeneity of a beta-galactoside alpha2 leads to 3 sialyltransferase and partial purification of an alpha-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase from porcine submaxillary glands.

Two different sialyltransferases (EC 2.4.99.1) have been resolved from Triton X-100 extracts of porcine submaxillary glands by affinity chromatography on CDP-hexanolamine agarose. The predominant sialyltransferase of this tissue, a CMP-N-acetylneuraminate: alpha-D-N-acetylgalactosaminide alpha2 leads to 6 sialyltransferase, has been obtained in a partially purified and stable form. A less abundant but highly active enzyme, a CMP-N-acetylneuraminate: beta-D-galactoside alpha2 leads to 3 sialyltransferase, was purified over 90,000-fold to homogeneity. Chromatography of the latter enzyme on Sephadex G-200 separated two noninterconverting forms, designated A and B, with Stokes radii of 51 A and 31 A, respectively. Both forms have equal specific activity toward lactose and contain a single polypeptide with a molecular weight of about 50,000 as estimated by gel electrophoresis. Form A appears to bind 1.18 g of Triton X-100 per g of protein, or nearly an entire detergent micelle per polypeptide, while Form B binds little or no detergent. The enzymatic properties of both forms are similar (Rearick, J.I., Sadler, J.E., Paulson, J.C., and Hill, R.L. (1979) J. Biol. Chem. 254, 4444-4451) supporting the conclusion that Form A may represent the native sialyltransferase with an intact membrane-binding site, and Form B may be a large proteolytic fragment of Form A.     

Human liver N-acetylglucosamine-6-sulphate sulphatase. Purification and characterization.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.     

Purification and characterization of five forms of glutathione transferase from human uterus

Five glutathione transferase (GST) forms were purified from human uterus by glutathione-affinity chromatography followed by chromatofocusing, and their structural, kinetic and immunological properties were investigated. Upon SDS/polyacrylamide slab gel electrophoresis all forms resulted composed of two subunits of identical molecular size. GST V (pI 4.5) is a dimer of 23-kDa subunits. GST I (pI 6.8) and GST IV (pI 4.9) are dimers of 24-kDa subunits whereas GST II (pI 6.1) and GST III (pI 5.5) are dimers of 26.5-kDa subunits. GST V accounts for about 85-90% of the activity whereas the other isoenzymes are present in trace quantities. On the basis of the molecular mass of the subunits, amino acid composition, substrate specificities, sensitivities to inhibitors, CD spectra and immunological studies, GST V appeared very similar to transferase pi. Structural and immunological studies provide evidence that GST IV is closely related to the less 'basic' transferase (GST pI 8.5) of human skin. Extensive similarities have been found between GST II and GST III. The comparison includes amino acid compositions, subunits molecular size and immunological properties. The two enzymes, however, are kinetically distinguishable. The data presented also indicate that GST II and GST III are related to transferase mu and to transferase psi of human liver. Even though GST I has a subunit molecular mass identical to GST IV, several lines of evidence, including catalytic and immunological properties, indicate that they are different from each other. GST I seems not to be related to any of known human transferases, suggesting that it may be specific for the uterus.     

Species distribution and properties of hepatic phenylalanine (histidine):pyruvate aminotransferase.

Hepatic phenylalanine(histidine):pyruvate aminotransferase activity is much higher in the mouse and rat than in other animal species (human, guinea-pig, rabbit, pig, dog and chicken). The activity is elevated in the mouse and rat by the injection of glucagon but not in other species (guinea-pig, rabbit and chicken). The enzyme was purified from the mitochondrial fraction of mouse liver to homogeneity as judged by polyacrylamide disc gel electrophoresis in the presence of dodecylsulphate. With histidine as amino donor, the enzyme was active with pyruvate, oxaloacetate and hydroxypyruvate as amino acceptors but not with 2-oxoglutarate. Effective amino donors were histidine, phenylalanine and tyrosine with pyruvate, and methionine, serine and glutamine with phenylpyruvate. The apparent Km for histidine was about 6.9 mM with pyruvate and that for pyruvate was 21 mM with histidine. The enzyme is probably composed of two identical subunits with a molecular weight of approximately 40000. The pH optimum was near 9.0. Isoelectric focusing of the purified enzyme resulted in the detection of four forms with pI 6.0, 6.2, 6.5 and 6.7, respectively, all of which were responsive to glucagon. These four forms were nearly identical with the purified enzyme before the focusing with respect to physical and enzymic properties. A possible mechanism of this multiplicity is discussed.     

Staphylococcal L-asparaginase: purification and properties of enzymic protein

Staphylococcal L-asparaginase has been purified 400-fold with 40% recovery. The procedure involves ammonium sulphate precipitation and a column chromatography on Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. protein (pI 4.4) with the approximate molecular weight of 125,000 (estimated by Sephadex G-200 gel filtration). The enzyme is composed of not identical subunits. The polyacrylamide-SDS gel electrophoresis indicated two subunits with molecular weight 18,000 and 22,000.     

Purification and comparison of the structures of human liver acidic alpha-D-mannosidases A and B.

Human liver alpha-D-mannosidases A and B were purified 11 500-fold and 2000-fold respectively. Both showed microheterogeneity when analysed by isoelectric focusing. Alpha-D-Mannosidases A and B are immunologically identical but differ in their range of pI values, molecular masses, uptake into fibroblasts and subunit compositions. Alpha-D-Mannosidase A consists of equimolar proportions of subunits of molecular masses 62 kDa and 26 kDa, which are linked by disulphide bridges in the intact enzyme. Alpha-D-Mannosidase B also contains a small subunit, of molecular mass 26 kDa, and a variable mixture of larger subunits, of molecular masses 58 kDa and 62 kDa. The 62 kDa and 58 kDa subunits, but not the 26 kDa one, contain concanavalin A-recognizing glycans. The 58 kDa subunit has a lower pI, contains less high-mannose glycans but probably contains more mannose 6-phosphate than the 62 kDa subunit. It is postulated that the differences in structure and properties of alpha-D-mannosidases A and B are due to differences in the state of processing of the large subunit. This suggestion is consistent with a single locus on chromosome 19 for lysosomal alpha-D-mannosidase.     

Multiple forms of L1210 dihydrofolate reductase differing in affinity for methotrexate

Dihydrofolate reductase, purified from a Methotrexate-resistant subline (R6) of L1210 mouse leukemia cells, consists of two forms (designated 1 and 2) differing in affinity for the drug. Form 1 is more sensitive to inhibition by Methotrexate. Form 2 is more heat-labile, but it can be stabilized by bovine serum albumin or NADPH. Isoelectric focusing resolves 1 and 2; pI values are 7.4 and 8.2. Forms 1 and 2 comprise about 10 and 90% of the total protein, but 1 has at least a 2-fold higher specific activity. Binding measurements with [3H]Methotrexate provide KD values of ca. 1 and 27 nM for 1 and 2.     

Molecular weight of the functional unit of human leukocyte, fibroblast, and immune interferons

There is good agreement between the target molecular weight and the known molecular weight of human leukocyte interferons (about 20,000). The target molecular weight of fibroblast interferon, 31,000 to 42,000, is significantly larger than the monomer molecular weight of 21,000 to 24,000, suggesting that the dimer may be the predominant active functional unit in solution. A range from 63,000 to 73,000 for the target molecular weight of several different fractions of immune interferon (including natural crude as well as the recombinant form) indicates that the functional form of the immune interferon may be a trimer or tetramer. Thus, these studies indicate that the functional unit of leukocyte interferon is the monomer, that of fibroblast interferon is a dimer, and that of immune interferon is probably a tetramer (or trimer).     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Properties of two molecular forms of beta-glucuronidase from the mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.     

Purification and characterization of native and proteolytic forms of rabbit liver phosphorylase kinase

1. Two forms of phosphorylase kinase having mol. wt of 1,260,000 (form I) and 205,000 (form II) have been identified by gel filtration chromatography of rabbit liver crude extracts. 2. Form I was the majority when the homogenization buffer was supplemented with a mixture of proteinase inhibitors. This form has been purified through a protocol including ultracentrifugation, gel filtration and affinity chromatography on Sepharose-heparin. 3. Form II was purified by a combination of chromatographic procedures including ion exchange, gel filtration and affinity chromatography on Sepharose-Blue Dextran and Sepharose-histone. 4. Upon electrophoresis in the presence of sodium dodecyl sulfate two subunits of 69,000 and 44,000 were identified for this low molecular weight enzyme. Thus, a tetrameric structure comprising two subunits of each kind can be proposed. 5. Treatment of form I with either trypsin or chymotrypsin gave an active fragment having a molecular weight similar to that of form II. On the contrary, other dissociating treatments with salts, thiols and detergents failed in producing forms of lower molecular weight. 6. The similarities between proteolyzed forms I and II were stressed by their behavior in front of antibodies raised against the muscle isoenzyme of phosphorylase kinase. 7. The study of the effect of magnesium and fluoride ions on the activity of both forms showed an inhibitory effect of magnesium when its concentration exceeded that of ATP. 8. The inhibition could nevertheless be reverted by including 50 mM NaF in the reaction mixture. 9. Form I and form II could be distinguished by their pH dependence in the presence of an excess of magnesium ions over ATP, whereas the affinity for both substrates was not significantly different.     

The Interaction of Mammalian Interferons with Immobilized Cibacron Blue F3Ga: Modulation of Binding Strength

Mouse, hamster, rabbit, horse, and human interferons bind to immobilized Cibacron Blue F3GA under appropriate solvent conditions. Three forms of the immobilized ligand have been investigated: Cibacron Blue F3GA-Sepharose 4B, Blue Dextran-Sepharose 4B and Blue Sepharose CL-6B. The strength of binding of an interferon depends critically on the sorbent: Cibacron Blue F3GA-Sepharose 4B is the weakest in the series and Blue Sepharose CL-6B the strongest. The use of Blue Dextran-Sepharose 4B - a sorbent of intermediate binding properties - allows the complete separation of hamster, mouse and human fibroblast interferons in a single chromatographic step. Indeed, both the resolution, as well as the recovery, of those interferons is complete - regardless of the relative complexity of the chromatographed preparation (containing either crude or purified interferons). Thus, these ligands should prove of considerable use     

L-Phenylalanine ammonia-lyase from Phaseolus vulgaris. Characterisation and differential induction of multiple forms from elicitor-treated cell suspension cultures

L-Phenylalanine ammonia-lyase (EC 4.3.1.5) has been purified over 200-fold from cell cultures of bean (phaseolus vulgaris L.) exposed to elicitor heat-released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification. A preparation (purified 43-fold by ammonium sulphate precipitation, gel-filtration and ion-exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis-Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value. A preparation purified 200-fold and containing all four forms was used to immunise rabbits for the production of anti-(phenylalanine ammonia-lyase) serum. The antiserum was characterised by: immunotitration experiments; solid phase enzyme-linked immunosorbent assays; comparison of immunoprecipitates of 35S-labelled phenylalanine ammonia-lyase subunits (synthesized both in vivo and in vitro) on both one-dimensional and two-dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia-lyase preparations and immune blotting. SDS/polyacrylamide gels and SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia-lyase subunits is 77000; a 70000-Mr form is readily generated as a partial degradation product during purification. Immunoprecipitates of bean phenylalanine ammonia-lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10-fold increase in phenylalanine ammonia-lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high-pI, low-Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.     

Purification and subunit-structural and immunological characterization of five glutathione S-transferases in human liver, and the acidic form as a hepatic tumor marker

Five glutathione S-transferase (GST, EC 2.5.1.18) forms were purified from human liver by S-hexylglutathione affinity chromatography followed by chromatofocusing, and their subunit structures and immunological relationships to rat liver glutathione S-transferase forms were investigated. They were tentatively named GSTs I, II, III, IV and V in order of decreasing apparent isoelectric points (pI) on chromatofocusing. Their subunit molecular weights assessed on SDS-polyacrylamide gel electrophoresis were 27 (Mr X 10(-3)), 27, 27.7,27 and 26, respectively, (26, 26, 27, 26, and 24.5 on the assumption of rat GST subunit Ya, Yb and Yc as 25, 26.5 and 28, respectively), indicating that all forms are composed of two subunits identical in size. However, it was suggested by gel-isoelectric focusing in the presence of urea that GSTs I and IV are different homodimers, consisting of Y1 and Y4 subunits, respectively, which are of identical Mr but different pI, while GST II is a heterodimer composed of Y1 and Y4 subunits. This was confirmed by subunit recombination after guanidine hydrochloride treatment. GST III seemed to be identical with GST-mu with regard to Mr and pI. GST V was immunologically identical with the placental GST-pi. On double immunodiffusion or Western blotting using specific antibodies to rat glutathione S-transferases, GST I, II and IV were related to rat GST 1-1 (ligandin), GST III(mu) to rat GST 4-4 (D), and GST V (pi) to rat GST 7-7 (P), respectively. GST V (pi) was increased in hepatic tumors.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.