High performance purification of glycolytic enzymes and creatine kinase from chicken breast muscle and preparation of their specific immunological probes

We developed a novel procedure for isolation of the muscle isozymes of aldolase, triose phosphate isomerase (TPI), glyceraldehyde phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), phosphoglycerate mutase (PGM), enolase, pyruvate kinase (PK) and lactic dehydrogenase (LDH), and also creatine kinase (CK), at high purity, specific activity and yield. Protein was extracted from chicken breast muscle and glycolytic enzymes were purified by a three step procedure consisting of: Ammonium sulfate combined with pH fractionation. Phosphocellulose chromatography with performance of high pressure liquid chromatography, exploiting a pH gradient formed by a gradient of the buffering ion for protein elution. Affinity chromatography causing elution by substrate or pH. The enzymes, obtained at over 95% purity as judged by specific activity and silver stained electropherograms, were injected into sheep. Antibody for each enzyme was purified on specific immunosorbant and its specificity was verified by immunotransfer analysis.     

Multiple forms of human phosphoglycerate kinase.

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 anodically 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 sulfhydryl 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.     

Isolation of phosphoglycerate kinases by affinity chromatography

A variety of Sepharose derivatives containing DL-O-phosphorylserine or adenosine nucleotides with different points of attachment, has been synthesized and tested for affinity to phosphoglycerate kinase. The most effective gels contained periodate-oxidized ATP or ADP bound via the ribose by hydrazone formation to adipoyl-dihydrazo-Sepharose. The effect of pH, magnesium and buffer ions on the binding capacity of the ATP derivative of Sepharose has been examined. Optimal elution of phosphoglycerate kinase was investigated using different combinations of adenosine nucleotides, 3-phosphogylcerate and magnesium ions. A method is presented giving conditions for the purification of phosphoglycerate kinase from different sources (spinach, human erythrocytes, human, rabbit and trout muscle). It includes extract preparation, affinity chromatography and gel filtration. The method is greatly superior to known isolation procedures by virtue of its technical simplicity, excellent yield (85-100%) and reproducability. The capacity of the ATP-ribosyl-adipoyl-dihydrazo-Sepharose was 5 mg phosphoglycerate kinase per 1 g of matrix. Polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate indicated that the final products are homogeneous. The phosphoglycerate kinases from different sources appear to have the same affinity for this ATP derivative of Sepharose, the same molecular weight and the same specific activity.     

Multiple enzyme purifications from muscle extracts by using affinity-elution-chromatographic procedures.

1. Starting with (NH4)2SO4 fractions of muscle extracts, procedures for purifying four to six separate enzymes from each fraction by using affinity-elution-chromatographic techniques are described. 2. Schemes for purifying 12 separate enzymes from rabbit muscle, and eight from chicken muscle extracts, are included. In nearly all cases the overall procedure involves three steps: the initial (NH4)2SO4 fractionation, the ion-exchange chromatography with affinity elution of the enzyme, and gel filtration. The specific activities of the enzymes so purified are comparable with the highest values in the literature. 3. The five schemes described include illustrations of affinity elution of the separate enzymes at different pH values, at different ionic strengths and in combination with conventional gradient elution. They also include stepwise adsorption on columns at different pH values. 4. Separation of two electrophoretically differing forms of phosphoglycerate kinase was achieved by gradient affinity elution from CM-cellulose. The lower-pI form was eluted by a lower concentration of substrate than the higher-pI form.     

Isolation and properties of the glycolytic enzymes from Zymomonas mobilis. The five enzymes from glyceraldehyde-3-phosphate dehydrogenase through to pyruvate kinase.

The five glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase, enolase and pyruvate kinase were each purified from extracts of Zymomonas mobilis cells, by using dye-ligand chromatography as the principal step. Two procedures, producing three and two of the enzymes respectively, are described in detail. Z. mobilis glyceraldehyde-phosphate dehydrogenase was found to be similar in most respects to the enzyme from other sources, except for having a slightly larger subunit size. Phosphoglycerate kinase has properties typical for this enzyme; however, it did not show the sulphate activation effects characteristic of this enzyme from most other sources. Phosphoglycerate mutase is a dimer, partially independent of 2,3-bisphosphoglycerate, and has a high specific activity. Enolase was found to be octameric; otherwise its properties were very similar to those of the yeast enzyme. Pyruvate kinase is unusual in being dimeric, and not requiring K+ for activity. It is not allosterically activated by sugar phosphates, having a high activity in the absence of any effectors. Some quantitative differences in the relative amounts of these enzymes, compared with eukaryotic species, are ascribed to the fact that Z. mobilis utilizes the Entner-Doudoroff pathway rather than the more common Embden-Meyerhoff glycolytic route.     

Liver 3-phosphoglycerate kinase. Physico-chemical characterization of the bovine-liver enzyme.

Homogeneous phosphoglycerate kinase from bovine liver possesses a maximum ultraviolet absorption at 278 nm (A 1%,1Cm 280 equals 6.7; Amax/Amin equals 2.26; e280 equals 31.5 mM(-1) X cm(-1). The enzyme consists of about 420 amino-acid residues and is a slightly acidic protein with an isoelectric point of 6.5 as expected from amino-acid analysis. The most notable features of the chemical composition are two tryptophan, 12 methionine and four half-cystine residues per enzyme molecule. Although phosphoglycerate kinases from mammalian tissues are partially similar to each other, clear differences in serine, glutamic acid, glycine, cysteine, valine, leucine, tyrosine, tryptophan and arginine contents were found. Fingerprinting and column chromatography of tryptic digests of the S-carboxymethylated protein confirm the data of amino-acid analysis. Liver phosphoglycerate kinase is inactivated when modified with either p-chloromercuribenzoate or 5,5'dithio-bis(2-nitrobenzoic acid) (Nbs2). The enzyme has two thiol groups available for reaction with Nbs2 under denaturing conditions, one of which is essential for catalysis. After reduction by NaBH4 four cysteine residues per molecule were determined with Nbs2, sugessting the presence of a disulfide bridge. Using sedimentation equilibrium studies, the molecular weight was found to be 49600. Gel filtration yielded values of 43000-50000. By analytical dodecylsulfate-polyacrylamide gel electrophoresis a molecular weight of 45600 was estimated. Inconsistent with these results in the value 37500 obtained by thin-layer gel chromatography in 6 M guanidine-HCl. Sedimentation velocity experiments revealed a sedimentation coefficient s20,w equals 3.4 S. The Stokes radius was 2.77 nm, the partial specific volume v 0.747 ml x g(-1). The diffusion coefficient was found to be 76.9 mum2 x s(-1) by analytical gel filtration. From these data a molecular weight of 44000 was calculated. Other physical constants of bovine-liver phosphoglycerate kinase are: frictional ratio f/f0 equals 1.18, axial ratio equals 3.3, maximal degree of hydration equals 0.1 g per g of protein. Bovine-layer phosphoglycerate kinase could not be dissociated into smaller subunits by treatments which have caused dissociation of various other proteins (8 M urea, 6 M guanidine-HCl, dodecyl sulfate, carboxymethylation, maleylation). All experiments strongly support the lack of subunit structure of the enzyme. Some characteristics of bovine-liver phosphoglycerate kinase are compared with the corresponding proteins from rabbit muscle, yeast and human erythrocytes.     

Comparison of some physicochemical and kinetic properties of S-adenosylhomocysteine hydrolase from bovine liver, bovine adrenal cortex and mouse liver

S-Adenosyl-L-homocysteine hydrolase (EC 3.3.1.1) was purified to apparent homogeneity from bovine liver, bovine adrenal cortex and mouse liver. All enzymes were tetramers, composed of two types of subunit present in the proportion 1:1, as judged by SDS-polyacrylamide gel electrophoresis. The partition coefficient was exactly the same for these enzymes on high-performance gel permeation chromatography, and they co-sedimented in density gradients, suggesting the same molecular size and form of S-adenosylhomocysteine hydrolase from these sources. The bovine enzymes differed from the mouse liver enzyme with respect to isoelectric point (pI = 5.35, versus pI = 5.7), affinity for DEAE-cellulose, and migration of subunits on SDS-polyacrylamide gel electrophoresis with SDS from some commercial sources. The enzymes were not substrates for cAMP-dependent protein kinase. The apparent Km values for adenosine (0.2 microM) and S-adenosylhomocysteine (0.75 microM) were the same for all three enzymes. The ratio between Vmax for the synthesis and hydrolysis of S-adenosylhomocysteine was about 4 for the mouse liver enzyme, and about 6 for the bovine enzymes. It is concluded that only subtle kinetic and physicochemical differences exist between S-adenosylhomocysteine hydrolase from these bovine and mouse tissues. This suggests that differences in experimental procedures rather than species- and organ-differences of S-adenosylhomocysteine hydrolase are responsible for the variability in kinetic and physicochemical parameters reported for the mammalian hydrolase.     

Comparison of purified bovine heart and rat liver 6-phosphofructo-2-kinase. Evidence for distinct isoenzymes.

Rat liver and bovine heart 6-phosphofructo-2-kinase were purified by the same procedure. Compared with the liver enzyme, the heart enzyme had a smaller apparent Mr, different kinetic properties, was not inactivated by cyclic AMP-dependent protein kinase, and contained less fructose-2,6-bisphosphatase activity. These differences suggest that heart and liver 6-phosphofructo-2-kinase are distinct isoenzymes. Likewise, 6-phosphofructo-2-kinase from rat heart and skeletal muscle was not inactivated on treatment with cyclic AMP-dependent protein kinase.     

Structural and immunological similarities between high molecular weight zinc ion-dependent p-nitrophenylphosphatase and fructose-1,6-bisphosphate aldolase from bovine liver

High molecular weight zinc ion-dependent acid p-nitrophenylphosphatase (HMW-ZnAPase) was purified from bovine liver to homogeneity as judged by native and sodium dodecyl sulfate polyacrylamide gel electrophoresis. The partial sequence of the purified enzyme electroblotted on PVDF membrane reveals a 95% sequence homology with human and bovine liver fructose-1,6-bisphosphate aldolase isozyme B (FALD B). FALD B was isolated from bovine liver using an affinity elution from phosphocellulose column. FALD B from bovine liver shows a native and subunit molecular weight that is indistinguishable from that of HMW-ZnAPase. In addition, an affinity purified antiserum raised in rabbits against purified HMW-ZnAPase cross-reacts with bovine liver FALD B and rabbit muscle isozymes. Despite these similarities, HMW-ZnAPase does not show FALD activity and bovine liver FALD does not display any zinc ion-p-nitrophenylphosphatase activity. These results suggested the existence of structural and immunological similarities between bovine liver HMW-ZnAPase and FALD B. Differences in some amino acid residues in enzyme activity indicate that they may be involved in different biochemical functions.     

Induction of choline kinase by polycyclic aromatic hydrocarbons in rat liver. I. A comparison of choline kinases from normal and 3-methylcholanthrene-induced rat liver cytosol

Choline kinase in rat liver has been shown to be induced up to 2-fold by the administration of polycyclic aromatic hydrocarbon carcinogens such as 3-methylcholanthrene and 3,4-benzo[a]pyrene (Ishidate, K., Tsuruoka, M. and Nakazawa, Y., (1980) Biochem. Biophys. Res. Commun. 96, 946-952). In order to characterize the nature of choline kinase induction by these carcinogens, the 3-methylcholanthrene-induced form as well as the normal form of choline kinase were partially purified from rat liver cytosol through acid treatment, (NH4)2SO4 precipitation and DEAE-cellulose column chromatography with linear KCl-gradient elution, and the catalytic properties were compared between the two preparations. Both enzyme activities were purified about 17-fold with a yield of 50% through the purification steps and there appeared no detectable difference in the elution pattern from either DEAE-cellulose column or Sephadex G-200 gel filtration. On the other hand, some differences were observed in catalytic properties between the two enzyme preparations; (1) the induced form showed a higher apparent Km value for choline (0.19 mM) when compared to the normal form (0.11 mM) and (2) the addition of polyamines caused a considerable increase in the maximum reaction velocity for the normal form whereas no remarkable change for the induced form, when the activities were plotted as a function of choline concentration. The overall results suggest that the 3-methylcholanthrene-induced form of choline kinase in rat liver could be different from the normal form, or that there exist several isoenzymes of choline kinase in rat liver, and one or some of them are inducible by the administration of polycyclic aromatic hydrocarbons.     

Phosphorylase kinase from bovine stomach smooth muscle: a Ca2(+)-dependent protein kinase associated with an actin-like molecule

Phosphorylase kinase was purified (110-fold) from bovine stomach smooth muscle by a procedure involving DEAE-cellulose chromatography, ammonium sulfate fractionation and glycerol density ultracentrifugation. On sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) the final enzyme preparation shows a single protein band of 43 kDa. The purified protein exhibits a close similarity with bovine aortic actin, as revealed by amino acid analysis and sequencing of a tryptic decapeptide fragment, although it differs widely from actin in several respects. In our effort to separate phosphorylase kinase activity from the 43 kDa protein we used a variety of chromatographic procedures, but in all cases the catalytic activity (when eluted) was accompanied by the 43 kDa protein band. Bovine stomach phosphorylase kinase exhibits an apparent molecular mass of 950 kDa, it shows a low Vmax value for phosphorylase b (85 nmol.min-1.mg-1), a pH 6.8/8.2 activity ratio of 0.23, it has an absolute requirement for Ca2+ and it is activated 1.8-fold by Ca2+/calmodulin. Furthermore, the protein kinase activity is neither inhibited by antibodies against rabbit skeletal muscle phosphorylase kinase nor activated by protein phosphorylation. These results suggest that bovine stomach phosphorylase kinase is tightly bound to an aggregate of actin-like molecules.     

Phosphoglycerate kinase B from ram testis. Purification, characterisation and comparison with the muscle isoenzyme

1. The testis-specific isoenzyme of phosphoglycerate kinase (phosphoglycerate kinase B) has been isolated from ram testes using a procedure which separates it from 'normal' phosphoglycerate kinase which is also present in testis tissue. The purification procedure is described. 2. The best preparations had no detectable impurity on electrophoresis, and had specific activities comparable with the same enzyme from other sources. 3. Kinetic studies indicated that the two isoenzymes have identical properties, within experimental error, for substrate affinity (for MgATP, 3-phosphoglycerate and MgADP), energy of activation and thermal denaturation. 4. The molecular weights of both isoenzymes were not distinguishably different from those previously reported, as measured by polyacrylamide/dodecylsulphate electrophoresis. The amino acid compositions showed only slight differences, and tryptic peptide maps showed that there was close homology of sequence. Starch gel electrophoresis at pH 6.5 indicates that the B isoenzyme has 1--2 more positive charges than the A. 5. Phosphoglycerate kinase A isolated from sheep muscle was shown, within experimental error, to be identical to the phosphoglycerate kinase A isolated from testis. 6. The results further substantiate the suggestion that the B isoenzyme is coded by a gene which was duplicated from the phosphoglycerate kinase A gene.     

Purification by affinity chromatography of 2,4-dienoyl-CoA reductases from bovine liver and Escherichia coli

1. Dye-ligand chromatography using immobilized Cibacron blue F3GA (blue Sepharose CL-6B) and Procion red HE3B (Matrex gel red A) as matrices and general ligand chromatography employing immobilized 2',5'-ADP (2',5'-ADP-Sepharose 4B) and immobilized 3',5'-ADP (3',5'-ADP-Agarose) were employed for purification of NADPH-dependent 2-enoyl-CoA reductase and 2,4-dienoyl-CoA reductase from bovine liver (formerly called 4-enoyl-CoA reductase [Kunau, W. H. and Dommes, P. (1978) Eur. J. Biochem. 91, 533-544], as well as 2,4-dienoyl-CoA reductase from Escherichia coli. 2. The NADPH-dependent 2-enoyl-CoA reductase from bovine liver mitochondria was separated from 2,4-dienoyl-CoA reductase by dye-ligand chromatography (Matrex gel red A/KCl gradient) as well as by general ligand affinity chromatography (2',5'-ADP-Sepharose 4B/NADP gradient). The enzyme was obtained in a highly purified form. 3. The NADPH-dependent 2,4-dienoyl-CoA reductase from bovine liver mitochondria was purified to homogeneity using blue Sepharose CL-6B, Matrex gel red A, and 2',5'-ADP-Sepharose 4B chromatography. 4. The bacterial 2,4-dienoyl-CoA reductase was completely purified by ion-exchange chromatography on DEAE-cellulose followed by a single affinity chromatography step employing 2',5'-ADP-Sepharose 4B and biospecific elution from the column with a substrate, trans,trans-2,4-decadienoyl-CoA. 5. The application of dye-ligand and general ligand affinity chromatography for purification of NADPH-dependent 2,4-dienoyl-CoA reductases taking part in the beta-oxidation of unsaturated fatty acids is discussed. It is concluded that making use of coenzyme specificity for binding and substrate specificity for elution is essential for obtaining homogeneous enzyme preparations.     

Isolation and characterization of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.     

Effect of poly(vinyl alcohol) treatment of the dyematrix on the chromatography of pyruvate kinase

Summary The utility of poly(vinyl alcohol) as a shielding polymer in dye-affinity chromatography was studied. Difference spectroscopy was used to estimate the strength of the polymer-dye complex. The target enzyme, pyruvate kinase (E.C. 2.7.1.40) from porcine muscle was purified on a Red HE3B-Sepharose column with 95% recovery by salt elution and 85% recovery with specific eluent.     

Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.     

Large-scale preparation of phosphoglycerate kinase from Saccharomyces cerevisiae using Cibacron Blue-Sepharose 4B pseudoaffinity chromatography.

A reproducible procedure for the large-scale preparation of phosphoglycerate kinase frombaker's yeast is described. This method includes autolysis of dried yeast in 0.75 m ammonia, heat treatment, ammonium sulfate fractionation, ion-exchange chromatography on DEAE-cellulose, Cibacron blue 3 G-A-Sepharose 4B pseudoaffinity chromatography, and Sephadex G-100 gel filtration. Approximately 1.7 g of homogeneous phosphoglycerate kinase can be obtained from 1 kg of air-dried bakers' yeast (yield 52%, specific activity 890 units/mg at 25°C). In a few cases further purification was achieved by reversible salting out on Sepharose CL-4B, hydroxylapatite chromatography, or ATP-Sepharose 4B affinity chromatography. Differences in the preparation of phosphoglycerate kinase from yeast with those from pig liver and pig muscle are discussed, especially concerning the interaction of the three enzymes with the chromophores of Cibacron blue- and dextran blue-Sepharose.     

Purification and properties of phosphoglycerate kinase from Thermus thermophilus strain HB8.

(1) A glycolytic enzyme, phosphoglycerate kinase [EC 2.7.2.3], was purified from cells of an extreme thermophile, Thermus thermophilus strain HB8. The enzyme was resistant to heat, and no loss of activity was observed after incubation for 10--20 min at 79 degrees C. (2) Catalytic properties such as pH optimum (pH 6--8.5), kinetic parameters (Km=0.28 mM for ATP, 1.79 mM for glycerate 3-phosphate), substrate specificity and inhibitors of the enzyme were investigated and compared with those of phosphoglycerate kinase from other sources. (3) The enzyme protein consists of a single polypeptide chain of molecular weight 44,600. The isoelectric point is 5.0 The amino acid composition of the enzyme was studied. The contents of ordered secondary structures were estimated to be 29% alpha-helix and 11% pleated sheet from the circular dichroic spectrum of the enzyme protein. (4) The fluorescence spectrum of the enzyme protein showed an emission maximum at 320 nm when excited at 280 nm. The quantum yield was 0.19. Tryptophyl fluorescence was not quenched, in contrast to the fluorescence reported for yeast phosphoglycerate kinase.     

Studies on a possible phosphoryl-enzyme intermediate in the catalytic reaction of yeast phosphoglycerate kinase

A phosphoryl-enzyme intermediate as part of the mechanism of phosphoglycerate kinase has been suggested for the rabbit muscle enzyme (6) and the yeast enzyme (7,8). ATP in the binary enzyme-substrate complexes appeared to phosphorylate these enzymes and ADP-ATP exchange activities were observed (6,7,8). The present report shows, however, that highly purified yeast enzyme cannot be phosphorylated by ATP. On the other hand ADP-ATP exchange activity was obtained but this was proportional to trace amounts of adenylate kinase activity, which was found to contaminate the enzyme preparations. Thus a Ping Pong mechanism as an alternative to a mechanism including a ternary complex between the enzyme and its two substrates appears very improbable. Whether the enzyme or the phosphoryl-group-accepting substrate is responsible for the primary nucleophilic attack occurring in the ternary complex is still an open question, however. Yeast phosphoglycerate kinase appears to have no ATPase activity.     

Purification and properties of glycogen synthase I from bovine polymorphonuclear leucocytes

Glycogen synthase I was purified from bovine polymorphonuclear leucocytes (PMNs) by a procedure involving concanavalin A-Sepharose affinity chromatography. The purified glycogen-bound glycogen synthase I had a specific activity of 9.83 U/mg protein and the glycogen free enzyme 21 U/mg protein. Molecular ratio of the native enzyme and the subunit were 340 K and 85 K respectively. After phosphorylation by the catalytic subunit of cAMP-dependent protein kinase the phosphorylated sites were studied using high-performance liquid chromatography (HPLC) of the tryptic 32P-peptides. The enzyme was phosphorylated at three different sites with retention times identical to site 1a, site 1b, and site 2 from rabbit skeletal muscle glycogen synthase.