A study of phosphoglycerate kinase in human erythrocytes. I. Enzyme isolation, purification and assay.

The enzyme ATP-3-phospho-D-glycerate-1-phosphotransferase (EC 2.7.2.3) (phosphoglycerate kinase) has been isolated from human red cells in crystalline form by a modification of the method of Yoshida and Watanabe (1972) J. Biol. Chem. 247, 440-445). The crystalline enzyme was further purified by electrofocusing using carrier ampholytes (pH 7-9). The isoelectric point of phosphoglycerate kinase was estimated to be 8.75. The specific activity of purified phosphoglycerate kinase from electrofocusing was 2200 units per mg of protein at pH 8.3 (37 degrees C). Enzyme activity was assayed in the forward direction leading from 1,3-diphosphoglycerate to a 3-phosphoglycerate using a fluorimetric procedure for NAD-coupled enzymes for the measurement of the reaction rate at very low substrate concentrations. The auxiliary indicator enzymes were added in excess to yield true initial velocity kinetics, i.e. with no time lag upon addition of substrate (1,3-diphosphoglycerate). This was established theoretically using a mathematical model and confirmed experimentally. Further phosphoglycerate kinase was shown to be the rate-limiting step when the assay conditions were varied.     

Purification, characterisation and mutagenesis of highly expressed recombinant yeast pyruvate kinase.

Recombinant yeast pyruvate kinase has been purified from a strain of Saccharomyces cerevisiae expressing the enzyme to very high levels. Expression was from a multicopy plasmid under the control of the yeast phosphoglycerate kinase promoter. The gene was expressed in the absence of the genomically encoded pyruvate kinase, using a strain of yeast in which the pyruvate kinase gene has been disrupted by the insertion of the yeast Ura3 gene. The purification procedure minimised proteolytic artefacts and enabled the convenient purification of 15-20 mg enzyme from 11 culture. The purified enzyme was characterised by a high specific activity and by a lack of proteolytic degradation. Two active-site mutants of yeast pyruvate kinase have been produced, expressed and characterised in this system and preliminary results are described.     

Purification and properties of protein kinase C from bovine polymorphonuclear leucocytes

A calcium-activated, phospholipid-dependent protein kinase (protein kinase C) was purified to near homogeneity from bovine polymorphonuclear leucocytes. The purified enzyme had a specific activity of 10 000 U/mg protein and on SDS gelelectrophoresis the Mr was 88 000. The enzyme activity was almost totally dependent upon phosphatidylserine and could be strongly activated by Ca2+ concentrations in the micromolar range. At lower concentrations of calcium (less than 1 X 10(-7) M) the enzyme was only activated by the simultaneous presence of phosphatidylserine and diolein. Phorbol 12-myristate 13-acetate mimicked the effect of diolein and partially activated the enzyme. Protein kinase C activity and the phorbolester binding protein co-purified throughout all the purification steps.     

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.     

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 and characterization of human muscle pyruvate kinase.

The M1 isozyme of pyruvate kinase has been purified from human psoas muscle in a seven-step procedure. Fractionation by ammonium sulfate precipitation, heat treatment, acetone precipitation, diethylaminoethyl cellulose batchwise treatment followed by chromatography on carboxymethyl cellulose and Sephadex G-200 gave a product with a specific activity of 383 U/mg representing a 294-fold purification with a yield of 11%. The product formed orthorhombic crystals and was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate, sedimentation velocity, sedimentation equilibrium, and immunodiffusion. The purified enzyme has a molecular weight of 240700 and has a sedimentation coefficient (S20,W) of 10.04S. It contains four subunits with identical molecular weights of 61000. No free N-terminal amino acids could be detected. Antibody prepared against the purified human M1 isozyme does not cross-react by immunodiffusion or enzyme inactivation with the human erythrocyte isozyme and in the reverse experiment antibody prepared against human erythrocyte pyruvate kinase does not cross-react with the purified M1 isozyme. The amino acid composition of the M1 isozyme is presented.     

Expression of Torpedo californica creatine kinase in Escherichia coli and purification from inclusion bodies

The pET17 expression vector was used to express creatine kinase from the electric organ of Torpedo californica as inclusion bodies in Escherichia coli BL21(DE3) cells. The insoluble aggregate was dissolved in 8M urea and, following extraction with Triton X-100, the enzyme was refolded by dialysis against Tris buffer (pH 8.0) containing 0.2M NaCl. After two buffer changes, chromatography on Blue Sepharose was used as a final step in the purification procedure. Approximately 54mg active protein was recovered from a 1L culture and the refolded enzyme had a specific activity of 75U/mg. The molecular mass of the purified protein was consistent with that predicted from the amino acid sequence and the CD spectrum of the refolded enzyme was essentially identical to that of creatine kinase from human muscle (HMCK). The K(m) values of ATP and ADP were also similar to those of HMCK, while the K(m) values for both phosphocreatine and creatine were approximately 5-10-fold higher. The purification described here is in marked contrast with earlier attempts at purification of this isozyme where, in a process yielding less than 1mg/L culture, enzyme with a specific activity of ca. 5U/mg was obtained.     

Rat liver phosphoglycerate kinase. I. Purification and kinetic properties in the biosynthesis of 1-3 diphosphoglycerate

Phosphoglycerate kinase (MgATP 3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) has been isolated from rat liver with a purification ratio of 960 and a specific activity of 300 IU/mg of protein. The purity of the enzyme preparations was estimated by polyacrylamide gel electrophoresis. The molecular weight, determined by gel filtration is 42 000. The "subunit" size of phosphoglycerate kinase as determined by sodium dodecyl sulfate gel electrophoresis is 46 000, indicating that the enzyme is monomeric. The rate of the enzyme reaction as a function of the concentration of D-3-phosphoglycerate indicated the usual Michaelis Menten relationship. The rate of the enzyme reaction as a function of the concentration of MgATP2- did not fit the usual Michaelis Menten relationship: two distinct regions can be fitted with different straight lines and suggest the presence of two sites for the Mg ATP2-. This hypothesis seems to be confirmed by the study of the action of the free and complexed nucleotides.     

Purification of L-type pyruvate kinase from human liver by affinity chromatography on Blue-Dextran-Sepharose column

L-type pyruvate kinase (ATP: pyruvate 2-O-phosphotransferase; EC 2.7.1.40) was purified from human liver by an original method. This purification included toluene extraction, a-monium sulphate fractionation, DEAE-Sephadex bactchwise, CM-Sephadex batchwise with elective elution by ATP and affinity chromatography on a Blud Dextran-Sepharose column with specific elution by fructose 1, 6-diphosphate. This purification procedure allowed us to obtain 6 mg protein with a specific activity of 420 IU/mg protein, i.e. 2,690-fold purification with an overall yield of 34%. This preparation was homogeneous as judged by immuno-diffusion, acrylamide and sodium dodecyl sulphate acrylamide-gel electrophoresis.     

Nucleotide catabolism and nucleoside cycles in human thymocytes. Role of orthophosphate.

Phosphatidylinositol 4-phosphate (PtdIns4P) kinase was purified from cytosolic and particulate material of rat brain. The purification procedure of the enzyme from cytosol consisted of (NH4)2SO4 precipitation. DEAE-cellulose column chromatography and preparative isoelectric focusing. Other methods after DEAE-cellulose column chromatography failed to achieve further purification of the PtdIns4P kinase, probably caused by the tendency of the enzyme to aggregate with contaminating proteins. The final purification was 67-fold, and the recovery was 0.6%. After isoelectric focusing the fraction containing the highest PtdIns4P kinase activity showed only one protein as visualized by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and silver staining. The apparent Mr of this protein was 45 kDa and the isoelectric point about 5.8. The activity of PtdIns4P kinase was dependent on the concentration of divalent cations in the incubation medium. PtdIns4P kinase activity was found to be optimal at 10-30 mM-Mg2+. In an attempt to compare the cytosolic with the membrane-derived kinase activity, a Triton/KCl extract from synaptic membranes was subjected to the same purification procedure as the cytosolic enzyme. A difference in isoelectric focusing was observed, possibly due to a higher tendency to form aggregates. However, we tend to conclude that also in the membranes the PtdIns4P kinase activity is present as a 45 kDa protein, identical with that found in the cytosol.     

Purification of 3-phosphoglycerate kinase from diverse sources by affinity elution chromatography.

1. Affinity elution chromatography was used to purify phosphoglycerate kinase from a variety of sources. The choice of buffer pH for the chromatography was made according to the relative electrophoretic mobility of the enzyme from the species concerned. 2. Outlines of the methods used to isolate the enzyme from over 20 sources are presented. The enzyme was purified from the muscle tissue of a variety of mammals, fish and birds, from liver of several animals, from yeast, Escherichia coli, and plant leaves. The more acidic varieties of the enzymes were purified by conventional gradient elution from ion-exchangers as affinity elution procedures were not applicable. 3. The structural and kinetic parameters investigated show that phosphoglycerate kinase is evolutionarily a highly conservative enzyme; there were few differences in properties regardless of source or function (glycolytic, gluconeogenic or photosynthetic). 4. A detailed comparison of the enzyme preparations purified from bovine muscle and bovine liver failed to detect any significant differences between them; the evidence indicates that they are genetically identical.     

Purification of 3-hydroxy-3-methylglutaryl coenzyme A reductase.

This paper describes a rapid purification procedure for 3-hydroxy-3-methylglutaryl coenzyme A reductase, the major regulatory enzyme in hepatic cholesterol biosynthesis. A freeze-thaw technique is used for solubilizing the enzyme from rat liver microsomal membranes. No detergents or other stringent conditions are required. The purification procedure employs Blue Dextran-Sepharose-4B affinity chromatography, and purification can be carried out from microsomal membranes to purified enzyme in 8 to 10 hours. The purified enzyme has a specific activity of 517 nmoles/min/mg protein, and it is 975-fold purified with respect to the original microsomal membrane suspension. SDS polyacrylamide gel electrophoresis of the purified enzyme shows only trace impurities; the subunit molecular weight for the enzyme measured by this technique is 47,000.     

Purification and characterization of a protein tyrosine phosphatase which dephosphorylates the nicotinic acetylcholine receptor.

The nicotinic acetylcholine receptor (nAChR) is phosphorylated to a high stoichiometry on tyrosine residues both in vitro and in vivo. Moreover, tyrosine phosphorylation has been shown to regulate the functional properties of the receptor. We report here the purification and characterization of a protein tyrosine phosphatase that dephosphorylates tyrosine-phosphorylated nAChR from Torpedo electroplax, a tissue highly enriched in the nAChR. The 32P-labeled tyrosine phosphorylated nAChR was used as a substrate to monitor the enzyme activity during purification. The protein tyrosine phosphatase activity was purified using three consecutive cation-exchange columns (phosphocellulose, S Sepharose Fast Flow, Bio-Rex 70), followed by two affinity matrices (p-aminobenzylphosphonic acid-agarose and thiophosphotyrosyl nAChR-Sepharose 4B). The enzyme activity was purified to homogeneity, with an overall purification of 25,000-fold and a yield of 20%. The purified enzyme had an apparent molecular mass of 43 kDa on sodium dodecyl sulfate-polyacrylamide gels and migrated as a monomer during Superose 12 chromatography. It had a neutral pH optimum and a specific activity of 18 mumol/mg of protein/min, with a Km of 4.7 microM for tyrosine-phosphorylated nAChR. The phosphatase was specific for tyrosine phosphorylated nAChR; it showed no activity towards the nAChR phosphorylated on serine residues by cAMP-dependent protein kinase. The enzyme also dephosphorylated 32P-labeled poly(Glu-Tyr) (4:1). However, it did not dephosphorylate p-nitrophenylphosphate. The tyrosine phosphatase was inhibited by ammonium molybdate (IC50 of 2 microM), sodium vanadate (IC50 of 150 microM) and the divalent cations Mg2+, Mn2+, and Ca2+ at millimolar concentrations, but not by 100 microM ZnCl or 10 mM NaF. Poly-(Glu, Tyr) (4:1) and heparin inhibited the enzyme activity at micromolar concentrations. These unique properties of the purified enzyme suggest that it may be a novel protein tyrosine phosphatase that specifically dephosphorylates the nAChR.     

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.     

Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms

The substrate analog alpha-D-galactosylamine was synthesized, linked to 6-aminohexanoic acid, and coupled to carboxyhexyl-Sepharose. This affinity support permitted the purification of human alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from spleen, placenta, and plasma. When used in conjunction with conventional procedures, affinity chromatography enabled the rapid and specific purification of alpha-galactosidase A from each source. Significantly, pyrogenic endotoxins were eliminated from enzyme preparations by the use of the affinity column. Splenic alpha-galactosidase A was purified in high yield (38%) with a specific activity of 1.9 X 10(6) units/mg. The purified enzyme was a homodimer with a native molecular weight of 101,000 and a subunit weight of 49,800. The UV absorption coefficient was E280 1% = 18 and the lambda max was 282 nm. The plasma form was purified with a markedly improved yield to a specific activity (229,000 units/mg) which was 3 times greater than that achieved previously. The enzymes from plasma, spleen, and placenta were immunologically identical. The physical and kinetic properties of the purified enzymes were consistent with and confirmed previous findings.     

Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base.     

Purification and characterization of mouse kidney beta-glucuronidase.

Beta-Glucuronidase has been purified from mouse kidneys previously induced by gonadotrophin to a specific enzyme activity 15 times higher than the non-induced kidney. The purification procedure includes ultrasonication to solubilize the enzyme, acid and ammonium sulfate precipitations, gel filtration in Sephadex G-200, DEAE-ion exchange chromatography, and isoelectric focusing. The resulting product has a specific activity of 284,000 Fishman units/mg of protein, representing a 1,090-fold purification and is 17,000-fold higher than the level in the non-induced kidney. The purified beta-glucuronidase is apparently homogeneous by criteria of gel filtration, sodium dodecyl sulfate gel electrophoresis, and immunodiffusion. Characterization of the purified enzyme showed that it is identical with the lysosomal isoenzymic from electrophoretically, has subunit molecular weight of 74,000 (estimated by sodium dodecyl sulfate gel electrophoresis) and oligomer molecular weight of 300,000. The purified enzyme is stable at high temperature (up to 55 degrees) and at wide range of pH (from 4 to 11). It has a pH optimum for its activity at 4.7 and a Km of 1.18 times 10- minus 4 M. The purification and characterization of this enzyme from mouse kidney will have significance in the understanding of the molecular nature of the isoenzymes of beta-glucuronidase and will be useful in future studies on the mechanism of intracellular transport and distribution of this hydrolase.     

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.     

Specific protein kinase from Saccharomyces cerevisiae cells phosphorylating 60S ribosomal proteins.

A protein kinase, specific for 60S ribosomal proteins, has been isolated from Saccharomyces cerevisiae cells, purified to almost homogeneity and characterized. The isolated enzyme is not related to other known protein kinases. Enzyme purification comprised three chromatography steps; DEAE-cellulose, phosphocellulose and heparin-Sepharose. SDS/PAGE analysis of the purified enzyme, indicated a molecular mass of around 71 kDa for the stained single protein band. The specific activity of the protein kinase was directed towards the 60S ribosomal proteins L44, L44', L45 and a 38 kDa protein. All the proteins are phosphorylated only at the serine residues. None of the 40S ribosomal proteins were phosphorylated in the presence of the kinase. For that reason we have named the enzyme the 60S kinase. An analysis of the phosphopeptide maps of acidic ribosomal proteins, phosphorylated at either the 60S kinase or casein kinase II, showed almost identical patterns. Using the immunoblotting technique, the presence of the kinase has been detected in extracts obtained from intensively growing cells. These findings suggest an important role played by the 60S kinase in the regulation of ribosomal activity during protein synthesis.     

Yeast glyceraldehyde-3-phosphate dehydrogenase. Evidence that subunit cooperativity in catalysis can be controlled by the formation of a complex with phosphoglycerate kinase

Sepharose-bound tetrameric, dimeric and monomeric forms of yeast glyceraldehyde-3-phosphate dehydrogenase were prepared, as well as immobilized hybrid species containing (by selective oxidation of an active center cysteine residue with H2O2) one inactivated subunit per tetramer or dimer. The catalytic properties of these enzyme forms were compared in the forward reaction (glyceraldehyde-3-phosphate oxidation) and reverse reaction (1,3-bisphosphoglycerate reductive dephosphorylation) under steady-state conditions. In the reaction of glyceraldehyde-3-phosphate oxidation, immobilized monomeric and tetrameric forms exhibited similar specific activities. The hybrid-modified dimer contributed on half of the total activity of a native dimer. The tetramer containing one modified subunit possessed 75% of the activity of an unmodified tetramer. In the reaction of 1,3-bisphosphoglycerate reductive dephosphorylation, the specific activity of the monomeric enzyme species was nearly twice as high as that of the tetramer, suggesting that only one-half of the active centers of the oligomer were acting simultaneously. Subunit cooperativity in catalysis persisted in an isolated dimeric species. The specific activity of a monomer associated with a peroxide-inactivated monomer in a dimer was equal to that of an isolated monomeric species and twice as high as that of a native immobilized dimer. The specific activity of subunits associated with a peroxide-inactivated subunit in a tetramer did not differ from that of a native immobilized tetramer; this indicates that interdimeric interactions are involved in catalytic subunit cooperativity. A complex was formed between the immobilized glyceraldehyde-3-phosphate dehydrogenase and soluble phosphoglycerate kinase. Three monomers of phosphoglycerate kinase were bound per tetramer of the dehydrogenase and one per dimer. Evidence is presented that if the reductive dephosphorylation of 1,3-bisphosphoglycerate proceeds in the phosphoglycerate kinase - glyceraldehyde-3-phosphate dehydrogenase complex, all active sites of the latter enzyme act independently, i.e. subunit cooperativity is abolished.