Anomalous fluorescence of yeast 3-phosphoglucerate kinase.

The 3-phosphoglycerate kinase (EC 2.7.2.3) of yeast which contains two tryptophyl and eight tyrosyl residues per molecule, displayed an unusualy fluorescence emission spectrum with a maximum at 308 nm when excited at 280 nm. The emission peak shifted to 329 nm when excited at 295 nm. We could confirm that it was due to the efficient quenching of tryptophyl fluorescence as well as to the incomplete energy transfer from tyrosyl to tryptophyl residues. The average fluorescence quantum yield of this protein was 0.076 (excitation at 280 nm) and that of tryptophyl residues was 0.046 (excitation at 295 nm). As the pH of the solution was lowered, the fluorescence intensity of phosphoglycerate kinase at 329 nm dramatically increased between pH 5 and 4, while the position of the peak remained unchanged. When denatured in 4 M guanidine hydrochloride, the protein showed two emission peaks, one at 343 nm and the other at 303 nm.     

Kinetic studies of the unfolding-refolding of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The kinetics of the unfolding and refolding of horse muscle phosphoglycerate kinase were studied with three different signals: fluorescence emission intensity at 336 nm (excitation at 292 nm), ellipticity at 220 nm, and enzyme activity. The results corroborate the conclusion on the existence of intermediates in the folding pathway obtained from equilibrium studies. Kinetic studies showed at least two phases of refolding, as revealed by fluorescence as well as by circular dichroism measurements. During the fast phase, an intermediate was formed with a fluorescence intensity higher than that of the native protein, but devoid of enzyme activity. The fluorescence emission spectrum of this intermediate was determined. Only the slow phase was detected for the unfolding process; it was not attributable to proline isomerization. Several models were assumed, and simulated kinetics derived from these models were compared with the experimental results. A plausible one accounting for most of the data is proposed.     

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.     

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.     

Substrate binding to phosphoglycerate kinase monitored by 1-anilino-8-naphthalenesulfonate

The interaction between 1-anilino-8-naphthalenesulfonate (ANS) and yeast phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.2.3) and the use of ANS as a probe for studying the structure and function of phosphoglycerate kinase has been investigated. The interaction has been studied by kinetic methods, equilibrium dialysis, and fluorometric titrations. ANS inhibits the activity of the enzyme. More than one inhibitor site exists. ANS is competitive with MgATP and noncompetitive with 3-phosphoglycerate at the first detected inhibitor binding site. The Ki value is 1-2 mM. Several ANS molecules bind to the enzyme. By fluorometric titrations the first detected site has a dissociation constant that is in the same range as Ki or bigger. When ANS interacts with phosphoglycerate kinase its fluorescence is increased and a blue shift occurs. ANS appears to bind to a strongly hydrophobic site. The fluorescence is sensitive to the addition of substrates. ADP, ATP, or combinations of Mg2+ and nucleotide decreases the fluorescence as does free Mg2+. 3-Phosphoglycerate, on the other hand, increases the fluorescence giving evidence for conformational changes upon 3-phosphoglycerate binding.     

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.     

Purification and characterization of arginine kinase from the American cockroach (Periplaneta americana)

The isolation and characterization of homogeneous arginine kinase from the cockroach is reported. The purification protocol produces 6.6 mg of pure enzyme from 6.8 g of whole cockroach. The purified enzyme cross-reacts with a heterologous antibody and monoclonal antibody against arginine kinase from the shrimp. Both antibody preparations also cross-react with extracts from several species known to contain monomeric arginine kinase, but fail to react with extracts from organisms containing dimeric arginine kinase. Cockroach arginine kinase has a molecular mass of approximately 43,000 determined from measurements by gel filtration and gel electrophoresis. Compared with other arginine kinases, the enzyme from the cockroach is relatively thermostable (50% activity retained at 50 degrees C for 10 min) and has a pH optima of 8.5 and 6.5-7.5, for the forward and reverse reactions, respectively. Treatment with 5,5'dithiobis[2-nitrobenzoic acid] indicates that arginine kinase has a single reactive sulfhydryl group and, interestingly, the reaction is biphasic. The Michaelis constants for the phosphagen substrates, arginine: 0.49 mM, phosphoarginine: 0.94 mM, and nucleotide substrates MgATP: 0.14 mM, MgADP: 0.09 mM, are in the range reported for other arginine kinases. A 1% solution of pure enzyme has an absorbance of 7.0 at 280 nm. Calculations based on circular dichroic spectra indicate that arginine kinase from the cockroach has 12% alpha-helical structure. The intrinsic protein fluorescence emission maximum at 340 nm suggests that tryptophan residues are below the surface of the protein and not exposed to solvent. Arginine kinase from the cockroach and shrimp are known to be deleterious immunogens towards humans. The availability of pure protein, its characterization and potential regulation of activity, will be useful in developing agents to control the cockroach population and its destructive role in agriculture and human health.     

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.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

The unfolding-refolding transition of horse muscle phosphoglycerate kinase induced by guanidine hydrochloride was studied under equilibrium conditions using four different signals: fluorescence intensity at 336 nm, UV difference absorbance at 286 and 292 nm, ellipticity at 220 nm, and enzyme activity. From the following arguments, we found that the process deviates from a two-state model and intermediates are significantly populated even at equilibrium: (1) the noncoincidence of the transition curves and (2) the asymmetry of the transition curve obtained from CD measurements. From these different data and the thermodynamic analysis, it was suggested that the two domains of the horse muscle phosphoglycerate kinase refold independently of one another with different equilibrium constants, the most favorable constant referring to the folding of the C-terminal domain which contains all tryptophans.     

Regulatory properties of phosphorylase from chicken skeletal muscle

The main kinetic parameters for purified phosphorylase kinase from chicken skeletal muscle were determined at pH 8.2: Vm = 18 micromol/min/mg; apparent Km values for ATP and phosphorylase b from rabbit muscle were 0.20 and 0.02 mM, respectively. The activity ratio at pH 6.8/8.2 was 0.1-0.4 for different preparations of phosphorylase kinase. Similar to the rabbit enzyme, chicken phosphorylase kinase had an absolute requirement for Ca2+ as demonstrated by complete inhibition in the presence of EGTA. Half-maximal activation occurred at [Ca2+] = 0.4 microM at pH 7.0. In the presence of Ca2+, the chicken enzyme from white and red muscles was activated 2-4-fold by saturating concentrations of calmodulin and troponin C. The C0.5 value for calmodulin and troponin C at pH 6.8 was 2 and 100 nM, respectively. Similar to rabbit phosphorylase kinase, the chicken enzyme was stimulated about 3-6-fold by glycogen at pH 6.8 and 8.2 with half-maximal stimulation occurring at about 0.15% glycogen. Protamine caused 60% inhibition of chicken phosphorylase kinase at 0.8 mg/ml. ADP (3 mM) at 0.05 mM ATP caused 85% inhibition with Ki = 0.2 mM. Unlike rabbit phosphorylase kinase, no phosphorylation of the chicken enzyme occurred in the presence of the catalytic subunit of cAMP-dependent protein kinase. Incubation with trypsin caused 2-fold activation of the chicken enzyme.     

Physico-chemical properties of thiamine kinase from Saccharomyces carlsbergensis yeasts

The amino acid composition and intrinsic fluorescence were studied in thiamine kinase (ES 2.7.6.2) of brewer's yeast. The enzyme molecule is characterized by higher concentrations of amino acids which promote alpha-helix formation of the protein globule, the amount of residues (cysteine, proline) either binding or folding polypeptide chains being considerably high. Amino acids of middle and low hydrophobicities were the most frequent among the amino acid residues with nonpolar R-groups. The value for the protein isoelectric point was 6.21. The eigen pH value and isoionic point were in good agreement with the isoelectric point value and amounted to 6.28. The fluorescence spectrum has a maximum at 328 nm, half-width at 53 nm and a quantum yield at 0.14 nm. The tryptophane residues were located in hydrophobic surroundings, unexposed to anion quenchers and almost unexposed to cation ones. The fluorescence and phosphofluorescence parameters were sensitive to the conformational changes in the molecule. At pH of 5-9 the protein conformation remained unchanged. The temperature rise above 40 degrees C resulted in a disturbance in the nativity of the globule. The elevation of the enzyme concentration from 0.05 to 1 mg/ml increased the polarization degree from 0.115 to 0.194, the quantum yield and the spectrum position remaining unchanged. The results obtained develop knowledge of the equilibrium system of oligomerous forms of thiamine kinase with different catalytic properties.     

Spectroscopic study on the structure and stability of beef liver arginase

The secondary and tertiary structure of the oligomeric arginase (EC 3.5.3.1) from beef liver was investigated by circular dichroism (CD) and fluorescence measurements. The far-ultraviolet CD spectrum of the enzyme at neutral pH is indicative of high helical content. The intrinsic fluorescence emission of the protein is due to tryptophan, the contribution of tyrosine being small. Upon excitation at 295 nm, the maximum of emission occurs at 330 nm, implying that the tryptophan residues are rather buried in a hydrophobic interior of the protein. Ethylenediaminetetraacetic acid (EDTA), which inactivates the enzyme by removing the functional Mn2+-ion from the enzyme, does not dissociate the enzyme into subunits, nor affect noticeably its secondary and tertiary structure. Inactivation occurs in the acid pH range, being complete at pH below 4. However, acidification up to pH 1.5 produced only limited changes in the far-ultra-violet CD spectrum and intrinsic fluorescence emission properties. The enzyme shows noteworthy thermal stability, as shown by measuring the residual activity after heating and by evaluating the temperature dependence of the CD signal at 220 nm and the intensity of emission fluorescence. A temperature of half inactivation (Tm) of 77 degrees was determined upon heating the enzyme at pH 7.5 in the presence of Mn2+-ions for 10 min; in the presence of EDTA, Tm is shifted to 55 degrees. Taken together, these observations indicate that the structural stability of beef liver arginase arises from a clustering of hydrophobic amino acids and from Mn2+-ion binding.     

Studies of the structure--function relationships of Neurospora crassa pyruvate kinase: interaction with blue dextran--sepharose and Cibacron blue 3G-A

Blue dextran--Sepharose and Cibacron blue 3G-A interact with pyruvate kinase of Neurospora crassa. The enzyme is readily released from the substituted Sepharose column by elution with 0.17 M potassium phosphate buffer (pH 7.9), or 2 mM fructose 1,6-diphosphate (FDP), but not with either of the substrates, ADP and phosphoenolpyruvate (PEP), at 2 mM. Cibacron blue 3G A is a noncompetitive inhibitor of pyruvate kinase with respect to both substrates. It appears to compete with the allosteric effector, FDP, for binding to the enzyme surface. A lack of elution of the enzyme from the immobilized blue dextran matrix by adenine nucleotides and the absence of a difference spectrum in the 650- to 700-nm range suggest that a "dinucleotide-fold" substructure is not implicated in the dye binding sites on pyruvate kiase. The interaction of Cibacron blue 3G-A and this enzyme can be followed fluorometrically; incremental additon of the dye to the enzyme solution results in a progressive decrease in the fluorescence of surface tryptophanyl residues. The quenching of fluorescence of exposed aromatic groups is subject to reversal following addition of FDP to the pyruvte kinase--Cibacron blue complex.     

Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex

A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20 degrees C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329-nm maximum and a 18.4-kJ/mol activation energy, Ea, for thermal quenching. The long-lifetime (3.5 ns) component has a 338-nm maximum and an Ea of only 7.9 kJ/mol. Tentatively we assign the short and long-lifetime components to Trp-333 and Trp-308. Binding of the substrates ATP and 3-phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the Ea for both components. Acrylamide-quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.     

Kinetic studies of the refolding of yeast phosphoglycerate kinase: comparison with the isolated engineered domains.

Unfolding and refolding kinetics of yeast phosphoglycerate kinase were studied by following the time-dependent changes of two signals: the ellipticity at 218 nm and 222 nm, and the fluorescence emission at 330 nm (following excitation at 295 nm). The protein is composed of two similar-sized structural domains. Each domain has been produced by recombinant DNA techniques. It has been previously demonstrated that the engineered isolated domains are able to fold into a quasinative structure (Minard, P., et al., 1989b, Protein Eng. 3, 55-60; Missiakas, D., Betton, J.M., Minard, P., & Yon, J.M., 1990, Biochemistry 29, 8683-8689). The behavior of the isolated domains was studied using the same two conformational probes as for the whole enzyme. We found that the refolding kinetics of each domain are multiphasic. In the whole protein, domain folding and pairing appeared to be simultaneous events. However, it was found that some refolding steps occurring during the refolding of the isolated C-domain are masked during the refolding of yeast phosphoglycerate kinase. The N-domain was also found to refold faster when it was isolated than when integrated.     

Fluorimetric and spectrophotometric studies of DPN-linked isocitrate dehydrogenase from bovine heart. Properties of tyrosyl and tryptophyl residues.

The emission maximum of DPN-linked isocitrate dehydrogenase in pH 7.07 buffer is shifted from 317 to 324 nm and fluorescence intensity is decreased when the excitation wave-length is varied from 270 to 290 nm; in 0.2 M KOH, where the fluorescence of tyrosyl residues is almost completely quenched, a further substantial decline in quantum yield of protein fluorescence and a red shift of the emission peak to 339 nm occur. The latter should be due mainly to tryptophyl residues. The enzyme contains 9.4 tyrosyl residues per subunit of molecular weight 42,000 determined spectrophotometrically (295 nm) at pH 13, in good agreement with a tyrosine content of 9.7 by amino acid analysis. No more than 1.1 tyrosyl residues per subunit can be detected up to pH 10.6 at 7 degrees upon prolonged incubation. The increase in absorption at 295 nm with increasing pH is related to loss of enzyme activity and results in a red shift of the emission maximum, and decreased fluorescence intensity. Treatment of the enzyme in a Li+-containing buffer at pH 7.5 with an excess of N-acetylimidazole results in (a) modification of 1.1 tyrosyl residues per subunit, (b) a 30% decrease in enzyme activity, (c) a 6-nm red shift in emission maximum, and (d) a decrease in fluorescence intensity. Manganous DL-isocitrate (1.06 mM) prevents the acetylation of the enzyme. Deacetylation of the O-acetylated enzyme by hydroxylamine completely restores the enzyme activity and reverses the spectral changes. The acetylation studies indicate that the reactive tyrosyl residue does not participate directly in catalysis but may be involved in maintaining the proper conformation of the active enzyme center. A net of 1 of the 2 tryptophyl residues per subunit is perturbed immediately by a number of solvents. This perturbation is not affected by manganous isocitrate, whereas exposure of tyrosyl residues occurs only with time and is prevented by the substrate. The perturbation of the tryptophyl residue is accompanied by a red shift of the fluorescence emission maximum. The more exposed tryptophyl residue may contribute to the energy transfer from protein to nucleotides since the quenching of protein fluorescence upon binding of DPN+, DPNH, or ADP by enzyme results in a blue shift of the emission maximum. Manganous DL-isocitrate (1.06 mM) quenches protein fluorescence by 16% without a shift in emission peak and does not affect the relative extent of fluorescence quenching induced by the nucleotides.     

Purification and characterization of trypanothione reductase from Crithidia fasciculata, a newly discovered member of the family of disulfide-containing flavoprotein reductases

Trypanothione reductase from Crithidia fasciculata has been purified ca. 1400-fold to homogeneity in an overall yield of 60%. The pure enzyme showed a pH optimum of 7.5-8.0 and was highly specific for its physiological substrates NADPH and trypanothione that had Km values of 7 and 53 microM, respectively. Trypanothione reductase was found to be a dimer of identical subunits with Mr 53 800 each. The enzyme displayed a visible absorption spectrum that was indicative of a flavoprotein with a lambda max at 464 nm. The flavin was liberated by thermal denaturation of the protein and identified, both by high-performance liquid chromatography (HPLC) and by fluorescence studies, as FAD. The extinction coefficient of pure enzyme at 464 nm was determined to be 11.3 mM-1 cm-1. Upon titration with 5,5'-dithiobis(2-nitrobenzoic acid), oxidized enzyme was found to contain 2.2 (+/- 0.1) free thiols, whereas NADPH-reduced enzyme showed 3.9 (+/- 0.3). Furthermore, whereas oxidized enzyme was stable toward inactivating alkylation by 2.0 mM iodoacetamide, NADPH-reduced enzyme was inactivated with a half-life of 14 min. These data suggested that a redox-active cystine residue was present at the enzyme active site. Upon reduction of the enzyme with 2 electron equiv of dithionite, a new peak in the absorption spectrum was observed at 530 nm, thus indicating that a charge-transfer complex between one of the newly reduced thiols and the oxidized FAD had formed.(ABSTRACT TRUNCATED AT 250 WORDS)     

Some properties of glycine aminotransferase purified from Rhodopseudomonas palustris No. 7 concerning extracellular porphyrin production

Glycine aminotransferase (EC 2.6.1.4; GlyAT) was presumed to be an enzyme concerning the supply of glycine for the extracellular porphyrin production by Rhodopseudomonas palustris No. 7. GlyAT was purified from strain No. 7 as an electrophoretically homogenous protein. The enzyme was a monomer protein with the molecular weight of about 42,000. From the absorption spectrum of the enzyme (350 nm, 410 nm), it was indicated that the enzyme had pyridoxal phosphate as a prosthetic group. The enzyme showed high substrate specificity for glutamate as an amino group donor. Apparent Kms for glutamate and glyoxylate were 6.20 mM and 3.75 mM, respectively. The Vmax and Kcat for glutamate were 66.8 mumol/min/mg protein and 46.8 s-1, respectively. The Vmax and Kcat for glyoxylate were 68.8 mumol/min/mg protein and 48.2 s-1. The optimum temperature and pH were 40-45 degrees C and 7.0-7.5, respectively. The enzyme activity lowered to about 50% in the presence of 15 mM ammonium chloride.     

Sensitivity of yeast glycolytic enzymes to chloroquine

Chloroquine at pH 8.0 and 10 mM concentration inhibits about 30% glucose consumption and ethanol formation in yeast cells. Out of the 11 glycolytic enzymes assayed, phosphoglycerate kinase and pyruvate decarboxylase have been found to be most sensitive to chloroquine. Next sensitive are hexokinase, glyceraldehyde-3-phosphate dehydrogenase and pyruvate kinase. Kinetic studies with the three kinases studied revealed competitive inhibition of chloroquine with ATP (hexokinase, phosphoglycerate kinase) or ADP (pyruvate kinase).