Rabbit Muscle Phosphoglycerate Research Articles

Photodegradation of Tryptophan Residues and Attenuation of Molecular Chaperone Activity in α-Crystallin Are Correlated

The major eye-lens protein α-crystallin is known to possess a remarkable sequence homology to the low molecular weight heat-shock proteins and has been shown to protect several proteins against thermally induced aggregation. In this work we demonstrate that the rapid aggregation of rabbit muscle phosphoglycerate kinase during incubation at 52°C is completely inhibited in presence of 1/3 moles α-crystallin monomer per mole enzyme. Upon irradiation by UV light, tryptophan fluorescence intensity of α-crystallin declines, reflecting the destruction of these residues. A remarkable correlation is revealed between the reduction in α-crystallin fluorescence during UV-irradiation and the loss of its ability to protect phosphoglycerate kinase against aggregation. Since a loss of tryptophan fluorescence in intact eye lenses in vivo has been demonstrated to occur upon exposure to UV light, as well as during aging, it is proposed that the enhanced rate of lens opacification and cataract formation, as well as the increased levels of damaged lens proteins, which accumulate under these conditions, are the result of the gradual loss of the chaperone-protein efficacy of α-crystallin.

Phosphoglycerate-kinase-glyceraldehyde-3-phosphate-dehydrogenase interaction. Molecular mass studies.

When rabbit muscle phosphoglycerate kinase (PGK; a 48-kDa monomeric protein) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH; a 145-kDa homotetrameric protein) are present together in solution in the proportion of 1 mol PGK/1 mol GraPDH monomer (total protein 0.2-1.0 mg/ml), an 80--82-kDa protein species is observed by gel-penetration (dilution factor) method and by the conventional procedure of elution from a gel column. Individually, PGK and GraPDH do not exhibit any self association or dissociation in the concentration range employed. Electrophoresis of the 80-82-kDa peak eluted from the gel column shows a single protein band with mobility intermediate between those of GraPDH and PGK. In titration experiments by the gel-penetration method, plots of dilution factor of PGK (or GraPDH) activity versus GraPDH (or PGK) concentration shows two linear portions intersecting at approximately 1 mol GraPDH monomer/1 mol PGK. From the molecular-mass values and the titration experiments, it has been suggested that, in solution, these enzymes form a complex consisting of 1 molecule of PGK and one monomeric subunit of GraPDH (expected molecular mass 84 kDa). Its dissociation constant has been estimated to be equal to or less than 13 nM. The complex is dissociated in the presence of KCl or NADH, with approximately half dissociation at 0.1 M salt or 0.25 mM NADH. At 0.1 M KCl, the complex is completely dissociated by adding ATP, NADH or 3-phosphoglycerate. AMP, ADP, NAD+, glyceraldehyde-3-phosphate, phosphate ions and fructose-1,6-bisphosphate reverse the effect of KCl.

2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate.

The binding of inorganic vanadate (Vi) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [Vi]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 X 10(-6) M. The relevance of this result to other studies which have shown that the Vi-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [Vi] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 microM Vi has little effect on the phosphatase activity. However, in the presence of 25 microM Vi, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed.

Organothallium(III) reagents for modification of biomacromolecules: irreversible labelling of phosphoglycerate kinase from rabbit muscle

Organothallium(III) reagents, by analogy with organomercurials, have been found to rapidly label phosphoglycerate kinase from rabbit muscle. By use of a radio-labelled version of p-methylphenylthallium(III) bis-trifluoroacetate (MPT) the inhibition was shown to be irreversible by the criterion of gel filtration desalting. The rate of labelling was shown to depend on the temperature, enzyme and thallium reagent concentrations, and the presence or absence of the various substrates of the enzyme. The structure and oxidation state of the thallium reagent used affected the extent of modification by the compounds MPT, o-carboxyphenylthallium(III) bis-trifluoroacetate, thallic trifluoroacetate and thallous acetate. A number of pieces of evidence implicate cysteine residues in the labelling, including changes in the free thiol titre of the enzyme on thalliation, model studies on the interaction of thiols (e.g. glutathione) with thallium(III) and thallous materials, the lack of inactivation of phosphoglycerate kinase from yeast (which has only one thiol residue distant from the active site), and the partial restoration of enzymic activity by treatment of thalliated enzyme with sulphydryl reducing agents. Substrate protection studies showed that modification of rabbit muscle phosphoglycerate kinase by MPT was fully prevented by 3-phosphoglycerate and partially by MgATP. The latter protected only against the fast phase of thallic modification, the slower phase being unaffected. The presence of MgADP potentiated the labelling by MPT. No evidence of an MgADP-induced conformational change in the enzyme could be obtained from fluorescence or circular dichroic spectroscopies. although changes of the native spectra were noted on thalliation by MPT alone. The cross-linking potential of these arylthallium(III) reagents is discussed along with conformational changes required to trigger the hinge-movement between the N- and C-domains of the protein.

The catalytic bimodality of mammalian phosphoglycerate mutase.

Rabbit muscle phosphoglycerate mutase, presumed to manifest an absolute cofactor requirement for activity, has been found to express catalysis (3 +/- 1% of optimum) in the absence of added D-glycerate-2,3-P2. Isotope experiments indicate that this catalysis proceeds through a binary phosphoryl enzyme-glycerate intermediate which dissociates into free enzyme and monophosphoglycerate. 32P-Labeled phosphoglycerate mutase is formed by reaction with either D-32P-glycerate-3-P or D-U32P-glycerate-2,3-P2. In each case, the acid lability and alkali stability of the covalent adduct, phosphoenzyme, is consistent with a phosphohistidyl residue having been formed within the active site. D-[U-14C]Glycerate reacts with phosphoenzyme to generate D-[U-14C]monophosphoglycerate which, in turn, can react further with phosphoenzyme to yield D-[U-14C]glycerate-2,3-P2. The pH profile for the cofactor-independent activity exhibits an optimum at 6.0 as opposed to 7.0 when D-glycerate-2,3-P2 is present in the reaction medium. Bisubstrate kinetics (pH 7.0, 23 degrees C) with D-glycerate-3-P concentration as the variable, yields a family of reciprocal plots which is in accord with a modified ping-pong mechanism when D-glycerate-2,3-P2 concentrations are greater than 10(-1) Km (where Km = 0.33 microM). Progressively diminishing concentrations (much less than Km) of D-glycerate-2,3-P2 produce curvilinear reciprocal plots that approach linearity as a limit in accordance with single substrate kinetics.

Inactivation of rabbit muscle phosphoglycerate mutase by limited proteolysis with thermolysin.

Rabbit muscle phosphoglycerate mutase is inactivated by proteolysis with thermolysin. Inactivation is correlated with the breakage of one (or a few) bond(s) near one end of the polypeptide chain. There is no change in the overall conformation, quaternary structure or binding to Cibacron Blue on proteolysis. The possible analogy with the existence of a flexible tail in the yeast enzyme is discussed.

Metabolism of glycerate-2,3-P 2—VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5′-phosphate produces the concurrent loss of the three activites of the enzyme: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. 3. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P 2. 4. 4. Substrate (glycerate-3-P) protects against pyridoxal-5′-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P 2) does not prevent inactivation. 5. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. 6. 6. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.

Metabolism of glycerate-2,3-P 2—V. Histidine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Both treatment with diethylpyrocarbonate and photo-oxidation with rose bengal produces the loss of the three enzymatic activities of rabbit muscle phosphoglycerate mutase: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. The synthase and the phosphatase activities are less affected than the mutase activity. 3. 3. Glycerate-2,3-P 2 and glycerate-3-P protect against diethylpyrocarbonate inactivation, but do not protect against inactivation produced by photo-oxidation. 4. 4. Hydroxylamine reactivates the diethylpyrocarbonate-treated enzyme. 5. 5. Chemical modification of phosphoglycerate mutase markedly reduces its ability to form the functionally active phosphoenzyme. 6. 6. These results provide evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase. In addition, they support the existence of two separate binding sites for monophosphoglycerates and for bisphosphoglycerates.

Metabolism of glycerate-2,3-P 2—III. Arginine-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase activities of rabbit muscle phosphoglycerate mutase

1. 1. Treatment of rabbit muscle phosphoglycerate mutase with diketones (2,3-butanedione and 1,2-cyclohexanedione) and with glyoxal derivatives (methylglyoxal and phenylglyoxal) produces the loss of the three activities of the enzyme: phosphoglycerate mutase, glycerate-2,3-P 2 synthase and glycerate-2,3-P 2 phosphatase. 2. 2. Hydroxylamine reactivates all the activities of the modified enzyme. 3. 3. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P 2. 4. 4. Both substrate and cofactor protect against inactivation. 5. 5. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favor their location at the same active site. 6. 6. In addition, they suggest that arginine is involved in the binding of the cofactor to the enzyme.

Phylogeny and ontogeny of the phosphoglycerate mutases—III. Inactivation of rabbit muscle phosphoglycerate mutase (type M isozyme) by the sulfhydryl group reagents

1. 1. The three phosphoglycerate mutase isozymes from mammals (types M, B and MB isozymes) differ in their sensitivity to the —SH group reagents. 2. 2. Rabbit muscle phosphoglycerate mutase (type M isozyme) is reversibly inactivated by tetrathionate, p-chloromercuribenzoate and Hg 2+. 3. 3. Titration with p-chloromercuribenzoate shows the existence of two sulfhydryl groups per enzyme subunit, the modification of which produces a progressive decline in enzyme activity. 4. 4. The apparent K m values for substrate and cofactor are not affected by tetrathionate treatment. 5. 5. Phosphoglycerate mutase inactivated by tetrathionate and by p-chloromercuribenzoate is unable to form the functionally active phosphorylenzyme when mixed with glycerate-2,3-P 2, and is not protected by the cofactor against heating. 6. 6. Glycerate-2,3-P 2 protects against tetrathionate treatment, but fails to protect against Hg 2+ and p-chloromercuribenzoate inactivation.

Phylogeny and ontogeny of the phosphoglycerate mutases—v. inactivation of phosphoglycerate mutase isozymes by histidine-specific reagents

1. 1. The three isozymes of glycerate-2,3-P 2 dependent phosphoglycerate mutase present in tissues of mammals and reptiles were inactivated by both treatment with diethylpyrocarbonate and photooxidation with rose bengal. 2. 2. Inactivation of type M isozyme purified from rabbit muscle was complete when two histidine residues per enzyme subunit were carboethoxylated. Hydroxylamine removed the carboethoxy groups, with partial recovery of the enzymatic activity. The cofactor protected the enzyme against inactivation. 3. 3. The inactivation of rabbit muscle phosphoglycerate mutase by photooxidation with methylene blue and rose bengal was sharply pH dependent. The pH profile of enzyme inactivation followed the titration curve of histidine, suggesting that this amino acid was critical for enzyme activity. Glycerate-2,3-P 2 did not protect phosphoglycerate mutase against photoinactivation.

Amino acid sequences of active-site histidine peptides from rabbit muscle phosphoglycerate mutase

Monophosphogiyc~rate mutase (MPGM) catalyses the interconversion of 3-phosphoglycerate and 2-phosphoglycerate, in a reaction mechanism involving a pl~osphohistidine illter~nediate [ 1 ,I?]. A correlation of the 2.8 _& resolution electron-density map ffom X-ray crystallographic studies ofyeast MPGM with its amino acid sequence shows 2 histidines at the active site [3]. Structural studies of muscle MGP~I are much less complete, although the enzyme has been used extensively in kinetic work, e.g. [4,5]. The muscle and yeast enzymes have been shown to differ in several respects. The muscle enzyme is a dimer with identical subunits of mol. wt 28 000 [6], whereas the yeast enzyme is isolated as a tetramer composed of 4 identical subunits of mol. wt 27 000 [7]. The activity of the muscle enzyme is sensitive to modification of cysteinyl and arginyl residues [8,9], in contrast to the yeast enzyme that has no thiol [lo], and is only moderately sensitive to butanedione [l l]. In addition, a phosphohistidine peptide isolated from chicken breast muscle MGPM has been sequenced [ 121, and shows no homology with any of the 4 histidine sequences of the yeast enzyme (see table 2). We report here the purification and determination of the amino acid sequences of histidine-containing peptides from rabbit muscle MPGM to obtain an estimate of the extent of structural similarity at the active sites of the muscle and yeast enzymes.

Interaction of phosphoglycerate kinase with human erythrocyte membranes.

The purpose of this study was to investigate the interaction of phosphoglycerate kinase with the human erythrocyte ghost membrane. Ghosts prepared in 0.1 mM EDTA and 17 mM Tris buffer (pH 7.5) have about 230 molecules of phosphoglycerate kinase/ghost. No additional binding is observed after incubating soluble enzyme with these leaky ghosts. This binding is tight but reversible with Kd = 7.1 X 10(-10) M. The enzyme can be eluted significantly from the membrane by incubation with 0.15 M NaCl and it rebinds to the membrane when the depleted ghosts are incubated with rabbit muscle phosphoglycerate kinase. Ligand binding studies show that NADH and NAD have opposite effects on the binding of the enzyme to the membrane; NAD (1.0 MM) favors binding while NADH (0.25 MM) does not. Similarly, ADP (0.2 mM) favors binding while ATP does not. ATP elutes the membrane-bound enzyme with Kd = 0.058 mM. MgSO4 also stimulates dissociation of the membrane-bound phosphoglycerate kinase (Kd = 0.36 mM), an effect which appears to be due to the magnesium ion. ADP (0.2 mM) can counteract the negative effect of MgSO4 (1.0 mM) on binding of phosphoglycerate kinase to the membrane. We have been unable so far to find tight coupling of the (Na+-K+)-ATPase with the membrane-bound phosphoglycerate kinase.

Detection of an essential sulfhydryl group in phosphoglycerate mutase with an affinity-labeling reagent.

N-Bromoacetylethanolamine phosphate rapidly and irreversibly inactivates rabbit muscle phosphoglycerate mutase. At high molar ratios of reagent to enzyme, loss of activity (both mutase and phosphatase) approximates pseudo-first order kinetics. A rate-saturation effect is observed with half-maximal rate of inactivation occurring at 0.32 mM reagent, a value close to the Km for 3-phosphoglyceric acid. This datum and the dissociation constant of the 2,3-bisphosphoglycerate-enzyme complex, as determined from inactivation kinetics in the presence of the bisphosphate, suggest that the reagent reacts at the substrate binding site. Inactivation results from the covalent incorporation of about 0.8 mol of reagent/mol of catalytic subunit as determined with 14C-labeled reagent. Incorporation is negligible in the presence of substrate and is reduced 8-fold in the presence of 6 M urea. From amino acid analyses on acid hydrolysates of the inactivated enzyme, we have identified a sulfhydryl group as the site of alkylation. A peptide containing the essential sulfhydryl group has been isolated from a tryptic digest of the enzyme inactivated with labeled reagent; its amino acid composition is Trp1, Lys1,-Cys(Cm)1, Asp1, Ser1, Glu2, Gly1, Ala1, Leu1, Phe2.

Inhibition of glycolytic enzymes by 2-phosphotartronate

2-Phosphotartronate has been synthesized by permanganate oxidation of glycerol 2-phosphate and has been tested as an inhibitor of five glycolytic enzymes that bind phosphoglycerate or phosphoglycollate. Competitive inhibition of rabbit muscle phosphoglycerate mutase, enolase and pyruvate kinase was observed. Triose phosphate isomerase and 3-phosphoglycerate kinase were not inhibited.

A Phosphoenzyme Intermediary in Phosphoglycerate Kinase Action

Rabbit muscle phosphoglycerate kinase (EC 2.7.2.3) has been phosphorylated with γ-32P-ATP and then isolated by gel filtration. Phosphorylation was favored by the presence of phosphoenolpyruvate and a catalytic amount of pyruvate kinase, which appeared to remove ADP from the phosphorylation equilibrium. The phosphoenzyme was catalytically active, transferring its phosphoryl group to ADP to synthesize ATP. The phosphoenzyme also transferred its phosphoryl group to 3-phosphoglycerate to form 1,3-diphosphoglycerate, which, being unstable, was not isolated. It was converted instead to the corresponding hydroxamate or transformed enzymatically to fructose 1,6-diphosphate. Inorganic phosphate was released from the phosphoenzyme by neutral hydroxylamine at room temperature within 10 min. The pH stability profile of the phosphoenzyme is U-shaped. These facts accord with a covalent binding of the phosphoryl group to a carboxyl of the enzyme.

The effects of mercury and other resgents on Phosphoglycerate mutase−2,3−diphosphoglycerate Phosphatase from kidney, muscle and other tissues

Phosphoglycerate mutase (EC 2.7.5.3) was purified 2000-fold from pork kidney. The molecular weight estimated by Sephadex gel filtration was approx. 65000. This is in close agreement with other animal phosphoglyceromutases. The previously observed Hg 2+−induced reversible transformation of chicken and rabbit muscle phosphoglycerate mutase (EC 2.7.5.3) into 2,3-diphosphoglycerate mutase but does not occur with purified pork kidney phosphoglycerate mutase or with preparations of phosphoglycerate metase from beef liver, beef brain and human red cells. The uniqueness of the bahavvior of muscle phosphoglycerate mutase from a number of species is illustrated. On the other hand, the insensitivity of phosphoglycerate mutase from other tissues to Hg 2+ is typified by the kidney enzyme. Pyrophosphate and phosphoglycolate stimulated the 2,3-diphosphoglycerate phosphatase activity of purified preparations of phosphoglycerate mutase from pork kidney and chicken breast. The mutase activity was unaffected by these reagents.

The Purification and Properties of Phosphoglycerate Mutase from Chicken Breast Muscle

Abstract A very rapid procedure for the preparation of phosphoglycerate mutase from chicken breast muscle is presented. Commercially available frozen chicken breasts may be used. The enzyme is obtained in high yields, and the specific activity of the crystalline homogenous preparations is equal to that of the crystalline enzymes from yeast and rabbit muscle. The chicken breast enzyme requires 2,3-diphosphoglycerate for activity, and it is very similar in kinetic and molecular properties to the rabbit muscle phosphoglycerate mutase. Similarly, it also possesses 4 sulfhydryl groups per mole of enzyme. The molecular weight of the enzyme is 65,690. With the use of a gel filtration technique, it has been demonstrated that 2 moles of 2,3-diphosphoglycerate may be bound per mole of the enzyme. It appears that the binding of this phosphoglycerate may entail phosphoenzyme formation. Again, as with other pure phosphoglycerate mutases, the chicken breast muscle mutase shows some 2,3-phosphoglycerate phosphatase activity (about 1/40,000 the mutase activity).