Reaction of phenylglyoxal with chicken gizzard myosin

Modification of chicken gizzard myosin with phenyl[2-14C]-glyoxal inhibited the K+-ATPase (ATP phosphohydrolase, EC 3.6.1.32) activity as a function of time. During the 2.5 and 15 min interval 3.2 mol of the reagent were incorporated per 4.7 X 10(5) g protein and the K+-ATPase activity was 50% inhibited. Phenylglyoxal reacted with arginine residues of gizzard myosin in a mol ratio of two to one, phenylglyoxal to arginine as determined spectrophotometrically. The modification was limited to the subfragment 1 heavy chain and rod-like regions and none of the light chains were lost. The inhibition of the ATPase activity occurred when the subfragment 1 region was modified predominantly. The same results were obtained when the myosin was phosphorylated and then incubated with phenylglyoxal. Substrate MgATP2- or MgADP enhanced the inactivation of gizzard myosin; there was an increase in the incorporation of the reagent and a change in the distribution into the heavy chains. Approx. 0.5 mol of the nucleotide was bound to 4.7 X 10(5) g of phenylglyoxal myosin. Conformational changes, induced by these modifications, were responsible for the inhibition of enzymic activity. Arginine residues of gizzard myosin are necessary for the maintenance of the ATPase activity of this contractile protein.     

Characterization of an essential arginine residue in the plasma membrane H+-ATPase of Neurospora crassa

Treatment of the plasma membrane H+-ATPase of Neurospora crassa with the arginine-specific reagents phenylglyoxal or 2,3-butanedione at 30 degrees C, pH 7.0, leads to a marked inhibition of ATPase activity. MgATP, the physiological substrate of the enzyme, protects against inactivation. MgADP, a competitive inhibitor of ATPase activity with a measured Ki of 0.11 mM, also protects, yielding calculated KD values of 0.125 and 0.115 mM in the presence of phenylglyoxal and 2,3-butanedione, respectively. The excellent agreement between Ki and KD values makes it likely that MgADP exerts its protective effect by binding to the catalytic site of the enzyme. Loss of activity follows pseudo-first order kinetics with respect to phenylglyoxal and 2,3-butanedione concentration, and double log plots of pseudo-first order rate constants versus reagent concentration yield slopes of 0.999 (phenylglyoxal) and 0.885 (2,3-butanedione), suggesting that the modification of one reactive site/mol of H+-ATPase is sufficient for inactivation. This stoichiometry has been confirmed by direct measurements of the incorporation of [14C]phenylglyoxal. Taken together, the results support the notion that one arginine residue, either located at the catalytic site or shielded by a conformational change upon nucleotide binding, plays an essential role in Neurospora H+-ATPase activity.     

Modification of myosin subfragment 1 by carbodiimide in the presence of a nucleophile. Effect on adenosinetriphosphatase activities

The modification of myosin subfragment 1 by N-cyclohexyl-N'-[2-(4-morpholinyl)ethyl]carbodiimide methyl p-toluenesulfonate in the presence of the nucleophile nitrotyrosine ethyl ester was investigated. For elimination of interference of the thiol groups, the two most reactive thiols were protected by cyanylation with 2-nitro-5-(thiocyanato)benzoic acid. The ATPase activity of the cyanylated myosin subfragment 1 was not lost, but had changed. At pH 5.9, carbodiimide in the presence of the nucleophile rapidly inactivated the cyanylated enzyme. The inactivation followed first-order kinetics. The K+(EDTA)--, Ca2+--, and Mg2+--ATPase activities decreased at the same rate. Inactivation and incorporation of nucleophile occurred simultaneously. A full loss of activity resulted from the incorporation of 1 mol of nitrotyrosine per mol of myosin subfragment 1. Pyrophosphate, ITP, ADP, and ATP protected against inactivation, and the efficiency of the protection was parallel to the ligand binding strength. These results suggested that one carboxyl group was essential for the active conformation of myosin.     

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5''-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.     

Modification of the (Ca2+ + Mg2+)-ATPase protein of sarcoplasmic reticulum with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole

The (Ca2+ + Mg2+)-ATPase (ATP phosphohydrolase (Ca2+-transporting), EC 3.6.1.38) protein of rabbit skeletal sarcoplasmic reticulum (SR) rapidly incorporated 2 mol of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) per 10(5) g of protein with little change in the Ca2+-dependent ATPase activity. When 2 additional mol of the reagent were bound the Ca2+-ATPase, activity was inhibited. The same pattern was found for modified intact SR and the Ca2+ uptake ability was inhibited. MgATP, CaATP and MgADP protected the Ca2+-ATPase activity concurrent with a decrease of about 1 mol of the NBD group per 10(5) g protein, but the Ca2+ uptake ability was not protected. Calcium alone had no effect on the modification. The modified ATPase protein or SR formed non-serial oligomers or aggregates, but the ATPase protein remained the predominant species present. In the presence of MgATP, oligomer formation was reduced partially but the major changes in the Ca2+-ATPase activity were due to the modification of the ATPase monomer. Thiolysis of the NBD-ATPase protein with dithiothreitol did not restore the Ca2+-ATPase activity, although more than 1 mol of the NBD group was removed from cysteine residues. Cysteine residues were modified in the NBD-ATPase protein or SR when the enzyme activity was inhibited. Trypsin digestion of NBD-SR or its ATPase protein released the A, B, A1, and A2 fragments. The A fragment and its subfragment A2 contained most of the label. Substrate MgATP protection studies showed that the A1 and A2 fragments were involved in maintaining the Ca2+-ATPase activity. Reagent-induced conformational changes of these fragments rather than direct active site group labeling accounted for the loss of ATPase activity.     

Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole

The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) reacted specifically with 1.9 lysyl residues/mol of the myosin subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+- and Ca2+-ATPase activities were almost completely inhibited, whereas the Mg2+-ATPase activity was increased to 180% of original activity. The actin-activated Mg2+-ATPase activity was decreased to 30% of original activity by this modification. However, affinity of S-1 for actin in the presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was quenched on addition of ATP, suggesting that ATP induced conformational changes around the NBD groups attached to S-1. Tryptic digestion of the modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa peptide of S-1, more precisely the 45-kDa peptide. These results confirm the recent reports that the 50-kDa peptide of S-1 is involved in the myosin ATPase reaction (K?rner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).     

Chemical modification of the calmodulin-stimulated phosphatase, calcineurin, by phenylglyoxal

Chemical modification of calcineurin by phenylglyoxal was used to probe for the presence of arginine at, or in close proximity to, the catalytic site of this phosphatase. Phenylglyoxal inactivated calcineurin with a second-order rate constant of 1.5 M-1 min-1 at pH 7.5 and 30 degrees C. The inactivation reaction was extremely sensitive to Ca2+-induced conformational changes on calcineurin; removal of this metal ion from the reaction medium increased the rate of inactivation by almost 1 order of magnitude. Furthermore, significant protection of calcineurin by ADP was observed only in the presence of Ca2+, which suggests either that distinct sites are modified by phenylglyoxal in the absence and presence of Ca2+ or that the metal ion promotes binding of ADP to calcineurin. Inactivation of calcineurin by phenyl[2-14C]glyoxal resulted in the incorporation of more than 12 eq of the reagent. However, a kinetic analysis of the order of the inactivation reaction and complete protection of calcineurin by p-nitrophenyl phosphate suggest that only one of the modified residues is responsible for the loss of enzymatic activity. Protection of calcineurin by ADP was enhanced severalfold by calmodulin, which correlated well with a calmodulin-stimulated decrease in the Ki for this ligand. Protection of calcineurin from inactivation by phenylglyoxal was also observed in the presence of various other nucleotides; half-maximal protection by these poor substrates and competitive inhibitors was observed at concentrations near their respective inhibition constants. Thus, the results of this modification study indicate that at least 1 arginine residue is essential for the expression of catalytic activity of the calmodulin-regulated phosphatase.     

Evidence for an essential role for arginyl residues for yeast phosphoglycerate kinase.

Reaction of yeast phosphoglycerate kinase with either butanedione or cyclohexanedione can result in modification of up to all 13 arginyl residues with total loss of activity; however, extrapolation to zero activity for partially modified preparations indicates that up to 7 arginyls are essential. Whereas 20 mm 3-phosphoglycerate affords partial protection of activity toward both reagents, 20 mm MgATP affords complete protection of activity and protects 2 arginyls against modification by butanedione and 1 arginyl against modification by cyclohexanedione. With butanedione the modification could be reversed with total recovery of activity, suggesting that only arginyl groups were modified, which is consistent with the amino acid analysis of the modified protein. Only at high cyclohexanedione concentrations or long reaction times was a yellow product obtained that showed loss of lysyl residues. Circular dichroism spectra show that even when all the arginyls are modified by butanedione or up to 10 modified by cyclohexanedione there is no change observed in the far or near ultraviolet, indicating that there is no detectable conformational change concomitant with modification, which is confirmed by hydrodynamic studies. It is concluded that at least one, possibly two, arginyls of yeast phosphoglycerate kinase are essential for its action on ATP.     

Chemical modification of thiol groups of mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe. Involvement of alpha- and gamma-subunits in the enzyme activity

Mitochondrial F1-ATPase from the yeast Schizosaccharomyces pombe has been prepared under a stable form and in relatively high amounts by an improved purification procedure. Specific chemical modification of the enzyme by the thiol reagent N-ethylmaleimide (NEM) at pH 6.8 leads to complete inactivation characterized by complex kinetics and pH dependence, indicating that several thiols are related to the enzyme activity. A complete protection against NEM effect is afforded by low concentrations of nucleotides in the presence of Mg2+, with ADP and ATP being more efficient than GTP. A total binding of 5 mol of [14C]NEM/mol of F1-ATPase is obtained when the enzyme is 85% inactivated: 3 mol of the label are located on the alpha-subunits and 2 on the gamma-subunit. Two out of the 3 mol on the alpha-subunits bind very rapidly before any inactivation occurs, indicating that the two thiols modified are unrelated to the inactivation process. Complete protection by ATP against inactivation by NEM prevents the modification of three essential thiols out of the group of five thiols labeled in the absence of ATP: one is located on a alpha-subunit and two on the gamma-subunit. These two essential thiols of the gamma-subunit can be differentiated by modification with 6,6'-dithiodinicotinic acid (CPDS), another specific thiol reagent. A maximal binding of 4 mol of [14C]CPDS/mol of enzyme is obtained, concomitant to a 25% inhibition. Sequential modification of the enzyme by CPDS and [14C]NEM leads to the same final deep inactivation as that obtained with [14C]NEM alone. One out of the two thiols of the gamma-subunit is no longer accessible to [14C]NEM after CPDS treatment. When incubated at pH 6.8 with [3H]ATP in the presence of Mg2+, F1-ATPase is able to bind 3, largely exchangeable, mol of nucleotide/mol of enzyme. Modification of the three essential thiols by NEM dramatically decreases the binding of 3H-nucleotide down to about 1 mol/mol of enzyme. Partial modification modifies the cooperative properties, the enzyme being no longer sensitive to anion activation.     

Evidence for an essential arginine residue at the active site of ATP citrate lyase from rat liver.

Rat liver ATP citrate lyase was inactivated by 2, 3-butanedione and phenylglyoxal. Phenylglyoxal caused the most rapid and complete inactivation of enzyme activity in 4-(2-hydroxyethyl)-1-piperazine-ethanesulphonic acid buffer, pH 8. Inactivation by both butanedione and phenylglyoxal was concentration-dependent and followed pseudo- first-order kinetics. Phenylglyoxal also decreased autophosphorylation (catalytic phosphate) of ATP citrate lyase. Inactivation by phenylglyoxal and butanedione was due to the modification of enzyme arginine residues: the modified enzyme failed to bind to CoA-agarose. The V declined as a function of inactivation, but the Km values were unaltered. The substrates, CoASH and CoASH plus citrate, protected the enzyme significantly against inactivation, but ATP provided little protection. Inactivation with excess reagent modified about eight arginine residues per monomer of enzyme. Citrate, CoASH and ATP protected two to three arginine residues from modification by phenylglyoxal. Analysis of the data by statistical methods suggested that the inactivation was due to modification of one essential arginine residue per monomer of lyase, which was modified 1.5 times more rapidly than were the other arginine residues. Our results suggest that this essential arginine residue is at the CoASH binding site.     

Modification of myo-inositol monophosphatase by the arginine-specific reagent phenylglyoxal.

myo-Inositol monophosphatase is inhibited by the arginine-specific reagent phenylglyoxal. The rate of inactivation is decreased in the presence of Pi, a competitive inhibitor of the enzyme. The effect of Pi is dependent on the presence of Mg2+, but is unaffected by Li+, an uncompetitive inhibitor. In the absence of Mg2+, the substrate, Ins(1)P, binds to the enzyme but is not converted into products, and affords only a small degree of protection against inactivation by phenylglyoxal. Li+ had no further effect under these conditions, but in the presence of Mg2+ caused a marked potentiation of the protective effect of substrate alone. In the absence of substrate, Li+ had no effect on activation by phenylglyoxal. Incorporation of 14C-labelled phenylglyoxal showed that inactivation was associated with modification of a single arginine residue per monomer in the dimeric enzyme. These findings support a mechanism in which Li+ inhibits monophosphatase by trapping a phosphorylated enzyme intermediate and preventing its hydrolysis.     

A functional arginine residue in the vacuolar H(+)-ATPase of higher plants.

The arginine-specific reagent phenylglyoxal inactivated the vacuolar H(+)-ATPase of red beet. Inactivation by phenylglyoxal followed pseudo-first-order kinetics and a double log plot of the t1/2 of inactivation versus phenylglyoxal concentration yielded a slope of 1.18. Neither inorganic anions nor DIDS protected from phenylglyoxal-mediated inactivation of the H(+)-ATPase. Indeed, Cl- stimulated the rate of phenylglyoxal-mediated H(+)-ATPase inactivation relative to SO4(2-). ATP, but not MgATP or ADP, protected from phenylglyoxal-mediated inactivation and inactivation resulted in a decrease in the Vmax of the H(+)-ATPase with little effect on the Km. Collectively, these results are consistent with phenylglyoxal-mediated inactivation of the vacuolar H(+)-ATPase resulting from modification of a single arginine residue in the catalytic nucleotide binding site of the vacuolar H(+)-ATPase. Stimulation of phenylglyoxal-mediated inactivation by Cl- indicates that exposure of the phenylglyoxal-sensitive functional arginine residue is enhanced in the presence of Cl-. The failure of MgATP to protect from phenylglyoxal inactivation suggests that ATP, rather than MgATP, binds directly to the catalytic site and that Mg2+ may act to promote catalysis subsequent to ATP binding.     

Inactivation of carbonyl reductase from human brain by phenylglyoxal and 2,3-butanedione: a comparison with aldehyde reductase and aldose reductase

Aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and carbonyl reductase (secondary-alcohol:NADP+ oxidoreductase, EC 1.1.1.184) constitute the enzyme family of the aldo-keto reductases, a classification based on similar physicochemical properties and substrate specificities. The present study was undertaken in order to obtain information about the structural relationships between the three enzymes. Treatment of human aldehyde and carbonyl reductase with phenylglyoxal and 2,3-butanedione caused a complete and irreversible loss of enzyme activity, the rate of loss being proportional to the concentration of the dicarbonyl reagents. The inactivation of aldehyde reductase followed pseudo-first-order kinetics, whereas carbonyl reductase showed a more complex behavior, consistent with protein modification cooperativity. NADP+ partially prevented the loss of activity of both enzymes, and an even better protection of aldehyde reductase was afforded by the combination of coenzyme and substrate. Aldose reductase was partially inactivated by phenylglyoxal, but insensitive to 2,3-butanedione. The degree of inactivation with respect to the phenylglyoxal concentration showed saturation behavior. NADP+ partially protected the enzyme at low phenylglyoxal concentrations (0.5 mM), but showed no effect at high concentrations (5 mM). These findings suggest the presence of an essential arginine residue in the substrate-binding domain of aldehyde reductase and the coenzyme-binding site of carbonyl reductase. The effect of phenylglyoxal on aldose reductase may be explained by the modification of a reactive thiol or lysine rather than an arginine residue.     

Ca2+ bindings of pig cardiac myosin, subfragment-1, and g2 light chain.

Ca2+ binding to pig cardiac myosin, subfragment-1 (S-1), and g2 light chain were investigated by the equilibrium dialysis method. Two different S-1s, one of which can bind Ca2+ and another which cannot, were prepared. In order to calculate the free Ca2+ concentrations adequately, the amounts of Ca2+ included in various chemicals and proteins were measured by atomic absorption spectroscopy. Ca2+ contamination was greatest in KCl among the chemicals tested. In addition, the Ca2+ strongly bound to myosin and S-1 was released in the presence of Mg2+. When Mg2+ was not added, the Ca2+-binding constant of myosin was 4 x 10(5) M-1 and the maximum binding number was 1.8 mol per mol of myosin. Cooperativity between the 2 Ca2+ bindings could not be demonstrated. Mg2+ strongly inhibited the Ca2+ binding: at a free Ca2+ concentration of 1 x 10(-5) M, 1.3 mol Ca2+ was bound to myosin in the absence of Mg2+, but 0.6 and 0.2 mol were bound in the presence of 0.3 and 4.5 mM Mg2+, respectively. The Ca2+-binding constant of S-1, which contained a 15,000 dalton component, was 8.6 x 10(5) M-1, and the maximum binding number was 0.7 mol per mol of S-1. The 15,000 dalton component could be exchanged with extraneous g2. S-1 which lacked the 15,000 component could not bind Ca2+ at free Ca2+ concentrations less than 0.1 mM. The Ca2+ binding to free g2 light chain was about 100 times weaker than the binding to myosin, as indicated previously for skeletal myosin (Okamoto, Y. & Yagi, K. (1976) J. Biochem. 80, 111--120). The Ca2+-binding constant was obtained as 4.1 x 10(3) M-1 in the absence of added Mg2+. Phosphorylation of g2 light chain did not affect the Ca2+ binding to the free g2 light chain or to myosin. Ca2+ binding to cardiac native tropomyosin was also measured.     

Dense precipitate of brain tubulin with skeletal muscle myosin

Purified tubulin from porcine brain formed a dense precipitate at 37 degrees C with muscle myosin filaments from rabbit skeletal muscle; this effect was greater than that with partially purified tubulin. ATP or GTP, which prevented the myosin filaments from precipitating, inhibited the formation of the dense precipitate, but did not dissociate the dense precipitate once formed. The dense precipitate was found by thin-section electron microscopy to be composed to side-by-side aggregates of myosin filaments whose projections might be decorated by tubulin. The decoration was also seen by negative-stain electron microscopy. The binding of tubulin to myosin filaments decreased the Mg2+- and Ca2+-GTPase activity of the myosin by about half, but did not affect either Mg2+- or Ca2+-ATPase activity. The binding ratio of tubulin to myosin in the presence of 5 mM MgCl2 was 2.2 mol/mol using purified tubulin and 1.8 mol/mol using partially purified tubulin. Five mM ATP and GTP in the presence of 5 mM MgCl2 decreased the tubulin binding by 1.6-2.0 and 1.1-1.3 mol/mol, respectively, when added before an encounter of tubulin with myosin filaments, but did not cause any decrease when added after such an encounter.     

The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin.

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.     

The presence of functional arginine residues in phosphoenolpyruvate carboxykinase from Saccharomyces cerevisiae

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase (ATP:oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.49) is completely inactivated by phenylglyoxal and 2,3-butanedione in borate buffer at pH 8.4, with pseudo-first-order kinetics and a second-order rate constant of 144 min-1 X M-1 and 21.6 min-1 X M-1, respectively. Phosphoenolpyruvate, ADP and Mn2+ (alone or in combination) protect the enzyme against inactivation, suggesting that the modification occurs at or near to the substrate-binding site. Almost complete restoration of activity was obtained when a sample of 2,3-butanedione-inactivated enzyme was freed of excess modifier and borate ions, suggesting that only arginyl groups are modified. The changes in the rate of inactivation in the presence of substrates and Mn2+ were used to determine the dissociation constants for enzyme-ligand complexes, and values of 23 +/- 3 microM, 168 +/- 44 microM and 244 +/- 54 microM were found for the dissociation constants for the enzyme-Mn2+, enzyme-ADP and enzyme-phosphoenolpyruvate complexes, respectively. Based on kinetic data, it is shown that 1 mol of reagent must combine per enzyme active unit in order to inactivate the enzyme. Complete inactivation of the carboxykinase can be correlated with the incorporation of 3-4 mol [7-14C]phenylglyoxal per mol of enzyme subunit. Assuming a stoichiometry of 1:1 between phenylglyoxal incorporation and arginine modification, our results suggest that the modification of only two of the three to four reactive arginine residues per phosphoenolpyruvate carboxykinase subunit is responsible for inactivation.     

Requirement of phosphorylation of Physarum myosin heavy chain for thick filament formation, actin activation of Mg2+-ATPase activity, and Ca2+-inhibitory superprecipitation

In an attempt to elucidate the Ca2+-regulated mechanism of motility in Physarum plasmodia, we improved the preparation method for myosin B and pure myosin. The obtained results are as follows: 1. We obtained two types of myosin B which are distinguishable from each other with respect to their sensitivity to Ca2+. The inactive type of myosin B had low superprecipitation activities both in the presence and in the absence of Ca2+. The active type showed very high superprecipitation activity in EGTA, and the activity was conspicuously inhibited by Ca2+. The active type was converted into the inactive type by treatment with potato acid phosphatase. Also the inactive type or the phosphatase-treated active type was converted into the active type upon reacting with ATP-gamma-S. 2. In the reaction with ATP-gamma-S, only the myosin HC of myosin B was phosphorylated. The phosphorylation was independent of Ca2+ and calmodulin, and the extent was about 1 mol/mol HC. 3. The Ca2+ sensitivity in the superprecipitation of the active type was not decreased by adding an excess amount of F-actin. Besides, the actin-activated Mg2+-ATPase activity of purified phosphorylated myosin was not Ca2+-sensitive. Therefore, presence of a Ca2+-dependent inhibitory factor(s) that could bind to myosin was suggested. 4. The Mg2+-ATPase activity of purified phosphorylated myosin was 7-8 times enhanced by F-actin, but that of dephosphorylated myosin was hardly activated at all. 5. In a gel filtration in 0.5 M KCl, phosphorylated myosin was eluted behind dephosphorylated myosin. Electron microscopy applying the rotary-shadow method showed significant difference in flexibility in the tail between phosphorylated and dephosphorylated myosin molecules. 6. In 40 mM KCl and 5-10 mM MgCl2, phosphorylated myosin formed thick filaments, but dephosphorylated myosin did not, whether there was ATP or not. The above results clearly show that the phosphorylation of myosin HC is indispensable to ATP-induced superprecipitation, the actin-activated Mg2+-ATPase activity, and the formation of thick filaments of myosin. A myosin-linked factor(s) that inhibits an actin-myosin interaction in a Ca2+-dependent manner may exist.     

Arginine chemical modification of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase

Reaction of Petunia hybrida 5-enol-pyruvylshikimate-3-phosphate synthase (EPSPS) with the arginine reagents phenylglyoxal (PGO) and p-hydroxyphenylglyoxal (HPGO) leads to inactivation of the enzyme. Inactivation with HPGO leads to modification of approximately 3 mol of arginine per mole of enzyme. The modification reaction follows pseudo-first-order kinetics with a t1/2 of 1 min at 5 mM p-hydroxyphenylglyoxal in 0.1 M triethanolamine HCl, pH 7.8. By titration of HPGO-modified enzyme with 5,5'-bis(dithio-2-nitrobenzoic acid), the possibility of cysteine modification by the arginine reagent was ruled out. While shikimate 3-phosphate (S3P) afforded partial protection to the enzyme against inactivation by HPGO, complete protection could be obtained by using a mixture of S3P and glyphosate. Under the latter conditions, only 1 mol arginine was modified per mole of enzyme. This pattern of reactivity suggests that two arginines may be involved in the binding of S3P and glyphosate to EPSP synthase. A third reactive arginine appears to be nonessential for EPSPS activity. Labeling of EPSP synthase with [14C]phenylglyoxal, peptic digestion, HPLC mapping, and amino acid sequencing indicate that Arg-28 and Arg-131 are two of the reactive arginines labeled with [14C]PGO.     

Involvement of arginine residues in the allosteric activation and inhibition ofSynechocystis PCC 6803 ADPglucose pyrophosphorylase

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacteriumSynechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl₂ enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form.V _max at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.