Glycolytic enzyme interactions with tubulin and microtubules

Interactions of the glycolytic enzymes glucose-6-phosphate isomerase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, triose-phosphate isomerase, enolase, phosphoglycerate mutase, phosphoglycerate kinase, pyruvate kinase, lactate dehydrogenase type-M, and lactate dehydrogenase type-H with tubulin and microtubules were studied. Lactate dehydrogenase type-M, pyruvate kinase, glyceraldehyde-3-phosphate dehydrogenase, and aldolase demonstrated the greatest amount of co-pelleting with microtubules. The presence of 7% poly(ethylene glycol) increased co-pelleting of the latter four enzymes and two other enzymes, glucose-6-phosphate isomerase, and phosphoglycerate kinase with microtubules. Interactions also were characterized by fluorescence anisotropy. Since the KD values of glyceraldehyde-3-phosphate dehydrogenase, pyruvate kinase and lactate dehydrogenase for tubulin and microtubules were all found to be between 1 and 4 microM, which is in the range of enzyme concentration in cells, these enzymes are probably bound to microtubules in vivo. These observations indicate that interactions of cytosolic proteins, such as the glycolytic enzymes, with cytoskeletal components, such as microtubules, may play a structural role in the formation of the microtrabecular lattice.     

Evidence for absence of an interaction between purified 3-phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase

The possibility of a functional complex formation between glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) and 3-phosphoglycerate kinase (EC. 2.7.2.3), enzymes catalysing two consecutive reactions in glycolysis has been investigated. Kinetic analysis of the coupled enzymatic reaction did not reveal any kinetic sign of the assumed interaction up to 4 X 10(-6) M kinase and 10(-4) M dehydrogenase. Fluorescence anisotrophy of 10(-7) M or 2 X 10(-5) M glyceraldehyde-3-phosphate dehydrogenase labeled with fluorescein isothiocynate did not change in the presence of non-labeled 3-phosphoglycerate kinase (up to 4 X 10(-5) M). The frontal gel chromatographic analysis of a mixture of the two enzymes (10(-4) M dehydrogenase) could not reveal any molecular species with the kinase activity having a molecular weight higher than that of 3-phosphoglycerate kinase. Both types of physicochemical measurements were also performed in the presence of substrates of the kinase and gave the same results. The data seem to invalidate the hypothesis that there is a complex between purified pig muscle glyceraldehyde-3-phosphate dehydrogenase and 3-phosphoglycerate kinase.     

31P NMR magnetization-transfer measurements of flux between inorganic phosphate and adenosine 5'-triphosphate in yeast cells genetically modified to overproduce phosphoglycerate kinase

31P NMR magnetization-transfer measurements were used to measure flux between inorganic phosphate and ATP in the reactions catalyzed by phosphoglycerate kinase and glyceraldehyde-3-phosphate dehydrogenase in anaerobic cells of the yeast Saccharomyces cerevisiae. Flux between ATP and Pi and glucose consumption and ethanol production were measured in cells expressing different levels of phosphoglycerate kinase activity. Overexpression of the enzyme was obtained by transforming the cells with a multicopy plasmid containing the phosphoglycerate kinase coding sequence and portions of the promoter element. Fluxes were also measured in cells in which the glyceraldehyde-3-phosphate dehydrogenase activity had been lowered by limited incubation with iodoacetate. These measurements showed that both enzymes have low flux control coefficients for glycolysis but that phosphoglycerate kinase has a relatively high flux control coefficient for the ATP----Pi exchange catalyzed by the two enzymes. The Pi----ATP exchange velocities observed in the cell were shown to be similar to those displayed by the isolated enzymes in vitro under conditions designed to mimic those in the cell with respect to the enzyme substrate concentrations.     

Glycolytic enzymes and a GLUT-1 glucose transporter in the outer segments of rod and cone photoreceptor cells

The presence of glycolytic enzymes and a GLUT-1-type glucose transporter in rod and cone outer segments was determined by enzyme activity assays, glucose uptake measurements, Western blotting, and immunofluorescence microscopy. Enzyme activities of six glycolytic enzymes including hexokinase, phosphofructokinase, aldolase, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, pyruvate kinase, and lactate dehydrogenase, were found to be present in purified rod outer segment (ROS) preparations. Immunofluorescence microscopy of bovine and chicken retina sections labeled with monoclonal antibodies against glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and lactate dehydrogenase have confirmed that these enzymes are present in rod and cone outer segments and not simply contaminants from the inner segments or other cells. Rod outer segments were also found to contain glucose transport activity as detected by 3-O-[14C]methylglucose uptake and exchange. The glucose transporter had a Km of 6.3 mM and a Vmax of 0.15 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for net uptake and a Km of 29 mM and a Vmax of 1.06 nmol of 3-O-methylglucose/s/mg of ROS membrane protein for equilibrium exchange. These Km values for net uptake and equilibrium exchange are similar to values obtained for human red blood cells and are characteristic of GLUT-1-type glucose transporter. The transport was inhibited by both cytochalasin B and phloretin. Western blot analysis and immunofluorescence microscopy using type-specific glucose transporter antibodies indicated that both rod and cone outer segment plasma membranes have a GLUT-1 glucose transporter of Mr 45K as found in red blood cells and brain microsomal membranes. Solid-phase radioimmune competitive inhibition studies indicated that rod outer segment plasma membranes contained 15% the number of glucose transporters found in human red blood cell membranes and had an estimated density of 400 glucose transporter per micron2 of plasma membrane. These studies support the view that outer segments can generate energy in the form of ATP and GTP by anaerobic glycolysis to supply at least some of the energy requirements for phototransduction and other metabolic processes.     

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).     

Heteromerous interactions among glycolytic enzymes and of glycolytic enzymes with F-actin: effects of poly(ethylene glycol)

Interactions of glucose-6-phosphate isomerase (D-glucose-6-phosphate ketol-isomerase, EC 5.3.1.9), aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13), glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12), triose-phosphate isomerase (D-glyceraldehyde-3-phosphate ketol-isomerase, EC 5.3.1.1), phosphoglycerate mutase (D-phosphoglycerate 2,3-phosphomutase, EC 5.4.2.1), phosphoglycerate kinase (ATP:3-phospho-D-glycerate 1-phosphotransferase, EC 2.7.3), enolase (2-phospho-D-glycerate hydro-lyase, EC 4.2.1.11), pyruvate kinase (ATP:Pyruvate O2-phosphotransferase, EC 2.7.1.40) and lactate dehydrogenase [S)-lactate:NAD+ oxidoreductase, EC 1.1.1.27) with F-actin, among the glycolytic enzymes listed above, and with phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) were studied in the presence of poly(ethylene glycol). Both purified rabbit muscle enzymes and rabbit muscle myogen, a high-speed supernatant fraction containing the glycolytic enzymes, were used to study enzyme-F-actin interactions. Following ultracentrifugation, F-actin and poly(ethylene glycol) tended to increase and KCl to decrease the pelleting of enzymes. In general, the greater part of the pelleting occurred in the presence of both F-actin and poly(ethylene glycol) and the absence of KCl. Enzymes that pelleted more in myogen preparations than as individual purified enzymes in the presence of poly(ethylene glycol) and the absence of F-actin were tested for specific enzyme-enzyme associations, several of which were observed. Such interactions support the view that the internal cell structure is composed of proteins that interact with one another to form the microtrabecular lattice.     

A spectrophotometric assay of ATP synthesized by sarcoplasmic reticulum.

The problems encountered with a coupled enzyme assay for ATP using glucose, hexokinase and glucose-6-phosphate dehydrogenase are discussed and a modification where fructose and glucosephosphate isomerase were substituted for glucose is described. This modified assay was used successfully to measure the ATP synthesized by reversal of the sarcoplasmic reticulum ATPase. ATP synthesized by adenylate kinase contaminating the sarcoplasmic reticulum was easily corrected for by a subtraction procedure.     

Five enzymes of the glycolytic pathway serve as substrates for purified epidermal-growth-factor-receptor kinase.

Several enzymes of the glycolytic pathway are phosphorylated in vitro and in vivo by retroviral transforming protein kinases. These substrates include the enzymes phosphoglycerate mutase (PGM), enolase and lactate dehydrogenase (LDH). Here we show that purified EGF (epidermal growth factor)-receptor kinase phosphorylates the enzymes PGM and enolase and also the key regulatory enzymes of the glycolytic pathway, phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH), in an EGF-dependent manner. Stoichiometry of phosphate incorporation into GAPDH (calculated from native Mr) is the highest, reaching approximately 1. LDH and other enzymes of the glycolytic pathway are not phosphorylated by the purified EGF-receptor kinase. These enzymes are phosphorylated under native conditions, and the Km values of EGF-receptor kinase for their phosphorylation are close to the physiological concentrations of these enzymes in the cell. EGF stimulates the reaction by 2-5-fold by increasing the Vmax. without affecting the Km of this process. Phosphorylation is rapid at 22 degrees C and at higher temperatures. However, unlike the self-phosphorylation of EGF-receptor, which occurs at 4 degrees C, the glycolytic enzymes are poorly phosphorylated at this temperature. Some enzymes, in particular enolase, increase the receptor Km for ATP in the autophosphorylation process and thus may act as competitive inhibitors of EGF-receptor self-phosphorylation. On the basis of the Km values of EGF receptor for the substrate enzymes and for ATP in the phosphorylation reaction, these enzymes may also be substrates in vivo for the EGF-receptor kinase.     

Key enzymes of carbohydrate metabolism as targets of the 11.5-kDa Zn(2+)-binding protein (parathymosin)

The 11.5-kDa Zn(2+)-binding protein (ZnBP) was covalently linked to Sepharose. Affinity chromatography with a cytosolic subfraction from liver resulted in purification of a predominant 38-kDa protein. In comparable experiments with brain cytosol a 39-kDa protein was enriched. The ZnBP-protein interactions were zinc-specific. Both proteins were identified as fructose-1,6-bisphosphate aldolase. Experiments with crude cytosol showed zinc-specific interaction of additional enzymes involved in carbohydrate metabolism. From liver cytosol greater than 90% of the following enzymes were specifically retained: aldolase, phosphofructokinase-1, hexokinase/glucokinase, glucose-6-phosphate dehydrogenase, glycerol-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, and fructose-1,6-bisphosphatase. Glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, and most of triosephosphate isomerase remained unbound. From L-type pyruvate kinase only the phosphorylated form seems to interact with ZnBP. Using brain cytosol hexokinase, phosphofructokinase-1, and aldolase were completely bound to the affinity column, whereas glucose-6-phosphate isomerase, phosphoglycerate kinase, enolase, lactate dehydrogenase, pyruvate kinase, and most of triose-phosphate isomerase remained unbound. The behavior of glucose-6-phosphate dehydrogenase and glycerol-3-phosphate dehydrogenase from this tissue could not be followed. A possible function of ZnBP in supramolecular organization of carbohydrate metabolism is proposed.     

Combined glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase in catecholamine-stimulated guinea-pig cardiac muscle. Comparison with mass-action ratio of creatine kinase.

The steady-state reactant levels of triose-phosphate isomerase and the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea-pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3-phosphate were directly measured and compared with those of creatine kinase. Non-working Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near-equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP].[Pi])) was derived from creatine kinase and the free [NAD+]/([NADH].[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near-zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose-phosphate isomerase was near-equilibrium only in control or potassium-arrested Langendorff hearts as well as in postischemic 'stunned' hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure-volume work the enzyme was displaced from equilibrium. The alternative phosphorylation ratio [ATP]'/([ADP]).[Pi]) was derived from the magnesium-dependent glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1-2.0 mM). As predicted, [ATP]/([ADP].[Pi]) and [ATP]'/([ADP]'.[Pi]') were in excellent agreement when glycolysis was virtually halted by hyperkalemic arrest (flux approximately 0.2 mumol C3.min-1.g dry mass-1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux approximately 5-14 mumol C3.min-1.g dry mass-1). In contrast, when flux increased due to transition from no-work to physiological pressure-volume work (rate increase from approximately 3 to 11 mumol C3.min-1.g dry mass-1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial 'stunning', not due to hydraulic work load per se, glyceraldehyde-3-phosphate levels increased from about 4 microM to greater than or equal to 16 microM. Thus the guinea-pig cardiac glyceraldehyde-3-phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near-equilibrium catalysis at significant flux provided glyceraldehyde-3-phosphate levels rise, e.g., due to 'stunning' or adrenergic hormones.     

Regulation of the glucolytic enzymes inPseudomonas putida

Summary The synthesis of glucose catabolizing enzymes is under inductive control inPseudomonas putida. Glucose, gluconate and 2-ketogluconate are the best nutritional inducers of these enzymes. Mutants unable to catabolize gluconate or 2-ketogluconate synthesized relatively high levels of glucose dehydrogenase and gluconate-6P dehydrase activities when grown in the presence of these substrates. This identifies both compounds as true inducers of these enzymes. KDGP aldolase is induced by its substrate, as evidenced by the inability of mutant cells unable to form KDGP to produce this enzyme at levels above the basal one. A 3-carbon compound appears to be the inducer of glyceraldehyde-3P dehydrogenase. This pattern of regulation suggests that there is a low degree of coordinate control in the synthesis of the glucolytic enzymes byP. putida. This is also supported by the lack of proportionality found in the levels of two enzymes governed by the same inducers, glucose dehydrogenase and gluconate-6P dehydrase, in cells grown on different conditions.Abbrevitions P phosphate - KDGP 2-Keto-3-deoxygluconate-6-phosphate - GDH glucose dehydrogenase - GNDH gluconate dehydrogenase - GK glucokinase - GNK gluconokinase - KGK ketogluconokinase - KGR 2-Ketogluconate-6-phosphate reductase - GPDH glucose-6-phosphate dehydrogenase - GNPD gluconate-6-phosphate dehydrase - KDGPA 2-Keto-3-deoxygluconate-6-phosphate aldolase - GAPDH glyceraldehyde-3-phosphate dehydrogenase     

Glucose requirement for postischemic recovery of perfused working heart

The quantitative importance of glycolysis in cardiomyocyte reenergization and contractile recovery was examined in postischemic, preload-controlled, isolated working guinea pig hearts. A 25-min global but low-flow ischemia with concurrent norepinephrine infusion to exhaust cellular glycogen stores was followed by a 15-min reperfusion. With 5 mM pyruvate as sole reperfusion substrate, severe contractile failure developed despite normal sarcolemmal pyruvate transport rate and high intracellular pyruvate concentrations near 2 mM. Reperfusion dysfunction was characterized by a low cytosolic phosphorylation potential [( ATP]/[( ADP][Pi]) due to accumulations of inorganic phosphate (Pi) and lactate. In contrast, with 5 mM glucose plus pyruvate as substrates, but not with glucose as sole substrate, reperfusion phosphorylation potential and function recovered to near normal. During the critical ischemia-reperfusion transition at 30 s reperfusion the cytosolic creatine kinase appeared displaced from equilibrium, regardless of the substrate supply. When under these conditions glucose and pyruvate were coinfused, glycolytic flux was near maximum, the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction was enhanced, accumulation of Pi was attenuated, ATP content was slightly increased, and adenosine release was low. Thus, glucose prevented deterioration of the phosphorylation potential to levels incompatible with reperfusion recovery. Immediate energetic support due to maximum glycolytic ATP production and enhancement of the glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase reaction appeared to act in concert to prevent detrimental collapse of [ATP]/[( ADP][Pi]) during creatine kinase dysfunction in the ischemia-reperfusion transition. Dichloroacetate (2 mM) plus glucose stimulated glycolysis but failed fully to reenergize the reperfused heart; conversely, 10 mM 2-deoxyglucose plus pyruvate inhibited glycolysis and produced virtually instantaneous de-energization during reperfusion. The following conclusions were reached. (1) A functional glycolysis is required to prevent energetic and contractile collapse of the low-flow ischemic or reperfused heart (2). Glucose stabilization of energetics in pyruvate-perfused hearts is due in part to intensification of glyceraldehyde-3-phosphate dehydrogenase/3-phosphoglycerate kinase activity. (3) 2-Deoxyglucose depletes the glyceraldehyde-3-phosphate pool and effects intracellular phosphate fixation in the form of 2-deoxyglucose 6-phosphate, but the cytosolic phosphorylation potential is not increased and reperfusion failure occurs instantly. (4) Consistent correlations exist between cytosolic ATP phosphorylation potential and reperfusion contractile function. The findings depict glycolysis as a highly adaptive emergency mechanism which can prevent deleterious myocyte deenergization during forced ischemia-reperfusion transitions in presence of excess oxidative substrate.     

Effects of m-Cl-peroxy benzoic acid on glycolysis in Saccharomyces cerevisiae

Concentrations of m-Cl-peroxy benzoic acid (CPBA) higher than 0.1 mM decrease the ATP-content of Saccharomyces cerevisiae in the presence of glucose in 1 min to less than 10% of the initial value. In the absence of glucose, 1.0 mM CPBA is necessary for a similar effect. After the rapid loss of ATP in the first min in the presence of glucose caused by 0.2 mM CPBA, the ATP-content recovers to nearly the initial value after 10 min. Aerobic glucose consumption and ethanol formation from glucose are both completely inhibited by 1.0 mM CPBA. Assays of the activities of nine different enzymes of the glycolytic pathway as well as analysis of steady state concentrations of metabolites suggest that glyceraldehyde-3-phosphate dehydrogenase is the most sensitive enzyme of glucose fermentation. Phosphofructokinase and alcohol dehydrogenase are slightly less sensitive. Incubation for 1 or 10 min with concentrations of 0.05 to 0.5 mM CPBA causes a) inhibition of glyceraldehyde-3-phosphate dehydrogenase, b) decrease of the ATP-content and c) a decrease of the colony forming capacity. From these findings it is concluded that the disturbance of the ATP-producing glycolytic metabolism by inactivation of glyceraldehyde-3-phosphate dehydrogenase may be an explanation for cell death caused by CPBA.Abbreviations CPBA m-Chloro-peroxy benzoic acid - G-6-P glucose-6-phosphate - F-6-P fructose-6-phosphate - F-1,6-P₂ frnctose-1,6-bisphosphate - DAP dihydroxyacetone phosphate - GAP glyceraldehyde-3-phosphate - 2PGA 2-phosphoglycerate - PEP phosphoenol pyruvate - Pyr pyruvate - EtOH ethanol - PFK phosphofructokinase - GAPDH glyceraldehyde-3-phosphate dehydrogenase - ADH alcohol dehydrogenase Dedicated to Prof. Dr. Wolfgang Gerok at the occasion of his 60th birthday     

What controls glycolysis in bloodstream form Trypanosoma brucei?

On the basis of the experimentally determined kinetic properties of the trypanosomal enzymes, the question is addressed of which step limits the glycolytic flux in bloodstream form Trypanosoma brucei. There appeared to be no single answer; in the physiological range, control shifted between the glucose transporter on the one hand and aldolase (ALD), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and glycerol-3-phosphate dehydrogenase (GDH) on the other hand. The other kinases, which are often thought to control glycolysis, exerted little control; so did the utilization of ATP. We identified potential targets for anti-trypanosomal drugs by calculating which steps need the least inhibition to achieve a certain inhibition of the glycolytic flux in these parasites. The glucose transporter appeared to be the most promising target, followed by ALD, GDH, GAPDH, and PGK. By contrast, in erythrocytes more than 95% deficiencies of PGK, GAPDH, or ALD did not cause any clinical symptoms (Schuster, R. and Holzhütter, H.-G. (1995) Eur. J. Biochem. 229, 403-418). Therefore, the selectivity of drugs inhibiting these enzymes may be much higher than expected from their molecular effects alone. Quite unexpectedly, trypanosomes seem to possess a substantial overcapacity of hexokinase, phosphofructokinase, and pyruvate kinase, making these "irreversible" enzymes mediocre drug targets.     

Immobilized glyceraldehyde-3-phosphate dehydrogenase forms a complex with phosphoglycerate kinase

Yeast glyceraldehyde-3-phosphate dehydrogenase (GPDH) covalently attached to CNBr-activated Sepharose 4B was shown to be capable of binding soluble yeast phosphoglycerate kinase (PGK) in the course of incubation in the presence of an excess of 1,3-diphosphoglycerate. The association of the matrix-bound and soluble enzymes also occurred if the kinase was added to a reaction mixture in which the immobilized glyceraldehyde-3-phosphate dehydrogenase, NAD, glyceraldehyde-3-phosphate and Pi had been preincubated. Three kinase molecules were bound per a tetramer of the immobilized dehydrogenase and one molecule per a dimer. An immobilized monomer of glyceraldehyde-3-phosphate dehydrogenase was incapable of binding phosphoglycerate kinase. The matrix-bound bienzyme complexes were stable enough to survive extensive washings with a buffer and could be used repeatedly for activity determinations. Experimental evidence is presented to support the conclusion that 1,3-diphosphoglycerate produced by the kinase bound in a complex can dissociate into solution and be utilized by the dehydrogenase free of phosphoglycerate kinase.     

Kinetic misinterpretation of a coupled enzyme reaction can lead to the assumption of an enzyme-enzyme interaction. The example of 3-phospho-D-glycerate kinase and glyceraldehyde-3-phosphate dehydrogenase couple

The time course of the conversion of 3-phospho-D-glycerate (GriP) to glyceraldehyde-3-phosphate (GraP) catalyzed by 3-phospho-D-glycerate kinase (GriP kinase) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH) couple has been reinvestigated. The dependence of the steady-state rate on the dehydrogenase concentration is fully compatible with the consecutive nature of the reaction and therefore is not necessarily related to a complex formation of the two enzymes. To derive a Kd value of a bienzyme complex, as was done by Sukhodolets et al. [Sukhodolets, M. V., Muronetz, V. I. & Nagradova, N. K. (1987) Biochem. Int. 15, 373-379], is basically erroneous. In contrast with some previous reports, the maximal activity of GriP kinase is not influenced by the auxiliary enzyme present in the coupled assay system. Thus, no special accelerating effect can be attributed to GraPDH. 1,3-Bisphospho-D-glycerate (GriP2) bound to GriP kinase does not seem to be a substrate for GraPDH, providing evidence against channelling of GriP2 between the two enzymes.     

Double mixing technique for linked enzyme assays

A rapid double mixing technique has been applied to the reaction kinetics of enzymes usually analyzed by linked assay. The test enzyme is allowed to perform in the absence of the linking enzyme but then the convenience of enzymatic analysis of accumulated produce is exploited when a second mixing adds the linking enzyme. The analytical method is presented which enables the determination of the rate of product accumulation during the delay between mixings. Various advantages of the system in both steady-state and transient kinetic analyses are described. As an example, 3-phosphoglycerate kinase is assayed. The results address the proposal that the kinase interacts with the usual linking enzyme, glyceraldehyde-3-phosphate dehydrogenase, and also raise questions about product inhibition.     

Activation of Glyceraldehyde-Phosphate Dehydrogenase (NADP) and Phosphoribulokinase in Phaseolus vulgaris Leaf Extracts Involves the Dissociation of Oligomers

Phosphoribulokinase (EC 2.7.1.19, ATP: d-ribulose-5-phosphate-1-phosphotransferase) resembles the NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13, d-glyceraldehyde-3-phosphate: NADPH(+) oxidoreductase [phosphorylating]) of chloroplasts in that the activation of both of these enzymes involves the dissociation of oligomers (apparently tetrameric forms) with low catalytic activity to give protomers which possess higher catalytic activity. Gel filtration on Sepharose 6B has shown that the molecular weights of the oligomer and active protomer of phosphoribulokinase are, respectively, about 6.8 x 10(5) and 1.7 x 10(5), whereas the corresponding values for glyceraldehyde-3-phosphate dehydrogenase are 8.2 x 10(5) and 2.2 x 10(5). Activation of both enzymes occurs in response to either ATP, dithiothreitol, or cholate while the glyceraldehyde-3-phosphate dehydrogenase is also activated by NADPH. Activation/dissociation of these enzymes may involve conformational changes resulting from nucleotide binding, the reduction of sulfur bridges, and the cholate induced loosening of hydrophobic interactions.     

The activation of glycolysis performed by the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the model system

Influence of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) on glycolysis was investigated. The addition of GAPN-which oxidizes glyceraldehyde-3-phosphate directly to the 3-phosphoglyceric acid-led to the strong increase in the rate of lactate accumulation in the rat muscle extract with low ADP content. The lactate accumulation was also observed in the presence of GAPN in rat muscle extract, which contained only ATP and no ADP. This can be the evidence of the "futile cycle" stimulated by GAPN. Here ADP can be regenerated from ATP by the phosphoglycerate kinase reaction. The high resistance of GAPN from Streptococcus mutans towards inactivation by natural oxidant-H(2)O(2) was showed. This feature distinguishes GAPN from phosphorylating glyceraldehyde-3-phosphate dehydrogenase, which is very sensitive to modification by hydrogen peroxide. A possible role of the oxidants and non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase in the regulation of glycolysis is discussed.