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PubMed1979

Suicide inactivation of bacterial cystathionine gamma-synthase and methionine gamma-lyase during processing of L-propargylglycine.

L-Propargylglycine, a naturally occurring gamma, delta-acetylenic alpha-amino acid, induces mechanism-based inactivation of two pyridoxal phosphate dependent enzymes of methionine metabolism: (1) cystathionine gamma-synthease, which catalyzes a gamma-replacement reaction in methionine biosynthesis, and (2) methionine gamma-lyase, which catalyzes a gamma-elimination reaction in methionine breakdown. Biphasic pseudo-first-order inactivation kinetics were observed for both enzymes. Complete inactivation is achieved with a minimum molar ratio ([propargylglycine]/[enzyme monomer]) of 4:1 for cystathionine gamma-synthase and of 8:1 for methionine gamma-lyase, consistent with a small number of turnovers per inactivation event. Partitioning ratios were determined directly from observed primary kinetic isotope effects. [alpha-2H]Propargylglycine displays kH/kD values of about 3 on inactivation half-times. [alpha-3H]-Propargylglycine gives release of tritium to solvent nominally stoichiometric with inactivation but, on correction for the calculated tritium isotope discrimination, partition ratios of four and six turnovers per monomer inactivated are indicated for cystathionine gamma-synthase and methionine gamma-lyase, respectively. The inactivation stoichiometry, using [alpha-14C]-propargylglycine, is four labels per tetramer of cystathionine gamma-synthase but usually only two labels per tetramer of methionine gamma-lyase (half-of-the-sites reactivity). Two-dimensional urea isoelectrofocusing/NaDodSO4 electrophoresis suggests (1) that both native enzymes are alpha 2 beta 2 tetramers where the subunits are distinguishable by charge but not by size and (2) that, while each subunit of a cystathionine gamma-synthase tetramer becomes modified by propargylglycine, only one alpha and one beta subunit may be labeled in an inactive alpha 2 beta 2 tetramer of methionine gamma-lyase. Steady-state spectroscopic analyses during inactivation indicated that modified cystathionine gamma-synthase may reprotonate C2 of the enzyme--inactivator adduct, so that the cofactor is still in the pyridoxaldimine oxidation state. Fully inactivated methionine gamma-lyase has lambda max values at 460 and 495 nm, which may represent conjugated pyridoximine paraquinoid that does not reprotonate at C2 of the bound adduct. Either species could arise from Michael-type addition of an enzymic nucleophile to an electrophilic 3,4-allenic paraquinoid intermediate, generated initially by propargylic rearrangement upon a 4,5-acetylenic pyridoximine structure, as originally proposed for propargylglycine inactivation of gamma-cystathionase [Abeles, R., & Walsh, C. (1973) J. Am. Chem. Soc. 95, 6124]. It is reasonable that cystathionine gamma-synthase is the major in vivo target for this natural acetylenic toxin, the growth-inhibitory effects of which are reversed by methionine.

Alkynes↗

PubMed1979

Inactivation of delayed outward current in molluscan neurone somata.

1. Inactivation of delayed outward current was studied by voltage clamp of isolated neurone somata of the molluscs Archidoris and Anisodoris. During prolonged voltage clamp steps in normal artificial sea water delayed outward current rises to a peak and then declines to a non-zero steady-state. During repetitive clamp pulses at repetition rates slower than 2/sec, the amplitude of peak outward current in the second pulse is commonly less than the amplitude at the end of the preceding pulse, giving the impression of continued inactivation during the repolarized interval. We have termed this property cumulative inactivation. 2. Two components of delayed outward current were separated using tetraethyl ammonium ions (TEA) and cobalt ions (Co). External TEA blocks 90% of a voltage and time dependent outward current termed K current (IK). External Co blocks 85% of a Ca activated delayed outward current termed Ca current (ICa does not inactivate during prolonged or repetitive voltage clamp pulses. IK, however, inactivates during prolonged voltage clamp steps and shows cumulative inactivation during repetitive voltage clamp pulses. 3. Inactivation of IK is voltage and time dependent and does not require influx of Ca ions. 4. As measured by a prepulse method, the onset of inactivation is characterized by a two time constant process. Fast inactivation occurs with a time course comparable to the rate of rise of outward current and can account for 90% of total inactivation. 5. Recovery from inactivation is slow with a time constant approximately an order of magnitude slower than the onset of inactivation. 6. The current-voltage (I-V) curve for peak IK can be N-shaped, with a region of negative slope resistance in the range of +30 to +80 mV. The I-V curve for steady-state IK, however, shows little or no tendency to form a local maximum. 7. The pattern of delayed outward current varies considerably between cells. A major contributing factor to this variability appears to be the relative contributions of ICa and IK to delayed outward current.

Animals↗

PubMed1978

C-1 inactivator and cold-promoted activation of factor VII.

When exposed to temperatures between -5 degree and +5 degree C, plasma from pregnant women and from certain blood donors show shortening of the Thrombotest clotting time, kallikrein formation, and activation of blood-clotting factor VII. This phenomenon has been called cold-promoted activation of factor VII (CPA). In this study, it was found that CPA-positive plasma or serum samples which had been exposed to low temepratures showed spontaneous disappearance of C-1-inactivator activity in parallel to the shortening of the Thrombotest clotting time. C-1-inactivator antigen was not affected by storage at 4 degree C. In these CPA-possitive samples the loss of C-1-inactivator activity is caused partially by the formation of kallikrein at this temperature because when kallikrelin was added to C-1 inactivator, the latter was inactive when tested in the esterolytic assay. The formation of Hageman factor fragments may add to further loss. Purified C-1 inactivator effectively inhibited the CPA phenomenon, whereas alpha2-macroglobulin did so only weakly. This finding indicates that during exposure of CPA-positive plasma samples to low temperatures, Hageman factor fragments, which are inhibited only by C-1 inactivator, induce the activation of the kallikrein system and blood clotting factor VII. The reported lowered activity of C-1 inactivator in pregnancy is probably an artifact caused by generation of CPA during storage, since in fresh samples the levels were compeletely normal. Similarly, various subjects classified as belonging to the variant type of HANE (low C-1-inactivator activity with a normal antigen content) were found to have normal C-1-inactivator activity when determinations were made on fresh instead of frozen samples. It is recommended that plasma or serum samples should not be exposed to temperatures between -5degree and +5degree C prior to the determination of C-1-inactivator activity. Moreover, during purification procedures of kallikrein-binding antiproteases such as C-1 inactivator and also alpha2-macroglobulin, the occurrence of CPA should be avoided by the use of CPA-negative plasma as starting material.

Cold Temperature↗

PubMed1975

Cold inactivation of glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle.

Inactivation of apo-glyceraldehyde-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase(phosphorylating) (EC 1.2.1.12) from rat skeletal muscle at 4 degrees C in 0.15 M NaC1, 5 mM EDTA, 4 mM 2-mercaptoethanol pH 7.2 is a first-order reaction. The rate constant of inactivation depends on protein concentration. With one molecule of NAD bound per tetrameric enzyme, a 50 per cent loss in activity is observed and the rate constant of inactivation becomes independent of the protein concentration over a 30-fold range. Two moles of NAD bound per mole of enzyme fully protect it against inactivation. NADH affords a cooperative effect on enzyme structure similar to that of NAD. Inactivation of 7.8 S apoenzyme is reflected in its dissociation into 4.8-S dimers. In the case of enzyme-NAD1 complex, no direct relationship between the extent of inactivation and dissociation is observed, suggesting that these two processes do not occur simultaneously; we may say that dissociation is slower than inactivation. A mechanism in which the rate-limiting step for inactivation is a conformational change in the tetramer occurring prior to dissociation and affecting only the structure of the non-liganded dimer, is consistent with the experimental observations. Inorganic phosphate protects apoenzyme against inactivation. Its effect is shown to be due to the anion binding at specific sites on the protein with a dissociation constant of 2.6 plus or minus 0.4 mM. The NaC1-induced cold inactivation of glyceraldehyde-phosphate dehydrogenase is fully reversible at 25 degrees C in the presence of 20 mM dithiothreitol and 50 mM inorganic phosphate. The rate of reactivation is independent of protein concentration. Inactivated enzyme retains the ability to bind specific antibodies produced in rabbits, but diminishes its precipitating capability.

Animals↗

PubMed1977

Inactivation of sodium channels: second order kinetics in myelinated nerve.

1. Kinetics of inactivation of sodium channels in myelinated nerve from Rana pipiens were studied at 4.5 degrees C using the voltage clamp technique of Dodge & Frankenhaeuser (1958).2. Potassium currents were blocked by cutting the internodes in 20 mM-TEA-Cl + 100 mM-KCl and by adding 12 mM-TEA-Cl to the external Ringer. Leakage and capacitative currents were subtracted electronically.3. Kinetics of recovery from inactivation of the sodium channels were studied by inactivating the channels with a large depolarizing prepulse and allowing the channels to recover at different potentials; the extent of recovery was measured by applying a test pulse at various times after the prepulse.4. Kinetics of development of inactivation were studied by two different methods. The first was to measure the decay of sodium current under a maintained depolarization. The second method was to measure the decay of the peak sodium current in a test pulse as a function of time after the onset of a maintained depolarization. These two methods yielded similar results for the kinetics of inactivation development.5. Contrary to expectations of the Hodgkin-Huxley formalism, the time course of recovery from and development of inactivation is not strictly exponential. Rather, recovery from complete inactivation shows an initial delay which depends on recovery potentials. Development of inactivation at a fixed potential exhibits at least two exponentials.6. The steady-state inactivation curve h(infinity)(E) is asymmetrical and is fitted better by 1/[1+exp (A(1)E+B(1)) +exp (A(2)E+B(2))] than by 1/[1+exp (AE+B)].7. Most of the above kinetic observation on inactivation can be fitted by the following modification of the h system of the Hodgkin-Huxley formalism: [Formula: see text]8. In the analysis it was not necessary to modify the concept of two separate processes, activation and inactivation, governing the opening and closing of the sodium channels.

Animals↗

PubMed1978

Radiobiological inactivation of Epstein-Barr virus.

Lymphocyte transforming properties of B95-8 strain Epstein-Barr virus (EBV) are very sensitive to inactivation by either UV or X irradiation. No dose of irradiation increases the transforming capacity of EBV. The X-ray dose needed for inactivation of EBV transformation (dose that results in 37% survival, 60,000 rads) is similar to the dose required for inactivation of plaque formation by herpes simplex virus type 1 (Fischer strain). Although herpes simplex virus is more sensitive than EBV to UV irradiation, this difference is most likely due to differences in the kinetics or mechanisms of repair of UV damage to the two viruses. The results lead to the hypothesis that a large part, or perhaps all, of the EBV genome is in some way needed to initiate transformation. The abilities of EBV to stimulate host cell DNA synthesis, to induce nuclear antigen, and to immortalize are inactivated in parallel. All clones of marmoset cells transformed by irradiated virus produce extracellular transforming virus. These findings suggest that the abilities of the virus to transform and to replicate complete progeny are inactivated together. The amounts of UV and X irradiation that inactivate transformation by B95-8 virus are less than the dose needed to inactivate early antigen induction by the nontransforming P(3)HR-1 strain of EBV. Based on radiobiological inactivation, 10 to 50% of the genome is needed for early antigen induction. Inactivation of early antigen induction is influenced by the cells in which the assay is performed. Inactivation proceeds more rapidly in EBV genome-free cells than in genome carrier Raji or in P(3)HR-1 converted EBV genome-free cells clone B(1). These results indicate that the resident EBV genome participates in the early antigen induction process. Variation in radio-biological killing of B95-8 and P(3)HR-1 EBV is not attributable to variations in the repair capacities of the cells in which the viruses were assayed, since inactivation of HSV was the same in primary lymphocytes and in all lymphoid cell lines tested.

Animals↗

PubMed1977

Mechanisms of inactivation of bacteriophage phiX174 and its DNA in aerosols by ozone and ozonized cyclohexene.

The mechanisms of inactivation of aerosolized bacteriophage phiX174 in atmospheres containing ozone, cyclohexene, or ozonized cyclohexene were studied by using 32P-labelled phage. The inactivation of the aerosolized phage in clear air or in air containing cyclohexene is due to damage of the protein coat since the deoxyribonucleic acid (DNA) extracted from the inactivated phage retains its biological activity. Inactivation of the phage in air containing ozonized cyclohexene is due both to protein and DNA damage. Sucrose gradient analysis shows that aerosolized inactivated phiX174 releases unbroken DNA. In contrast, the DNA from phage phiX174 inactivated by ozonized cyclohexene is broken. The inactivation of aerosolized phage phiX174-DNA was studied in the same atmospheres using 32P-labelled DNA. phiX174-DNA aerosolized in clear air or air containing cyclohexene at 75% r.h. is inactivated by a factor of 2 in 30 min. The inactivated DNA is broken. Ozone as well as ozonized cyclohexene inactivates KNA very fast causing breaks in the molecule. This is in contrast with the intact bacteriophage in which ozone does not produce breaks in the DNA.

Aerosols↗

PubMed1975

Inactivation of rabbit, pig, and carp adenylate kinases by N6-o- and p-fluorobenzoyladenosine 5'-triphosphates.

N6-O- and p-fluorobenzoyladenosine 5'-triphosphates (IIIc and IIc, respectively) have been synthesized as potential adenosine 5'-triphosphate (ATP) site-directed reagents for enzymes. IIc and IIIc were substrates of yeast hexokinase; neither they nor the corresponding ADP derivatives inactivated yeast hexokinase or rabbit pyruvate kinase. IIc rapidly inactivated rabbit and carp muscle adenylate kinases; the effect is probably ATP site directed because N6-benzoyl-ATP did not inactivate and was a substrate (Vmax = 28 and 10%, respectively, that of ATP), and because of ATP retarded the inactivation. The inactivations followed pseudo-firsr-order kinetics; in the presence of 2.64 mM ATP at 0 degrees the half-life of the rabbit kinase was 210 min with 50 muM IIc and the half-life of the carp kinase was 130 min with 100 muM IIc. Adenylate kinase of pig muscle was inactivated by IIc in a manner similar to the rabbit and carp enzymes except that the rate of inactivation exhibited an inflexion. IIIc inactivated rabbit, pig, and carp adenylate kinases by pseudo-first-order kinetics; the rate constants for inactivation at 0 degrees were 9.1 X 10(-3), 1.3 X 10(-3), and 1.9 X 10(-3) min-1 and the apparent dissociation constants (K) of the IIIc-enzyme complexes were 710, 970, and 720 muM, respectively. From the substrate properties of IIIc alone and in admixture with ATP its dissociation constants (Ki) from the ATP sites of the enzymes were found to be 500, 700, and 845 muM, respectively. The similarity between the K and Ki values, together with marked retardation of the inactivations by ATP, indicates that IIIc is an ATP-site-directed reagent for the three adenylate kinases.

Adenine Nucleotides↗

PubMed1977

Inactivation of the sodium channel. II. Gating current experiments.

Gating current (Ig) has been studied in relation to inactivation of Na channels. No component of Ig has the time course of inactivation; apparently little or no charge movement is associated with this step. Inactivation nonetheless affects Ig by immobilizing about two-thirds of gating charge. Immobilization can be followed by measuring ON charge movement during a pulse and comparing it to OFF charge after the pulse. The OFF:ON ratio is near 1 for a pulse so short that no inactivation occurs, and the ratio drops to about one-third with a time course that parallels inactivation. Other correlations between inactivation and immobilization are that: (a) they have the same voltage dependence; (b) charge movement recovers with the time coures of recovery from inactivation. We interpret this to mean that the immobilized charge returns slowly to "off" position with the time course of recovery from inactivation, and that the small current generated is lost in base-line noise. At -150 mV recover is very rapid, and the immobilized charge forms a distinct slow component of current as it returns to off position. After destruction of inactivation by pronase, there is no immobilization of charge. A model is presented in which inactivation gains its voltage dependence by coupling to the activation gate.

Action Potentials↗

PubMed1979

Biphasic time course of inactivation of potassium contractures in single twitch muscle fibers of the frog.

The time course of the inhibition of peak tension (inactivation) of potassium contracture induced by conditioning depolarization with 10, 15, 20 and 30 mM K+ was examined, using single twitch fibers from the frog semitendinosus muscle. The time-dependence curve of the inactivation was biphasic, consisting of the first phase and the second phase. The first phase was characterized as follows: 1) the time course and the extent depend on the degree of conditioning depolarization; 2) the inactivation proceeds exponentially and finally reaches a steady level; and 3) the rate is markedly increased by lowering Ca2+ in the external medium. It could be considered that the first phase of the inactivation is an analogical phenomenon with the inactivation of sodium conductance in a squid giant axon. The second phase of the inactivation induced by conditioning with 20 and 30 mM K+ reached its full inactivation independently of the degree of conditioning depolarization. On the basis of these results, the terms inactivation 1 and inactivation 2 were given to the first phase and second phase, respectively, of the time-dependence curve of the inactivation of potassium contracture.

Animals↗

PubMed1976

Studies on the fatty acid inactivation of phosphofructokinase.

Investigation of phosphofructokinase in normal and regenerating livers led to the discovery of an inactivating factor in the extracts of these livers. The inactivating factor was found to be a mixture of free fatty acids. The fatty acid compositions of the normal and regenerating livers are the same, but the concentrations of most of the fatty acids are at least 3 to 4 times higher in the latter. Inactivation of phosphofructokinase by palmitate and oleate was investigated using purified rabbit muscle enzyme. Incubation of the enzyme with palmitate (250 muM) or oleate (50 muM) resulted in rapid inactivation of the enzyme with biphasic curves. The concentrations of oleate and palmitate required to produce 50% inactivation of the enzyme were 35 muM and 75 muM, respectively. Fructose-6-P (0.5 mM), MgATP, (1 mM), fructose-1,6-P2 (1 mM), AMP (1 mM), and cyclic adenosine 3':5'-monophosphate (20 muM) protected the enzyme against inactivation when these metabolites were incubated with the enzyme before the addition of fatty acid. Bovine serum albumin (100 muM) and beta-cyclodextrin (0.25 mM) also protected the enzyme against the inactivation. However, if the enzyme was inactivated by fatty acid, subsequent addition of the above metabolites or bovine serum albumin did not reactivate the enzyme. Binding studies with [3H]oleate revealed at least three types of binding sites. The first site binds 2 to 4 mol of oleate/mol of enzyme. Oleate binding to this site did not seem to affect the enzyme activity. The second binding site binds 5 to 15 mol of oleate/mol of enzyme resulting in complete loss of the activity. This is followed by an increase in oleate binding to the third site of the enzyme. Sucrose density gradient centrifugation of oleate-inactivated enzyme indicated that the enzyme dissociated to the dimeric form. Similarly, centrifugation of [3H]oleate-treated enzyme revealed that all polymeric forms of phosphofructokinase bound approximately 6 to 8 mol of oleate/mol of enzyme. In the presence of fructose-6-P, oleate is bound to the polymers to a lesser degree and therefore protects against the fatty acid inactivation. Various polymers which are cross-linked with dimethylsuberimidate are also inhibited by oleate.

Adenosine Monophosphate↗

PubMed1977

Inactivation of bacterial D-amino acid transaminases by the olefinic amino acid D-vinylglycine.

D-Vinylglycine (2-amino-3-butenoate) functions as a transamination substrate and irreversible inactivator of the homogeneous pyridoxal phosphate-dependent D-amino acid transaminases from Bacillus subtilis and Bacillus sphaericus. In the absence of alpha-ketoglutarate as co-substrate, vinyl-glycine causes little if any inactivation of either enzyme; in the presence of excess alpha-ketoglutarate, both enzymes are inactivated with pseudo-first order kinetics. The limiting rate constant for inactivation of the B. sphaericus enzyme is 1.9 min-1, for the B. subilis enzyme it is 0.36 min-1. The number of catalytic events before inactivation is about 450 for the B. sphaericus enzyme and about 800 for the B. subtilis enzyme; that is, about 0.2% inactivation in each catalytic cycle for the former enzyme and 0.15% for the latter. Comparisons are made with the L-aspartate amino-transferase from pig heart which is inactivated completely in one catalytic cycle and the L-alanine aminotransferase which is not inactivated in many cycles. Comparisons are also made between the likely mode of D-transaminase inactivation produced by vinylglycine and the mode of inactivation induced by beta-chloro-D-alanine.

Amino Acids↗

PubMed1976

Specific, reversible inactivation of phosphofructokinase by fructose-1,6-bisphosphatase. Involvement of adenosine 5'-triphosphate, oleate, and 3-phosphoglycerate.

Optimal conditions necessary for the reversible inactivation of crystalline rabbit muscle phosphofructokinase by homogeneous rabbit liver fructose-1,6-bisphosphatase have been studied. At higher enzyme levels (to 530 mug/ml of phosphofructokinase) the two proteins were mixed and incubated in a pH 7.5 buffer composed of 50 mM Tris-HC1, 2 mM potassium phosphate, and 0.2 mM dithiothreitol. Aliquots were removed at various times and assayed for enzyme activity. A time dependent inactivation of phosphofructokinase caused by 1-2.3 times its weight of fructose-1,6-bisphosphatase was observed at 30, 23, and 0 degree C. This inactivation did not require the presence of adenosine 5'-triphosphate or Mg2+ in the incubation mixture, but an adenosine 5'-triphosphate concentration of 2.7 mM or greater was required in the assay to keep phosphofructokinase in an inactive form. A mixture of activators (inorganic phosphate, (NH4)2SO4, and adenosine 5'-monophosphate), when added to the assay cuvette, restored nearly all of the expected enzyme activity. Incubations with other proteins, including aldolase, at concentrations equal to or greater than the effective quantity of fructose-1,6-bisphosphatase had no inhibitory effect on phosphofructokinase activity. Removal of tightly bound fructose 1,6-bisphosphate from phosphofructokinase could not explain this inactivation, since several analyses of crystalline phosphofructokinase averaged less than 0.1 mol of fructose 1,6-bisphosphate/320 000 g of enzyme. Furthermore, the inactivation occurred in the absence of Mg2+ where the complete lack of fructose-1-6-bisphosphatase activity was confirmed directly. At lower phosphofructokinase concentrations (0.2-2 mug/ml) the inactivation was studied directly in the assay cuvette. Higher ratios of fructose-1,6-bisphosphatase to phosphofructokinase were necessary in these cases, but oleate and 3-phosphoglycerate acted synergistically with lower amounts of fructose-1,6-bisphosphatase to cause inactivation. The inactivation did not occur when high concentrations of fructose 6-phosphate were present in the assay, or when the level of adenosine 5'-triphosphate was decreased. However, the inactivation was found at pH 8, where the effects of allosteric regulators on phosphofructokinase are greatly reduced. Experiments with rat liver phosphofructokinase showed that this enzyme was also subject to inhibition by rabbit liver fructose 1,6-bisphosphatase under conditions similar to those used in the muscle enzyme studies. Attempts to demonstrate direct interaction between phosphofructokinase and fructose-1,6-bisphosphate by physical methods were unsuccessful. Nevertheless, our results suggest that, under conditions which approximate the physiological state, the presence of fructose-1,6bisphosphatase can cause phosphofructokinase to assume an inactive conformation. This interaction may have a significant role in vivo in controlling the interrelationship between glycolysis and gluconeogenesis.

Adenosine Triphosphate↗

PubMed1975

Catabolite inactivation of biodegradative threonine dehydratase of Escherichia coli.

Incubation of Escherichia coli cells with glucose, pyruvate, and certain other metabolites led to rapid inactivation of inducible biodegradative threonine dehydratase. Analysis with several mutant strains showed that pyruvate, and not a metabolite derived from pyruvate, was capable of inactivating enzyme, and that glucose acted indirectly after being converted to pyruvate. Some other alpha-keto acids such as oxaloacetate and alpha-ketobutyrate (but not alpha-ketoglutarate) were also effective. Inactivation of threonine dehydratase by pyruvate was also observed with purified enzyme preparations. The rates of enzyme inactivation increased with increased concentrations of pyruvate and decreased with increased levels of AMP. Increasing protein concentrations lowered the rates of enzyme inactivation. Dithiothreitol had a large effect on the maximum extent of inactivation of the enzyme by pyruvate; high concentrations of AMP and DTT almost completely counteracted the effect of pyruvate. Gel filtration data showed that pyruvate influenced the oligomeric state of the enzyme by altering the association-dissociation equilibrium in favor of dissociation; the Stokes' radius of the pyruvate-inactivated enzyme was 32 A as compared to 42 A for the untreated enzyme. Reassociation of the dissociated form of the enzyme was achieved by removal of excess free pyruvate by dialysis against buffer supplemented with AMP and DTT. Incubation of threonine dehydratase with [14-C]pyruvate revealed apparent covalent attachment of pyruvate to the enzyme. Strong protein denaturants such as guanidine, urea, and sodium dodecyl sulfate failed to release bound radioactive pyruvate; the molar ratio of firmly bound pyruvate was approximately 1 mol/150,000 g of protein. Pretreatment of the enzyme with p-chloromercuribenzoate and 5,5'-dithiobis(2-nitrobenzoate) (Nbs2) did not reduce the binding of [14-C]pyruvate suggesting no active site SH was involved in the pyruvate-enzyme linkage. Titration of active and pyruvate-inactivated enzyme with Nbs2 indicated that the loss in enzyme activity was not due to oxidation of essential sulfhydryl groups on the enzyme. Based on these data we propose that the mechanism of enzyme inactivation by pyruvate involves covalent attachment of pyruvate to the active oligomeric form of the enzyme followed by dissociation of the oligomer to yield inactive enzyme.

Adenosine Monophosphate↗

PubMed1976

The structure and function of acid proteases. IV. Inactivation of the acid protease from Mucor pusillus by acid protease-specific inhibitors.

Mucor pusillus acid protease was rapidly inactivated with 1 : 1 stoichiometry by reaction with diazoacetyl-DL-norleucine methyl ester (DAN) in the presence of cupric ions. Cupric ions were essential for this inactivation. The rate of inactivation was maximal at around pH 6 when the enzyme was mixed with DAN and cupric ions without prior mixing of the reagents, and at pH 5.3 when DAN and cupric ions were mixed and incubated before addition to the enzyme solution. In both cases, the rate of inactivation decreased as the pH was either increased or decreased. The amino acid composition of an acid hydrolysate of the DAN-Modified enzyme was indistinguishable from that of the native enzyme except for the incorporation of about one norleucine residue per molecule of protein. The enzyme was also inactivated by reaction with 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). At the stage of about 90% inactivation, 1.50 residues of EPNP were incorporated per molecule of protein and the rate of inactivation followed pseudo-first order kinetics. The optimal pH for the inactivation was pH 3.0 and the rate of inactivation decreased as the pH was either increased or decreased. Furthermore, the enzyme was strongly inhibited by pepstatin, and the reactions of DAN and of EPNP was also inhibited significantly by prior treatment of the enzyme with pepstatin. These results suggest that the enzyme may have two essential carboxyl groups at the active site, one reactive with DAN in the presence of cupric ions and the other with EPNP, and that pepstatin binds part of the active site to inhibit the reactions with DAN and EPNP as well as the enzyme activity.

Aminocaproates↗

PubMed1976

The structure and function of ribonuclease T1. XX. Specific inactivation of ribonuclease T1 by reaction with tosylglycolate.

1. Ribonuclease T1 [EC 3.1.4.8] was inactivated by reaction with tosylglycolate (carboxymethyl rho-toluenesulfonate). At pH 5.5 and 8.0, alkylation of the gamma-carboxyl group of glutamic acid-58 appeared to be the predominant reaction and the major cause of inactivation by tosylglycolate, as in the case of the iodoacetate reaction, although the rate of inactivation was slower than that by iodoacetate. At pH 8.0, histidine residues were also alkylated to some extent. 2. The maximal rate of inactivation was observed at around pH 5.5 and the pH dependence of the rate of inactivation suggested the implication of two groups in the reaction, with apparent pKa values of about 3-4 (possibly histidine residue(s)). 3. In the presence of substrate analogs, ribonuclease T1 was markedly protected from inactivation by tosylglycolate at pH 5.5. The extent of protection corresponded to the binding strength of the substrate analog, except for guanosine. Ribonuclease T1 was much less protected from inactivation by guanosine than by 3'-AMP or 3'-CMP, which has a lower binding strength toward ribonuclease T1. This may indicate that glutamic acid-58 is situated in the catalytic site, at which the phosphate moiety of these nucleotides directly interacts. 4. Enzyme which had been extensively inactivated with tosylglycolate at pH 5.5 scarcely reacted with iodoacetate at pH 5.5, suggesting that these reagents react at the same site, i.e. glutamic acid-58. On the other hand, enzyme which had been inactivated almost completely with tosylglycolate at pH 8.0 still reacted with iodoacetate to some extent at pH 8.0, and the modes of reaction of tosylglycolate and iodoacetate toward ribonuclease T1 appeared to be somewhat different.

Adenosine Monophosphate↗

PubMed1977

Chemotactic factor inactivators of human granulocytes.

During phagocytosis, neutrophils release a variety of substances that include activators and inactivators of chemotactic factors. It is generally considered that these represent hydrolytic enzymes. Elastase and cathepsin G, major proteases released from lysosomal granules during phagocytosis, contain broad hydrolytic activity. This study examined granule elastase and cathepsin G for their role as inactivators of chemotactic factors. Elastase and cathepsin G were purified from human neutrophils by Trasylol-Sepharose and CM-cellulose chromatography. Small amounts (approximately equal to 3 microgram, 1 muM) of elastase and cathepsin G, comparable to quantities released by 10(6) neutrophils during phagocytosis, completely inactivated the C5 chemotactic factor generated in human serum. Larger concentrations were needed to inactivate the C3 chemotactic factor, and when the bacterial chemotactic factor from Escherichia coli was employed, five times more elastase or cathepsin G was ineffective against this chemotactic factor. Supernatant fluid from human neutrophils that had ingested complement-coated zymosan particles contained elastase and cathepsin G and had inactivator activity for both the C5 chemotactic fragment and the bacterial factor. A specific inhibitor of elastase largely abolished the inactivator activity in the phagocytic supernates that was directed against C5 factor but did not affect the inactivator activity for the bacterial factor. Similar results occurred in studies of granule lysates. These data indicate heterogeneity in the chemotactic factor inactivator activity released by phagocytosing neutrophils. The predominant inactivator activity of the C5 chemotactic fragment is attributable to elastase and cathepsin G.

Cathepsins↗

PubMed1976

N-substituted maleimide inactivation of the response to taste cell stimulation.

N-Ethylmaleimide (NEM) irreversibly inactivates the response of gustatory cells to stimulation by NaCl, sucrose and hydrogen ions. The rate of inactivation can be measured by monitoring the decay of NaCl-stimulated summated electrophysiological activity at the chorda tympani nerve in the presence of NEM. The observed pseudo first-order rate constants are linear with NEM concentration, and the second-order rate constant is 0.38 M-1 sec-1. Other N-substituted maleimides, such as N-methylmaleimide and N-butylmaleimide, which have ether:water partition coefficient and is essentially ineffective as an inactivator of the NaCl response. These results, together with the observation that the inactivation rate is independent of pH between 4.5 and 7.0, indicate the inactivation site is either intracellular or buried within the cell membrane at a locus inaccessible to most extracellular fluids. The rate of inactivation of the sucrose and HCl responses were measured indirectly and found to be comparable to the NaCl-stimulated inactivation rate, indicating the inhibited event is common to the transduction of the response for all of the stimuli examined. Possible sites of inactivation by N-substituted maleimides are considered in the context for and characterizing receptor-specific as well as other classes of taste cell inhibitors.

Animals↗