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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↗

PubMed1977

Conformational changes associated with the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase.

Crystalline ribulose-1,5-bisphosphate carboxylase-oxygenase (3-phospho-D-glycerate carboxy-lyase (dimerising), EC 4.1.1.39) isolated from tobacco (Nicotiana tabacum L.) leaf homogenates is partially inactivated by cold treatment and fully reactivated by simple heating in the absence of sulfhydryl reagents and effectors. Since the reversible cold inactivation of this bifunctional enzyme does not involve a gross change in the association state of subunits, a subtle conformational change induced by low temperatures was implicated (Chollet, R. and Anderson, L.L. (1976) Arch. Biochem. Biophys. 176, 344-351). Chemical modification of the cold-inactivated and heat-reactivated enzymes by 5,5'-dithiobis-(2-nitrobenzoate) and p-mercuribenzoate at 25 degrees C revealed no difference in the number of free -SH groups per mol protein. However, the reactivity of the sulfhydryl residues on the inactivated protein was considerably greater than that of the reactivated enzyme. Pretreatment of the two proteins with sodium dodecyl sulfate completely abolished the difference in -SH reactivity, indicating its dependence on protein conformation. Both the cold-inactivated and heat-reactivated enzymes enhanced the fluorescence intensity of 8-anilino-1-naphthalenesulfonate (ANS) and caused a blue shift of the emission maximum from 510 to 472 nm. When the inactivated enzyme was reactivated by heating, the increase in catalytic activity was closely paralleled by a concomitant decrease in the fluorescence intensity of the ANS - protein complex at 25 degrees C. Fluorescence titration experiments revealed that the decrease in fluorescence intensity accompanying heat reactivation of the inactivated enzyme was due to a reduction in the number of hydrophobic sites available for ANS binding rather than to a change in the dissociation constant of the ANS - protein complex. These results indicate that the reversible cold inactivation of ribulose-1,5-bisphosphate carboxylase-oxygenase is associated with a reversible change in the conformation of the protein. This cold-induced conformational change resluts in a greater exposure of sulfhydryl groups and hydrophobic regions to the external environment and is closely paralleled by changes in the catalytic activity of the protein. By analogy to other oligomeric enzymes also subject to reversible cold inactivation, perhaps low temperatures induce a partial dissociation of the octameric structure of the hydrophobic catalytic subunits, but complete dissociation is arrested in some unknown manner by the small hydrophilic subunits.

Anilino Naphthalenesulfonates↗

PubMed1977

Active site specific inactivation of chymotrypsin by cyclohexyl isocyanate formed during degradation of the carcinostatic 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea.

Prolonged incubation of 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea with chymotrypsin resulted in covalent modification and concomitant inactivation of chymotrypsin via degradation of the nitrosourea to form cyclohexyl isocyanate. Cyclohexyl isocyanate was shown to be an active-site-specific inactivator of chymotrypsin. A cyclohexyl isocyanate to enzyme molar ratio of 0.63 was required to produce 50% enzyme inactivation, thus demonstrating the high specificity of inactivation. At 2.38 X 10(-4) M chymotrypsin this near stoichiometric inactivation was not significantly affected by the presence of 1, 5, and 10 mM L-lysine. Degradation of an excess of 1-(2-chloroethyl)-3-([1-14C]-cyclohexyl)-1-nitrosourea in the presence of enzyme yielded 1.11 +/- 0.07 mol of covalently bound [14C]cyclohexyl moiety per mol of enzyme inactivated. Short-term incubation demonstrated that the nitrosourea neither inhibited nor protected the enzyme from cyclohexyl isocyanate inactivation. Treatment of chymotrypsin with less than stoichiometric amounts of cyclohexyl isocyanate or titration of the active-site serine with phenylmethanesulfonyl fluoride followed by in situ degradation of excess 1-(2-chloroethyl)-3-([1-14C]cyclohexyl)-1-nitrosourea resulted in a decreased amount of covalently bound 14C proportional to the extent of inactivation by these reagents prior to 14C labeling. These results strongly suggest that cyclohexyl isocyanate, whether added directly or generated by CCNU degradation, reacted almost exclusively with the active site of the enzyme. The extent of this inactivation indicates that 70% of the CCNU degraded in such a manner as to form cyclohexyl isocyanate.

Binding Sites↗

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↗

PubMed1976

Relative inactivation by Staphylococcus aureus of eight cephalosporin antibiotics.

These studies extend the recent observation that cefazolin is inactivated to a greater extent than cephaloridine by some strains of penicillinase-producing Staphylococcus aureus, whereas cephalothin undergoes little if any inactivation. In Mueller-Hinton broth (inoculum, 3 x 10(6)) 100 recently isolated strains had minimal inhibitory concentrations (MICs) </= 2 mug/ml for cephalothin and cephaloridine, whereas in Trypticase soy broth (TSB) 50% had MICs > 2 mug/ml and 10% (designated "resistant" strains) were >8 mug/ml for cephaloridine but remained </=2 mug/ml for cephalothin. A large inoculum (3 x 10(7)) of strains with high MICs in TSB almost completely inactivated 50 mug of cefazolin per ml in 6 h, with progressively less inactivation, in the following order, of cephaloridine, cephalexin, cephradine, cephapirin, and cefamandole; cefoxitin and cephalothin underwent little if any inactivation. The greater inactivation in TSB than in Mueller-Hinton broth appeared to be due to a greater production of beta-lactamases by each colony-forming unit, since the inoculum size in the two broths was not significantly different. In contrast, "susceptible" strains (MICs </= 2 mug/ml in both broths) inactivated cephaloridine more than cefazolin, and equal amounts of powdered bacterial extracts confirmed the fact that qualitatively different beta-lactamases were produced by the susceptible and resistant strains. Disk diffusion tests were unreliable in separating the two groups of staphylococci. The clinical significance of inactivation by strains with high MICs is not known but, unless susceptibility can be clearly established, cephalothin appears preferable for severe staphylococcal infections, since it undergoes little if any inactivation by any strains of staphylococci.

Cephalosporins↗

PubMed1975

Oxygen-dependent inactivation of glutamine phosphoribosylpyrophosphate amidotransferase in stationary-phase cultures of Bacillus subtilis.

Glutamine phosphoribosylpyrophosphate amidotransferase (ATase) activity is rapidly inactivated in stationary-phase cells of Bacillus subtilis. The inactivation of APase requires both the cessation of rapid cell growth and the presence of oxygen. ATase is inactivated in two protease-deficient mutant strains at a rate similar to that seen in the wild type, and is stable in anaerobic cell-free extracts of the parent strain. These results suggest that the inactivation of ATase is not the result of general proteolysis. The inactivation of ATase in stationary-phase cultures can be inhibited by oxygen starvation. This oxygen requirement does not reflect a dependence on the generation of metabolic energy, but appears to be a direct requirement for molecular oxygen. ATase synthesis is repressed by the addition of adenosine, and is inactivated only after the cessation of exponential growth. Addition of chloramphenicol or rifampin to exponential- and stationary-phase cells does not inhibit ATase inactivation, suggesting that protein or ribonucleic acid synthesis is not required for inactivation. ATase is inactivated at the end of exponential growth in cells that have exhausted a required amino acid.

Adenosine↗

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↗

PubMed1979

Catabolite inactivation of phosphoenolpyruvate carboxykinase in spheroplasts from Saccharomyces cerevisiae.

Catabolite inactivation of phosphoenolpyruvate carboxykinase was studied in yeast spheroplasts using 0.9 M mannitol or 0.6 M potassium chloride as the osmotic support. In the presence of potassium chloride the rate of catabolite inactivation was nearly the same as that occurring in intact yeast cells under different conditions of incubation. However, in the presence of mannitol, catabolite inactivation in spheroplasts was prevented. The mannitol inhibition of catabolite inactivation was released by addition of ammonium or phosphate ions. At a concentration of 0.3 M ammonium or 0.06 M phosphate ions, the maximum rate of catabolite inactivation in spheroplasts suspended in mannitol was achieved and was comparable with that observed in spheroplasts incubated in 0.6 M potassium chloride as the osmotic stabilizer. Sodium sulfate (0.04 and 0.4 M) or potassium chloride (0.06 and 0.6 M) did not release the mannitol inhibition of catabolite inactivation in spheroplasts. In intact yeast cells, 0.9 M mannitol, 0.08 M ammonium or 0.1 M phosphate ions did not influence the rate of catabolite inactivation. The nature of the effect of mannitol, ammonium and phosphate ions on catabolite inactivation in yeast spheroplasts is discussed.

Mannitol↗

PubMed1978

Differential inactivation of cephapirin vs. cephalothin by penicillin-resistant strains of Staphylococcus aureus: a preliminary study.

The bactericidal effectiveness of cephapirin and cephalothin against small (approximately 10(5)) and large (approximately 10(8)) inocula of penicillin-resistant Staphylococcus aureus was evaluated. With the smaller inoculum, no differences in bactericidal activity between the two drugs (tested at 2 and 40 microgram/ml) were seen after incubation for 2, 4, 6, or 24 hr. Neither cephalosporin effectively killed a larger inoculum in a concentration of 250 times the minimal bactericidal concentration for selected strains. Total inactivation of cephapirin (40 microgram/ml) by eight of 13 strains was demonstrated with the larger inoculum. These preliminary studies indicate that inactivation of cephapirin is pH-dependent. No strain inactivated cephapirin in less than or equal to 4 hr. Inactivation was independent of temperature at 37 C and 42 C. Although cephalothin was resistant to inactivation under the same conditions, the inoculum required to inactivate cephapirin was not killed by 100 microgram of cephalothin/ml. Although strains of S. aureus that slowly inactivate cephapirin appear to be prevalent, no strain that rapidly inactivates this cephalosporin was identified.

Cephalosporins↗

PubMed1978

Inactivation kinetics and steady-state current noise in the anomalous rectifier of tunicate egg cell membranes.

1. Inward K current through the anomalous rectifier in the tunicate egg (Halocynthis roretzi, Drashe) was studied under voltage clamp. The transient inward current in response to a step change of membrane potential was measured. The steady-state current fluctuations were analysed using the power density spectrum (p.d.s.). 2. The inward current showed time-dependent changes, which were described by a pair of the first order kinetic parameters, n and s for activation and inactivation, respectively. The steady-state channel open probability due to the activation process (n infinity) was assumed to be 1.0 for V more negative than about--100 mV, but that of the inactivation process (s infinity) and the time constant of inactivation (taus) were membrane potential dependent in the same potential range; both decreased with increasing hyperpolarization. 3. The inward currents in Na-free choline medium did not inactivate, but were decreased in size. In Na-free Li medium, inactivation was very small; the steady-state conductance was not affected significantly. 4. After exposure to high Ca media, an increase of the conductance was observed. This effect is probably caused by an increase of intracellular Ca due to Ca ions entering through the Na channels. Mg ions slightly decreased the conductance. 5. In the hyperpolarized membrane (-160 less than or equal to V less than or equal to -80mV), steady-state current noise was recorded and analysed using p.d.s. A p.d.s. of the 1/[1 + (f/fc)2] type as well a p.d.s. of the 1/f type was observed; f, frequency, fc, cut-off frequency. 6. fc was translated into time constant tauN (= 1/2pIfC) and compared with the time constant of inactivation, taus. There was a significant correlation betwen these values with a regression coefficient of 0.82. 7. Changing from 400 mM-Li abloshied inactivation and changed the p.d.s. from the 1/[1 + (f/fc)2] into the 1/f type. These results (paragraphs 5--7)suggest that the fluctuations in the steady-state currents originatte in the inactivation gatin kinetics of the an ofthe anomalous rectifier. 8. The number of anomalous rectifier channels and the unit channel conductance were estimated from the 1/[1 + (f/fc)2] type current noise according to the formula : (see text), where I infinity = gamma Nninfinity s infinity (V--VK), gamma the unit channel conductance, N the maximum number of channels that can be opened by a hyperpolarizing pulse per egg. The unit conductance was 6 pmho in standard artificial sea water and the channel density was 0.028/micrometer2. 9. The unit channel conductance (gamma) was dependent upon external K concentration, but the number ofchannels (N) was not. 10. The increase in chord conductance evoked by higher Ca concentrations was due to the increase of the channel number. By contrast, Mg ions seem to decrease the unit channel conductance slightly.

Animals↗

PubMed1976

Inactivation of carotenoid-producing and albino strains of Neurospora crassa by visible light, blacklight, and ultraviolet radiation.

Suspensions of Neurospora crassa conidia were inactivated by blacklight (BL) radiation (300 to 425 nm) in the absence of exogenous photosensitizing compounds. Carotenoid-containing wild-type conidia were less sensitive to BL radiation than albino conidia, showing a dose enhancement factor (DEF) of 1.2 for dose levels resulting in less than 10% survival. The same strains were about equally sensitive to shortwave ultraviolet (UV) inactivation. The kinetics of BL inactivation are similar to those of photodynamic inactivation by visible light in the presence of a photosensitizing dye (methylene blue). Only limited inactivation by visible light in the absence of exogenous photosensitizers was observed. BL and UV inactivations are probably caused by different mechanisms since wild-type conidia are only slightly more resistant to BL radiation (DEF = 1.2 at 1.0% survival) than are conidia from a UV-sensitive strain (upr-1, uvs-3). The BL-induced lethal lesions are probably no cyclobutyl pyrimidine dimers since BL-inactivated Haemophilus influenzae transforming deoxyribonucleic acid is not photoreactivated by N. crassa wild-type enzyme extracts, whereas UV-inactivated transforming deoxyribonucleic acid is photoreactivable with this treatment.

Carotenoids↗

PubMed1978

Modification of slow sodium inactivation in nerve after internal perfusion with trypsin.

Crayfish axons, internally perfused and held at depolarized membrane potentials, exhibit an inactivation of sodium conductance with slow kinetics. Restoration of maximum peak early currents requires prepulse hyperpolarizations of up to 1 s duration. Addition of trypsin to the internal perfusate at low concentrations (0.02 mg/ml) causes a rapid and irreversible loss of slow inactivation at the resting potential and a corresponding increase in Na currents to maximum values. After trypsin action, steady-state slow Na inactivation is shifted by 20--25 mV in the depolarizing direction, with no change in fast (h) inactivation. N-ethylmaleimide (NEM), a reagent with a high specificity for sulfhydryl groups, has been shown to induce slow inactivation, modify fast inactivation, and block a fraction of the Na conductance. After trypsin action NEM no longer increases slow Na inactivation but other effects remain. Prior exposure to NEM does not protect axons against the loss of slow inactivation caused by trypsin.

Animals↗

PubMed1979

Chemotactic factor inactivation by the myeloperoxidase-hydrogen peroxide-halide system.

Polymorphonuclear leukocytes may modulate the acute inflammatory response by the secretion of enzymes capable of inactivating mediators of inflammation. The ability of the myeloperoxidase-H(2)O(2)-halide system of the neutrophil to inactivate chemoattractants was examined using both a radioassay and a morphologic assay of chemotaxis. Incubation of either a complement-derived agent, C5a, or a synthetic formyl-methionyl peptide chemoattractant with the myeloperoxidase system for 15 min at 37 degrees C resulted in essentially complete loss of chemotactic activity. Inactivation was dependent on enzymatically active myeloperoxidase, H(2)O(2) or a peroxide-generating enzyme system, and a halide cofactor. It was blocked by agents which inhibit peroxidase (azide) or degrade H(2)O(2) (catalase). Inactivation of chemoattractants was time-dependent, reaching maximal levels within 1-5 min, and temperature-dependent with no significant inactivation occurring at 0 degrees C. H(2)O(2) alone had no significant inactivating ability at concentrations as high as 10 mM, whereas in the presence of myeloperoxidase and a halide, 0.1 muM H(2)O(2) showed significant activity and 10 muM H(2)O(2) caused complete inactivation. On a molar basis, the order of effectiveness of the halide cofactors was Br(-) > I(-) > Cl(-), although only chloride was fully active at physiologic concentrations. Neutrophils stimulated by phagocytosis or by membraneperturbing agents secrete enzymatic constituents, including myeloperoxidase, and metabolic products such as H(2)O(2). Thus, it is suggested that the myeloperoxidase system acting at an extracellular site serves as an inflammatory control mechanism by virtue of its ability to inactivate neutrophil chemoattractants.

Animals↗