Apparent Inactivation Rate Constants Research Articles

Trehalose and Sorbitol Alter the Kinetic Pattern of Inactivation of Glutamate Dehydrogenase During Drying in Levitated Microdroplets

A single-droplet acoustic levitator was used to determine the drying rate and the kinetics of inactivation of glutamate dehydrogenase in the presence of added trehalose or sorbitol. The solution was also spray dried under the same process condition of drying gas temperature on a bench-top machine. Both trehalose and sorbitol delay the point of onset of enzyme inactivation which lies after the critical point of drying. Both carbohydrates also reduce the apparent rate constant of inactivation calculated during the subsequent inactivation phase. The carbohydrates stabilise, therefore, the enzyme during droplet drying and particle formation mainly during the falling rate drying period. There is no difference between the stabilising effects of the two carbohydrates when examined as levitated single droplets. This suggests the importance of water replacement as a stabilising mechanism in the levitated droplets/particles. On spray drying, the trehalose stabilises the enzyme better than does the sorbitol at a drying gas (outlet) temperature of 60°C. This suggests glass formation with the trehalose but not the sorbitol during the very rapid drying process of small-atomised droplets in the spray dryer.

Impact of Virus Aggregation on Inactivation by Peracetic Acid and Implications for Other Disinfectants

Viruses in wastewater and natural environments are often present as aggregates. The disinfectant dose required for their inactivation, however, is typically determined with dispersed viruses. This study investigates how aggregation affects virus inactivation by chemical disinfectants. Bacteriophage MS2 was aggregated by lowering the solution pH, and aggregates were inactivated by peracetic acid (PAA). Aggregates were redispersed before enumeration to obtain the residual number of individual infectious viruses. In contrast to enumerating whole aggregates, this approach allowed an assessment of disinfection efficiency which remains applicable even if the aggregates disperse in post-treatment environments. Inactivation kinetics were determined as a function of aggregate size (dispersed, 0.55 and 0.90 μm radius) and PAA concentration (5-103 mg/L). Aggregation reduced the apparent inactivation rate constants 2-6 fold. The larger the aggregate and the higher the PAA concentration, the more pronounced the inhibitory effect of aggregation on disinfection. A reaction-diffusion based model was developed to interpret the experimental results, and to predict inactivation rates for additional aggregate sizes and disinfectants. The model showed that the inhibitory effect of aggregation arises from consumption of the disinfectant within the aggregate, but that diffusion of the disinfectant into the aggregates is not a rate-limiting factor. Aggregation therefore has a large inhibitory effect if highly reactive disinfectants are used, whereas inactivation by mild disinfectants is less affected. Our results suggest that mild disinfectants should be used for the treatment of water containing viral aggregates.

Kinetic analysis for suicide-substrate inactivation of microperoxidase-11: A modified model for bisubstrate enzymes in the presence of reversible inhibitors

Kinetics of microperoxidase-11 (MP-11) as a heme–peptide enzyme model in oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied in the presence of amino acids, taking into account the inactivation of MP-11 during reaction by its suicide substrate, H 2O 2. Reliability of the kinetic equation was evaluated by non-linear mathematical fitting. Fitting of experimental data into a new integrated kinetic relation showed a close match between the kinetic model and the experimental data. Indeed, it was found that the mechanism of suicide-peroxide inactivation of MP-11 in the presence of amino acids is different from MP-11 and/or horseradish peroxidase. In this mechanism, amino acids compete with hydrogen peroxide for the sixth co-ordination position of iron atom in the heme group through a competitive inhibition mechanism. The proposed model can successfully determine the kinetic parameters including inactivation by hydrogen peroxide as well as the inhibitory rate constants by the amino acid inhibitor. Kinetic parameters of inactivation including the initial activity of MP-11, α 0, the apparent inactivation rate constant, k i and the apparent inhibition rate constant for cysteine, k I were obtained 0.282 ± 0.006 min −1, 0.497 ± 0.013 min −1 and 1.374 ± 0.007 min −1 at [H 2O 2] = 1.0 mM, 27 °C, phosphate buffer 5.0 mM, pH 7.0. Results showed that inactivation and inhibition of microperoxidase as a peroxidase model enzyme occurred simultaneously even at low concentrations of hydrogen peroxide (0.4 mM). This kinetic analysis based on the suicide-substrate inactivation of microperoxidase-11, provides a tool and model for studying peroxidase models in the presence of reversible inhibitors. The introduced inhibition procedure can be used in designing activity tunable and specific protected enzyme models in the hidden and reversibly inhibited forms, which do not undergo inactivation.

The novel inhibitory effect of Pangdahai on fatty acid synthase

Recently, animal fatty acid synthase (FASN) is reported as a potential therapeutic target for obesity and cancer. Considerable interest has been developed in searching for novel inhibitors of this enzyme. An extract from Pangdahai has been found to inhibit FASN in both reversible and irreversible manners, with an IC(50) of 3.5 microg/ml and an apparent inactivation rate constant of k(obs) of 2.2 x 10(-3)/min. The kinetic study showed that the Pangdahai extract inhibited the overall FASN reaction uncompetitively with acetyl-CoA, but it presented in a mixed manner both with NADPH and with malonyl-CoA. Its major reacting site on this enzyme, as compared between two IC(50) values, is not in the beta-ketoacyl reduction domain. A weight reducing experiment in rats showed that the extract significantly reduced the adipose and food intake, but in view of statistics (P < 0.05), a correlation between the reductions in the adipose and in the food consumption and the inhibition of hepatic FASN could not be established. Three known flavonoid compounds were isolated from the extract and the structure-activity relationship was discussed.

Suicide-Peroxide inactivation of microperoxidase-11: A kinetic study

The kinetics of microperoxidase-11 (MP-11) in the oxidation reaction of guaiacol (AH) by hydrogen peroxide was studied, taking into account the inactivation of enzyme during reaction by its suicide substrate, H2O2. Concentrations of substrates were so selected that: 1) the reaction was first-order in relation to benign substrate, AH and 2) high ratio of suicide substrate to the benign substrate, [H2O2]>>[AH]. Validation and reliability of the obtained kinetic equations were evaluated in various nonlinear and linear forms. Fitting of experimental data into the obtained integrated equation showed a close match between the kinetic model and the experimental results. Indeed, a similar mechanism to horseradish peroxidase was found for the suicide-peroxide inactivation of MP-11. Kinetic parameters of inactivation including the intact activity of MP-11, αi, and the apparent inactivation rate constant, ki, were obtained as 0.282 ± 0.006 min− 1 and 0.497 ± 0.013 min− 1 at [H2O2] = 1.0 mM, 27°C, phosphate buffer 5.0 mM, pH = 7.0. Results showed that inactivation of microperoxidase as a peroxidase model enzyme can occur even at low concentrations of hydrogen peroxide (0.4 mM).

Interactions of Nitroaromatic Compounds with the Mammalian Selenoprotein Thioredoxin Reductase and the Relation to Induction of Apoptosis in Human Cancer Cells

Here we described novel interactions of the mammalian selenoprotein thioredoxin reductase (TrxR) with nitroaromatic environmental pollutants and drugs. We found that TrxR could catalyze nitroreductase reactions with either one- or two-electron reduction, using its selenocysteine-containing active site and another redox active center, presumably the FAD. Tetryl and p-dinitrobenzene were the most efficient nitroaromatic substrates with a k(cat) of 1.8 and 2.8 s(-1), respectively, at pH 7.0 and 25 degrees C using 50 muM NADPH. As a nitroreductase, TrxR cycled between four- and two-electron-reduced states. The one-electron reactions led to superoxide formation as detected by cytochrome c reduction and, interestingly, reductive N-denitration of tetryl or 2,4-dinitrophenyl-N-methylnitramine, resulting in the release of nitrite. Most nitroaromatics were uncompetitive and noncompetitive inhibitors with regard to NADPH and the disulfide substrate 5,5'-dithiobis(2-nitrobenzoic acid), respectively. Tetryl and 4,6-dinitrobenzofuroxan were, however, competitive inhibitors with respect to 5,5'-dithiobis(2-nitrobenzoic acid) and were clearly substrates for the selenolthiol motif of the enzyme. Furthermore, tetryl and 4,6-dinitrobenzofuroxan efficiently inactivated TrxR, likely by alkylation of the selenolthiol motif as in the inhibition of TrxR by 1-chloro-2,4-dinitrobenzene/dinitrochlorobenzene (DNCB) or juglone. The latter compounds were the most efficient inhibitors of TrxR activity in a cellular context. DNCB, juglone, and tetryl were highly cytotoxic and induced caspase-3/7 activation in HeLa cells. Furthermore, DNCB and juglone were potent inducers of apoptosis also in Bcl2 overexpressing HeLa cells or in A549 cells. Based on these findings, we suggested that targeting of intracellular TrxR by alkylating nitroaromatic or quinone compounds may contribute to the induction of apoptosis in exposed human cancer cells.

Catalytic inactivation of protein tyrosine phosphatase CD45 and protein tyrosine phosphatase 1B by polyaromatic quinones.

Polyaromatic quinones, such as the environmental pollutants 9,10-phenanthrenediones, elicit a wide range of responses including growth inhibition, immune suppression, and glucose normalization in diabetic models. Yet the molecular mechanisms behind these effects remain controversial. Here we report that many of them are oxygen-dependent and catalytic inactivators of protein tyrosine phosphatases (PTP). Under aerobic conditions, the PTP inactivation by 2-nitro-9,10-phenanthrenedione followed a pseudo-first-order process, with the rate of inactivation increasing nearly linearly with increasing inhibitor concentration, yielding apparent inactivation rate constants of 4300, 387, and 5200 M(-1) s(-1) at pH 7.2 against CD45, PTP1B, and LAR, respectively. The rate of CD45 inactivation increased approximately 25-fold from pH 6.0 to 7.5, with complete inactivation achieved using a catalytic amount (0.05 molar equiv) of the inhibitor. The quinone-catalyzed CD45 inactivation was prevented by catalase or superoxide dismutase. Inactivated CD45 after (125)I-9,10-phenanthrenedione treatment carried no radioactivity, indicating the absence of a stable inhibitor/enzyme complex. The activity of inactivated CD45 was partially restored ( approximately 10%) by hydroxylamine or dithiothreitol, supporting the presence of a small population of sulfenic acid or sulfenyl-amide species. Treatment of PTP1B with 2-nitro-9,10-phenanthrenedione resulted in the specific and sequential oxidation of the catalytic cysteine to the sulfinic and sulfonic acid. These results suggest that reactive oxygen species and the semiquinone radical, continuously generated during quinone-catalyzed redox cycling, mediate the specific catalytic cysteine oxidation. Naturally occurring quinones may act as efficient regulators of protein tyrosine phosphorylation in biological systems. Aberrant phosphotyrosine homeostasis resulting from continued polyaromatic hydrocarbon quinone exposure may play a significant role in their disease etiology.

Inactivation kinetics of mushroom tyrosinase by cetylpyridinium chloride.

Cetylpyridinium chloride (CPC) was found to inactivate tyrosinase from mushroom (Agaricus bisporus). CPC can bind to the enzyme molecule and induce the enzyme conformation changes. The fluorescence intensity (at 338.4 nm) of the enzyme decreased distinctly with increasing CPC concentrations, and a new little fluorescence emission peak appeared near 372 nm. The inactivation of the enzyme by CPC had first been studied by using the kinetic method of the substrate reaction described by Tsou. The results showed that the enzyme was inactivated by a complex mechanism that had not been previously identified. The enzyme first quickly binds with CPC reversibly and then undergoes a slow irreversible inactivation. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is a saturated trend being independent of CPC concentration if the concentration is sufficiently high. The micro rate constants of inactivation and the association constant were determined.

The Mechanism-based Inactivation of 2,3-Dihydroxybiphenyl 1,2-Dioxygenase by Catecholic Substrates

2,3-Dihydroxybiphenyl 1,2-dioxygenase (EC ), the extradiol dioxygenase of the biphenyl biodegradation pathway, is subject to inactivation during the steady-state cleavage of catechols. Detailed analysis revealed that this inactivation was similar to the O(2)-dependent inactivation of the enzyme in the absence of catecholic substrate, resulting in oxidation of the active site Fe(II) to Fe(III). Interestingly, the catecholic substrate not only increased the reactivity of the enzyme with O(2) to promote ring cleavage but also increased the rate of O(2)-dependent inactivation. Thus, in air-saturated buffer, the apparent rate constant of inactivation of the free enzyme was (0.7 +/- 0.1) x 10(-3) s(-1) versus (3.7 +/- 0.4) x 10(-3) s(-1) for 2,3-dihydroxybiphenyl, the preferred catecholic substrate of the enzyme, and (501 +/- 19) x 10(-3) s(-1) for 3-chlorocatechol, a potent inactivator of 2,3-dihydroxybiphenyl 1,2-dioxygenase (partition coefficient = 8 +/- 2, K(m)(app) = 4.8 +/- 0.7 microm). The 2,3-dihydroxybiphenyl 1,2-dioxygenase-catalyzed cleavage of 3-chlorocatechol yielded predominantly 2-pyrone-6-carboxylic acid and 2-hydroxymuconic acid, consistent with the transient formation of an acyl chloride. However, the enzyme was not covalently modified by this acyl chloride in vitro or in vivo. The study suggests a general mechanism for the inactivation of extradiol dioxygenases during catalytic turnover involving the dissociation of superoxide from the enzyme-catecholic-dioxygen ternary complex and is consistent with the catalytic mechanism.

Inhibition kinetics of green crab (Scylla serrata) alkaline phosphatase by zinc ions: a new type of complexing inhibition

The Tsou method was used to study the kinetic course of inactivation of green crab alkaline phosphatase by zinc ions. The results show that the enzyme was inactivated by a complexing scheme which has not been previously identified. The enzyme first reversibly and quickly binds Zn2+ and then undergoes a slow reversible course to inactivation and slow conformational change. The inactivation reaction is a single molecule reaction and the apparent inactivation rate constant is for a saturated reaction being independent of Zn2+ concentration if the concentration is sufficiently high. The microscopic rate constants of inactivation and the association constant were determined from the measurements.

A comparative study of the purity, enzyme activity, and inactivation by hydrogen peroxide of commercially available horseradish peroxidase isoenzymes A and C

Horseradish peroxidase (HRP) is a commercially important enzyme that is available from a number of supply houses in a variety of grades of purity and isoenzymic combinations. The present article describes a comparative study made on nine HRP preparations. Six of these samples were predominantly composed of basic HRP, pl 8.5, and three of acidic HRP, pl 3.5. Two of the basic preparations were of lower purity than the others. The apparent molar catalytic activity of basic HRP with 0.5 mMABTS and 0.2 mM H(2)O(2) was around 950 s(-1) (about 770 s(-1) for the less pure samples) and with a 5 mM guaiacol and 0.6 mM H(2)O(2) was about 180 s(-1) for all the samples. A similar value (approximately 1000 s(-1)) was observed for acidic HRP but only at higher concentrations of ABTS (20 mM). With 20 mM guaiacol the molar catalytic activity of the acid isoenzyme was 65 s(-1). The apparent K(M) for ABTS of the acidic isoenzyme was 4 mM whereas for the basic isoenzyme it was 0.1 mM. All the enzymes were inactivated by H(2)O(2) when it was supplied as the only substrate. Under these conditions the partition ratio (r = number of catalytic cycles given by the enzyme before its inactivation), apparent dissociation constant (K(l)), and apparent rate constant of inactivation (k(inact)) were about twice as large for the acidic samples (1350, 2.6 mM, 9 x 10(-3) s(-1)) as for the basic (650, 1.3 mM, 5 x 10(-3) s(-1)). The apparent catalytic constant (k(cat)) was 3-4 times larger, and the efficiency of catalysis (k(cat)/K(l)) was double for the acidic isoenzyme, but the efficiency of inactivation (k(inact)/K(l)) was similar. The data obtained provide useful information for those using HRP isoenzymes for biotechnological applications (e.g., biosensors, bioreactors, or assays).

Inactivation before significant conformational change during denaturation of papain by guanidine hydrochloride

During denaturation by GuHCl, papain shows a rapid decrease in activity with increasing concentrations of the denaturant followed by an intermediate stage of relatively little change from 1 to 2 M before complete inactivation at 4 M GuHCl. At GuHCl concentrations lower than 2 M, enzyme activity is more sensitive to GuHCl than noticeable conformation changes as followed by fluorescence and CD measurements. Kinetics of GuHCl inactivation were studied by following the substrate reaction in the presence of denaturant and the apparent rate constants thus obtained were found to be only slightly higher than those for conformational changes. However, apparent inactivation rate constants obtained in the presence of saturating concentration of substrate are actually inactivation constants for the ES complex. The inactivation rates at different substrate concentrations were, therefore, followed and the microscopic inactivation rate constants for the free enzyme obtained (Tsou, C.L. (1988) Adv. Enzymol. 61, 381–436).It was found that substrate protects strongly against inactivation and at the same GuHCl concentration, the inactivation rate of the free enzyme is 100-fold higher than that of unfolding. The above results show that the activity of papain is more sensitive to GuHCl than its overall conformation and like the enzymes previously studied in this laboratory, its active site is more flexible than the enzyme molecule as a whole.

Inactivation and phosphorylation of sarcoplasmic reticulum Ca 2+-ATPase by Mg · ATP analogues Rh(III)-ATP and Co(III)-ATP

The interactions of sarcoplasmic reticulum Ca 2+-ATPase with the Mg · ATP analogues Rh(H 2O) 4ATP and Co(NH 3) 4ATP have been examined. Co(NH 3) 4ATP slowly inactivates Ca 2+-ATPase in a first order process, with a rate constant of 1.13 × 10 −3 s −1 and an apparent inactivation constant, K I , of 32 m m. Rh(H 2O) 4ATP likewise inactivates sarcoplasmic reticulum Ca 2+-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H 2O) 4ATP] is biphasic. The x-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca 2+-ATPase by Rh(H 2O) 4ATP of K I1 = 30 μM and K I2 = 221 μM. The corresponding values of k 2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 × 10 −4 s −1. Tridentate Rh(H 2O) 3ATP also inhibits Ca 2+-ATPase, but only after much longer incubation times. Ca 2+-ATPase inactivation is accompanied by incorporation of radioactivity from γ- 32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca 2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca 2+-dependent ATPase activity in this experiment was 2.19 × 10 −4 s −1, and the first-order rate constant for Ca 2+-dependent E-P formation was 2.07 × 10 −4 s −1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca 2+-ATPase by Rh(H 2O) 4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca 2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H 2O) 4ATP is binding to the catalytic nucleotide site on Ca 2+-ATPase and producing a highly stable, phosphorylated intermediate.

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl α- d-glucopyranoside

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl α- d-glucopyranoside with an apparent rate constant of inactivation of 1.40 min −1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90μ m. Apparently two acceptor binding sites exist on dextransucrase as shown by ( i.) photolabeling the enzyme with p-azidophenyl-α- d-[5,6- 3H]glucopyranoside and ( ii.) fluorescence titration experiments.

Kinetics of trypsin inhibition by its specific inhibitors.

The kinetics of inhibition of trypsin by its specific inhibitors, pancreatic trypsin inhibitor, ovomucoid trypsin inhibitor, and soybean trypsin inhibitor, has been studied by following the hydrolysis of benzoylarginine ethyl ester in the presence of the inhibitor, and the results have been analyzed with the method described previously [Tian & Tsou (1982) Biochemistry 21, 1028]. The results obtained are consistent with the following: (a) The enzyme binds with the pancreatic inhibitor irreversibly to form an inactive complex. (b) The binding with the ovomucoid inhibitor to form the inactive complex is reversible. (c) An intermediate is formed before the relatively stable inactive complex with the soybean inhibitor, and both steps are reversible. The respective microscopic rate constants are determined by suitable plots of the apparent rate constants under different substrate and inhibitor concentrations. The second-order rate constants for the initial binding step thus obtained are in accord with the apparent inactivation rate constants determined by measuring the activity remaining with a stopped-flow apparatus equipped with a multimixing system after the enzyme-inhibitor mixture has been incubated for different time intervals.

Evidence for an essential arginine residue at the active site of Escherichia coli acetate kinase

Escherichia coli acetate kinase (ATP: acetate phosphotransferase, EC 2.7.2.1.) was inactivated in the presence of either 2,3-butanedione in borate buffer or phenylglyoxal in triethanolamine buffer. When incubated with 9.4 mM phenylglyoxal or 5.1 mM butanedione, the enzyme lost its activity with an apparent rate constant of inactivation of 0.079 min −1 and 0.051 min −1, respectively. The loss of enzymatic activity was concomitant with the loss of an arginine residue per active site. Phosphorylated substrates of acetate kinase, ATP, ADP and acetylphosphate as well as AMP markedly decreased the rate of inactivation by both phenylglyoxal and butanedione. Acetate neither provided any protection nor affected the protection rendered by the adenine nucleotides. However, it interfered with the protection afforded by acetylphosphate. These data suggest that an arginine residue is located at the active site of acetate kinase and is essential for its catalytic activity, probably as a binding site for the negatively charged phosphate group of the substrates.

Destruction of cytochrome P-450 by allylisopropylacetamide is a suicidal process

The destruction of cytochrome P-450 by allylisopropylacetamide (2-isopropyl-4-pentenamide) in microsomes from phenobarbital-pretreated rats has been shown to require oxygen, to be inhibited by NADP through inhibition of cytochrome P-450 reductase, and to be slightly stimulated by NADH. Glutathione (1 m m) does not inhibit destruction, but methyl 4,5-epoxy-2-isopropylpentanoate (5 m m), an analog of the epoxide of allylisopropylacetamide, does. The inactivation of cytochrome P-450 is both time dependent and saturable, although no more than approximately 40% of the microsomal enzyme appears to be normally destructible. However, mechanical perturbation of the microsomal suspension by rehomogenization initiates renewed destruction. Kinetic analysis shows that the destructive process is pseudo-first-order, with an apparent inactivation rate constant of 1.4 × 10 −3 s −1 and an apparent K m of 1.14 m m. Approximately 230 molecules of substrate are turned over for each destructive event. These results, in conjunction with previously reported data, clearly and unambiguously establish that inactivation of cytochrome P-450 by allylisopropylacetamide is a suicidal process.

Inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase activity in hepatoma tissue culture cells by pure cholesterol and several cholesterol derivatives. Evidence supporting two distinct mechanisms.20l.

Pure cholesterol associated in complexes with lipoproteins (whole serum and human low density lipoproteins) or esterified with succinic acid (cholesteryl succinate) and bound to albumin effectively suppresses 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activity in hepatoma tissue culture (HTC) cells grown in lipoprotein-poor serum medium during short 4-hour) incubation periods. Simultaneous measurments of the kinetics of uptake of radioactive unesterified cholesterol of whole serum and cholesteryl succinate, their conversion to lipid products, and the decay in enzyme activity, suggest that the cholesterol-induced suppression is mediated by the sterol itself rather than by inhibitory lipid products derived from its metabolism. Several cholesterol derivatives such as cholestenone, 7-ketocholesterol, and 7alpha-and 25-hydroxycholesterol also suppress reductase activiy in HTC cells and are significantly more inhibitory than the pure cholesterol preparations. The decrease in enzyme activity produced by cholesterol and its derivatives is concentration-dependent and specific. [1-14C]Oleate incorporation experiments indicate that cholesterol ester formation in HTC cells is not increased at inhibitory concentrations of the steroids. These data suggest that sterol ester formation is not an obligatory process in the feedback control of HMG-CoA reductase activity. The half-life of the reductase (3 to 4 hours) is not significantly changed by cycloheximide, plus or minus whole serum, and cholesteryl succinate. In contrast, the half-life is strongly reduced when HTC cells are incubated with cycloheximide plus maximal concentrations of 25-hydroxycholesterol, 7-ketocholesterol, or cholestenone, resulting in t1/2 values of 24, 36, and 60 min, respectively. Increasing concentrations of whole serum and cholesteryl succinate have no significant effect on the apparent rate constant of inactivation of the enzyme, whereas its apparent rate of synthesis is decreased 3- and 10-fold, respectively. These results are reversed with oxygenated steroid inhibitors. The rate of synthesis of reductase is essentially unchanged as the concentrations of 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone are increased in the culture medium, whereas the apparent rate constant for degradation is increased 9-, 7-, and 3-fold, respectively. HMG-CoA reductase activity in HTC cells thus appears to be modulated by two different mechanisms in which steroid structure is important. Whole serum and cholesteryl succinate specifically decrease the rate of enzyme synthesis, while 25-hydroxycholesterol, 7-ketocholesterol, and cholestenone increase the rate of inactivation of the reductase.

Inactivation of Soluble Bovine Milk Galactosyltransferase (Lactose Synthetase) by Sulfhydryl Reagents and Trypsin: PROTECTION BY SUBSTRATES AND PRODUCTS

Abstract Galactosyltransferase exhibited a time-dependent loss of activity when incubated with sulfhydryl reagents p-chloromercuribenzoate and N-ethylmaleimide. Though incubations were continued for periods 20-fold greater than the half-time of inactivation, neither reagent eliminated all catalytic activity, and generally 5 to 10% of the initial activity remained. p-Chloromercuribenzoate inactivated the enzme more rapidly then N-ethylmaleimide, but the inhibition by p-chloromercuribenzoate was reversed by mercaptoethanol. Substrates and substrate analogs protected against inhibition by p-chloromercuribenzoate and N-ethylmaleimide. Ligands giving the most protection against inhibition were analogs of the nucleotide substrate and product, uridine diphosphate galactose and UDP, respectively, in the presence of manganous chloride. When incubated with 10 mm N-ethylmaleimide, galactosyltransferase exhibited an apparent rate constant of inactivation of 0.023 min-1; adding analogs of UDP at concentrations of 2 mm reduced this rate by 30% and the same analogs in the presence of manganous chloride reduced the rate constant of inactivation by 98%. Inhibition of galactosyltransferase by N-ethylmaleimide resulted in a dramatic decrease in Vmax whereas the apparent Km for UDP-galactose remained constant. A similar pattern of protection against inactivation of galactosyltransferase by trypsin was observed suggesting that a conformational change in the protein is responsible for the observed protection.