Liver microsomal electron transport systems. Properties of a reconstituted, NADH-mediated benzo[a]pyrene hydroxylation system.

An enzyme system from rat liver microsomes which catalyzes the NADH-mediated hydroxylation of benzo[a]pyrene has been reconstituted. The essential microsomal components of this NADH-dependent pathway were NADH-cytochrome b₅ reductase, cytochrome b₅, cytochrome P-448 and, phosphatidyl choline. Highly purified NADPH-cytochrome c reductase containing small amounts of deoxycholate stimulated this NADH-mediated pathway supported by 0.2 mm NADH whereas boiled reductase had little effect. Part of this stimulation could be attributed to hydroxylation of benzo[a]pyrene via a second pathway; i.e., NADPH-cytochrome c reductase in combination with cytochrome P-448 and phosphatidylcholine also supported a low rate of NADH-dependent hydroxylation. The mechanism of the remaining stimulation is not known. However, the effect of NADPH-cytochrome c reductase on the reconstituted cytochrome b₅-dependent pathway was not unique; high concentrations of deoxycholate also stimulated this pathway, perhaps by facilitating the transfer of electrons from NADH-cytochrome b₅ reductase to cytochrome b₅. The addition of NADPH-cytochrome c reductase to the cytochrome b₅-dependent reconstituted system also affected the apparent K_m of NADH for benzo[a]pyrene hydroxylation. In the absence of NADPH-cytochrome c reductase, the apparent K_m of NADH was 1.3 μm while in its presence a low (1.3 μm) and a high (1700 μm) K_m were observed, consistent with the affinities of the two flavoproteins for NADH. Our results also indicate that the relative contribution of the pathway due to NADPH-cytochrome c reductase in combination with phosphatidyl choline and cytochrome P-448 to the overall rate of NADH-supported benzo[a]pyrene hydroxylation in microsomes would be greatly dependent on the concentration of NADH chosen. The rate of benzo[a]pyrene hydroxylation by these reconstituted components was almost 10-fold greater with 10 mm NADH than with 0.2 mm NADH, a result consistent with the reduction of NADPH-cytochrome c reductase by high concentrations of NADH.     

meso-α,ɛ-Diaminopimelate d-Dehydrogenase: Sulfhydryl Group Modification

meso-α,?-Diaminopimelate D-dehydrogenase was inhibited by sulfhydryl reagents such as p-chloromercuribenzoate and HgCl₂. Two sulfhydryl groups were titrated per molecule in the presence and absence of 6 M guanidine hydrochloride: the enzyme contained one sulfhydryl group per subunit. Modification of the sulfhydryl groups with p-chloromercuribenzoate, 5,5'-dithiobis(2-nitrobenzoic acid), 4,4'-dithiopyridine, N-ethylmaleimide, and iodoacetic acid was accompanied by a loss of enzyme activity. However, modification of sulfhydryl groups of the enzyme with cyanide did not affect the activity. Thus, the introduction of bulky or charged substituents to sulfhydryl groups decreased the catalytic activity of the enzyme, but modification of the groups with the small and uncharged group, a cyano group, did not. The sulfhydryl groups did not play an essential role in catalysis.     

Purification and characterization of the cytochrome P-448 component of a benzo(a)pyrene hydroxylase from Saccharomyces cerevisiae

Cytochrome P-448, a type of cytochrome P-450, from brewer's yeast (Saccharomyces cerevisiae) grown under conditions of glucose repression was isolated and purified. Triton X-100 in very low concentration proved to be very effective in stabilizing P-448 in the microsomal fraction and later prevented its conversion to cytochrome P-420 through solubilization with various ionic and nonionic detergents. Highest yields were obtained with 1% sodium cholate, in the presence of 0.1% Triton X-100 and reduced glutathione. A novel combination of hydrophobic adsorption and other chromatographic techniques was used for the purification of cytochrome P-448. These involve the use of amino octyl-Sepharose 4B, instead of the low-yielding aminohexyl derivative, followed by the fast-running hydroxyapatite-cellulose column. Finally, the use of DEAE-Sephacel was found to increase greatly the purity of the cytochrome P-448 obtained. The molecular weight of this preparation was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (M_r, 55,500). Using the known molar extinction coefficient of the carbon monoxide-difference spectrum the estimate of degree of purity of cytochrome P-448 obtained by this purification procedure was between 88 and 97%. Electrophoresis also showed that this preparation was completely homogeneous and assays showed that it was also completely free of cytochrome b_s, cytochrome c reductase and cytochrome P-420. Purified cytochrome P-448 reconstituted with cytochrome P-450 (cytochrome c) reductase, isolated from yeast, showed 10-fold higher aryl hydrocarbon hydroxylase activity with benzo[a]pyrene as a substrate than the corresponding microsomal fraction enzyme. Kinetics of benzo[a]pyrene hydroxylation were determined: K_m (33 μm) was comparable with that reported for purified hepatic cytochrome P-448. The number of binding sites of microsomal and purified cytochromes P-450 (from liver of phenobarbital-induced rats) and yeast cytochrome P-448 with benzo[a]pyrene has been determined using and equilibrium gel filtration method. There is one binding site in each case (contrast with six sites for microsomal enzymes). The Scatchard plot gives number of binding sites, apparent association constants (K), and the equivalent dissociation constants (K_s). Comparison is made with spectral dissociation constants for these enzymes and benzo[a]pyrene. Thus the proportion bound, dissociation constant (K_s), and stoichiometry of rat liver (phenobarbital induced) and yeast cytochrome P-448 with benzo[a]pyrene were compared with corresponding values for microsomal fractions of both systems. Purified enzymes had higher K_s values in both cases, and the proportion of enzyme that bound benzo[a]pyrene was high (53%) for liver and this value is 100% for purified enzyme from yeast, which is the same as the value obtained for the microsomal enzyme from yeast.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Distribution of Nuclease O Inhibitor among Aspergillus Species

Leucine dehydrogenase was inhibited by p-chioromercuribenzoate and HgCl₂, but not by 5,5′-dithiobis(2-nitrobenzoic acid), 4,4′-dithiopyridine and N-ethylmaleimide. Modification of sulfhydryl groups of the enzyme with p-chloromercuribenzoate and HgCl₂ was accompanied with a loss of the enzyme activity. The 6 reactive sulfhydryl groups per enzyme molecule play an essential role for catalysis. Approximately 12 sulfhydryl groups were titrated per molecule in the presence of 8 m urea: the enzyme contains 2 sulfhydryl groups per subunit, and one of them participates in the catalytic action. Fluorometric and gel filtration studies on binding of NADH to the enzyme revealed that the enzyme contains 6 coenzyme binding sites per molecule.

These results are compatible with the hexameric structure of leucine dehydrogenase composed of identical subunits, showing that each subunit has one catalytic site and one indispensable sulfhydryl group.     

Inhibition of lectin-induced lymphocyte activation by diamide and other sulfhydryl reagents

Inhibition of lectin-induced lymphocyte activation by five reagents capable of combining with or oxidizing free sulfhydryl groups was examined. Each of the reagents tested was capable of inhibiting [methyl-³H]thymidine or [¹⁴C]uridine incorporation into trichloroacetic acid-insoluble material. Four of these reagents, iodoacetamide and N-ethylmaleimide (alkylating agents) and 5,5′-dithiobis (2-nitrobenzoic acid) and p-hydroxymercuriphenylsulfonic acid (sulfhydryl binding agents), inhibited activation when added to lymphocyte cultures together with lectin or at any time thereafter through 48 hr. In contrast, the sulfhydryl oxidizing agent diazine dicarboxylic acid bis[N,N-dimethylamide] (diamide) was effective only when added within 30–60 min of lectin or when added after 24 hr. This inhibition of lymphocyte activation was not due to decreased intracellular levels of reduced glutathione or to inhibition of binding of lectin to the lymphocyte. These results suggest that maintenance of free sulfhydryl groups is important during the early induction of lymphocyte activation and suggest that an obligatory step or steps in the activation sequence may involve sulfhydryl interactions.     

The drug and carcinogen metabolism system of rat colon microsomes

The hydroxylation of N- and O-methyl drugs and polycyclic hydrocarbons has been demonstrated in microsomes prepared from colon mucosal cells. The hydroxylation of the drugs benzphetamine, ethylmorphine, p-nitroanisole, and p-nitrophenetole by colon microsomes is inducible two- to fourfold by pretreatment with phenobarbital/hydrocortisone. Colon microsomal benzo[α]pyrene hydroxylation is inducible 35-fold by pretreatment with β-naphthoflavone. Phenobarbital/hydrocortisone pretreatment also induces a fourfold increase in the specific content of colon microsomal cytochrome P-450, while β-naphthoflavone pretreatment causes a shift in the reduced CO difference spectrum peak to 448 nm and an eightfold increase in the specific content of this cytochrome. SKF 525-A inhibits the hydroxylation of the drug benzphetamine by colon microsomes or liver microsomes by 77% at a concentration of 2.0 mm. 7,8-Benzoflavone, on the other hand, inhibits the hydroxylation of the polycyclic hydrocarbon benzo[α]pyrene by colon microsomes by 76% and by liver microsomes by 44% at a concentration of 10 μm. Carbon monoxide, an inhibitor of oxygen interaction with cytochromes P-450 and P-448, inhibits benzphetamine hydroxylation and benzpyrene hydroxylation by colon microsomes 30 and 51%, respectively, at an oxygen to carbon monoxide ratio of 1:10. The K_m values of colon microsomal cytochrome P-450 reductase for the artificial electron acceptors cytochrome c, dichloroindophenol, and ferricyanide (10–77 μm) are in agreement with those for purified rat liver cytochrome P-450 reductase. These data support the conclusions that hydroxylation of drugs and polycyclic hydrocarbons is catalyzed by colon mucosal microsomes and that the hydroxylation activity is attributable to a cytochrome P-450-dependent drug metabolism system similar to that found in liver microsomes.     

Effect of Carbohydrates and Starvation on Nitrogen Sparing Action of Methionine and Threonine in Rats

Formaldehyde dehydrogenase from Pseudomonas putida C-83 was found to contain 7 halfcystine residues per subunit monomer, as checked by the method of performic acid oxidation. Approximately 7 sulfhydryl groups per subunit monomer were titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) after denaturation with 8 m urea. In the native enzyme, modification of three sulfhydryl groups per subunit with p-chloromercuribenzoate (PCMB) led to the complete loss of enzyme actiyities for both formaldehyde and n-butanol. Hydrogen-peroxide competitively inhibited the enzyme activity for formaldehyde, while it was only slightly inhibitory to the activity for n-butanol. Both formaldehyde and hydrogen-peroxide protected one sulfhydryl group per subunit monomer from modification with PCMB. Moreover, hydrogen-peroxide was hardly reactive to the enzyme which was preincubated with formaldehyde.

From these observations, we conclude that one of three PCMB-reactive sulfhydryl groups is essential for the binding of formaldehyde, and hydrogen-peroxide modifies this sulfhydryl group.     

Comparison of cytochromes P-450 with high activity toward benzo[a]pyrene purified from liver microsomes of beta-naphthoflavone and 3-methylcholanthrene-pretreated rats

Cytochromes P-450 with high activity toward benzo[a]pyrene were isolated from liver microsomes of rats treated with either β-naphthoflavone or 3-methylcholanthrene and examined for similarity using several physical and catalytic criteria. The β-naphthofla-vone-inducible cytochrome P-446 and the 3-methylcholanthrene-inducible cytochrome P-448 have the same subunit molecular weight (56,000 ± 1000) and electrophoretic mobility. Antibodies prepared to either form cross-react with each form without spurring in Ouchterlony double-diffusion experiments suggesting immunochemical identity. After proteolytic digestion with Staphylococcus aureus SV-8 protease and electrophoresis, both Cytochromes P-450 show the presence of the same bands. Both cytochromes have the same absorption maximum (446.5 ± 0.5 nm) in the CO-reduced absolute spectrum. The catalytic activity toward benzo[a]pyrene of cytochrome P-446 is somewhat greater than that of cytochrome P-448. Thus, all the physical evidence suggests identity of the two cytochromes. The significance of the difference in catalytic activity remains to be defined.     

The induction of cytochrome P-448 dependent benzo(a)pyrene hydroxylase in Saccharomycescerevisiae

When grown in high concentrations of glucose, the yeast $\text{Saccharomyces}\text{cerevisiae}$ produces a microsomal cytochrome P-450 monooxygenase system which is capable of hydroxylating benzo(a)pyrene. The addition of benzo(a)pyrene to the yeast during growth causes only a small increase in cytochrome P-448 levels but results in a dramatic improvement in the apparent kinetics of benzo(a)pyrene hydroxylation as measured by a decrease in the Michaelis constant and an increase in maximal velocity. Dimethylnitrosamine, phenobarbital and 3-methylcholanthrene also induce this enzyme to various degrees. Yeast pretreatment with β-naphthoflavone did not affect this enzyme, yet pretreatment with lanosterol resulted in a decreased affinity for benzo(a)pyrene. The addition of benzo(a)pyrene to yeast growing at low glucose concentration does not induce cytochrome P-448. The implications of these findings with regard to the presence of multiple forms of cytochromes $\text{P-448}\text{P-450}$ in yeast are briefly discussed.     

Covalent binding of benzo[a]pyrene to cytochrome P-450βNF-B2 and other proteins in reconstituted mixed-function oxidase systems

A reconstituted mixed-function oxidase system, containing the major β-naphthoflavone-induced isozyme of rat liver cytochrome P-450 bound benzo[a]pyrene covalently in the presence of NADPH. NADPH-cytochrome P-450 reductase was required for binding and a maximum rate of adduct formation was obtained at 8 units of reductase per nmol cytochrome P-450. Phosphatidylcholine inhibited this reaction. Benzo[a]pyrene was bound to the cytochrome, but not to the reductase, as shown by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Approximately 6 molecules of benzo[a]pyrene bound to each molecule cytochrome P-450 during prolonged incubations. No binding occurred when the β-naphthoflavone-induced isozyme of cytochrome P-450 was replaced by the major isozyme induced by phenobarbital, but both cytochromes incorporated benzo[a]pyrene to approximately the same extent when they were incubated together in the presence of the reductase and NADPH. Metabolically activated benzo[a]pyrene also bound covalently to purified epoxide hydrodrolase, when this enzyme was added to the reconstituted mixed-function oxidase system.     

Reactivity of cysteinyl,arginyl, and lysyl residues ofEscherichia coli phosphoenolpyruvate carboxykinase against group-specific chemical reagents

Calcium-activated phosphoenolpyruvate carboxykinase fromEscheria coli is not inactivated by a number of sulfhydryl-directed reagents [5,5′-dithiobis(2-nitrobenzoate), iodoacetate, N-ethylmaleimide, N-(1-pyrenyl)maleimide or N-(iodoacetyl)-N′-(5-sulfo-l-naphthylethylenediamine)], unlike phosphoenolpyruvate carboxykinase from other organisms. On the other hand, the enzyme is rapidly inactivated by the arginyl-directed reagents 2,3-butanedione and 1-pyrenylglyoxal. The substrates, ADP plus PEP in the presence of Mn²⁺, protect the enzyme against inactivation by the diones. Quantitation of pyrenylglyoxal incorporation indicates that complete inactivation correlates with the binding of one inactivator molecule per mole of enzyme. Chemical modification by pyridoxal 5′-phosphate also produces inactivation of the enzyme, and the labeled protein shows a difference spectrum with a peak at 325 nm, characteristic of a pyridoxyl derivative of lysine. The inactivation by this reagent is also prevented by the substrates. Binding stoichiometries of 1.25 and 0.30mol of reagent incorporated per mole of enzyme were found in the absence and presence of substrates, respectively. The results suggest the presence of functional arginyl and lysyl residues in or near the active site of the enzyme, and indicate lack of reactive functional sulfhydryl groups.     

Soybean (Glycine max) urease: Significance of sulfhydryl groups in urea catalysis

The soybean urease (urea amidohydrolase; EC 3.5.1.5) was investigated to elucidate the presence of sulfhydryl (–SH) groups and their significance in urea catalysis with the help of various –SH group specific reagents. The native urease incubated with 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) showed exponential increase in the absorbance, thereby revealing the presence of –SH groups. A total of 34 –SH groups per hexamer enzyme molecule were estimated from the absorption studies which represents nearly six –SH groups per subunit. The time-dependent inactivation of urease with DTNB, p-chloromercuribenzoate (p-CMB), N-ethylmaleimide (NEM) and iodoacetamide (IAM) showed biphasic kinetics, where half of the enzyme activity was lost more rapidly than the other half. This study reveals the presence of two categories of “accessible” –SH groups, one category being more reactive than the other. The inactivation of urease by p-CMB was largely reversed on treatment with cysteine, which might be due to unblocking of –SH group by mercaptide exchange reaction. Finally, when NEM inactivated urease was incubated with sodium fluoride, a time-dependent regain of activity was observed with higher concentrations of fluoride ion.     

Regulatory sulfhydryl groups, conformational change, and allosteric inhibition in rabbit muscle fructose diphosphatase

The 16 sulfhydryl groups of native, homogeneous rabbit muscle fructose diphosphatase can all react with 5,5′-dithiobis-(2-nitrobenzoic acid). High concentrations of substrate (1–2 mm) decrease the reaction rate of the sulfhydryl groups, while concentrations up to 70 μm have no effect. After titration of the four most rapidly reacting sulfhydryl groups there is a marked desensitization toward the allosteric inhibitor AMP. In the presence of 30 μm AMP only 4–5 sulfhydryl groups/tetramer react with 5,5′-dithiobis-(2-nitrobenzoic acid), and the enzyme again becomes desensitized toward AMP inhibition. Together with a 3.5-fold increase in the I₅₀ for AMP inhibition, the K_m for Mg²⁺ or Mn²⁺ ions is also increased. In the presence of 7 mm MgCl₂ or 0.28 mm MnCl₂ only 6–8 sulfhydryl groups are modified. The rapid reaction of 4 sulfhydryl groups again results in desensitization. There is neither a protection by the substrate against inactivation, nor a protection by the allosteric inhibitor against desensitization. It is concluded that AMP and the divalent cations induce conformational changes in the protein molecule making 11–12 or 8–10 sulfhydryl groups inert for 5,5′-dithiobis-(2-nitrobenzoic acid), respectively. The K_m for fructose-1,6-diphosphate is not changed after the modification of 4–5 sulfhydryl groups.     

Chemical modification of sulfhydryl residues of rabbit triosephosphate isomerase

Rabbit muscle triosephosphate isomerase (EC 5.3.1.1) is inactivated by maleimides, Na₂S₄O₆, organic mercurials, 5,5′-dithiobis (2-nitrobenzoic acid), Ag⁺, and Hg²⁺. Ag²⁺ and Hg²⁺ cause a decrease in the maximum velocity, and under specified conditions the other reagents induce an increase in the Michaelis constant.N-ethylmaleimide reacts with three sulfhydryl residues per mole of enzyme, and the maximum change in K_m is about threefold. Mercurials cause a greater change in K_m and react with more than three sulfhydryl groups, but subsequent precipitation prevents quantitative analysis after six residues have reacted (with p-hydroxymercuribenzoate).Experiments with several competitive inhibitors and the active-site affinity label, 3-chloroacetolphosphate, showed that the magnitude of the change in Michaelis constant was the same as the magnitude of the changes in the inhibition constants.The rabbit muscle and liver enzyme appear to have similar properties, but the chicken muscle enzyme is much less reactive, and the yeast enzyme does not become inactivated.Evidence is presented to show that the effects cannot be explained by assuming the hydrated substrates are bound to the enzyme as a result of sulfhydryl modification.     

Ketomethylthiobutyric acid formation from methylthioadenosine: A diffusion assay

Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H₂S derived from the cysteine. The yield of H₂S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + P_i → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H₂S, (5) H₂S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol.     

Liver microsomal DT diaphorase: noninvolvement in hydroxylation of benzo(a)pyrene.

A highly purified reconstituted system isolated from the microsomes of 3-methylcholanthrene-treated rats consisting of cytochrome P-448, NADPH-cytochrome $\text{c}$ reductase and synthetic dilauroyl phosphatidylcholine had no DT diaphorase activity, but hydroxylated benzo[a]pyrene at a faster rate than microsomes from 3-methylcholanthrene-treated rats. DT diaphorase purified from liver microsomes of 3-methylcholanthrene-treated rats when added to this reconstituted system did not stimulate or inhibit benzo[a]pyrene hydroxylation, nor could it replace or NADPH-cytochrome $\text{c}$ reductase in supporting the reaction. We therefore conclude that microsomal DT diaphorase is not involved in microsomal hydroxylation of benzo[a]pyrene to its phenolic products despite the observation that both DT diaphorase activity and the hydroxylation of benzo[a]pyrene are induced by 3-methylcholanthrene and 2,3,7,8-tetrachlorodibenzo-$\text{p}$-dioxin     

Hydroxymethylation of the benzene ring. Microsomal hydroxymethylation of benzo(alpha)pyrene to 6-hydroxymethylbenzo(alpha)pyrene

The formation of 6-hydroxymethylbenzo(α)pyrene from benzo(α)pyrene has been shown to be catalyzed by sonicates of rat liver microsomes. The biosynthetic pathway for the formation of the aryl hydroxymethyl groups appears to be a direct hydroxymethylation of the benzene ring and not involve 6-methylbenzo(α)pyrene as an intermediate, because the formation of 6-hydroxymethylbenzo(α)pyrene is not a cytochrome P-450-mediated reaction, whereas a model aryl side chain methyl group hydroxylation has been shown to be inhibited by cytochrome P-450 inhibitors.     

Anomalous electrophoretic behavior of creatine kinase: A thiol-dependent isomeric system with migration subject to kinetic control

Moving boundary electrophoresis of creatine kinase in 0.1 I diethylbarbiturate buffer, pH 8.9, has yielded anomalous migration behavior that indicates intereonversion between two coexisting states of the enzyme at a rate comparable with the rate at which the two enzymic forms tend to separate by differential migration. Whereas a single, symmetrical boundary is observed in the ascending limb, distinct bimodality of the descending pattern is evident in electrophoresis of enzyme isolated from skeletal muscle of either rabbit or fish (Mugil cephalus): pronounced changes in the nature of this bimodality with time are observed in the case of fish muscle creatine kinase. The abnormal migration behavior is eliminated by inclusion of dithiothreitol in the electrophoresis medium or by covalent modification of the enzyme with 5,5′-dithiobis(2-nitrobenzoic acid). Velocity and equilibrium sedimentation studies have been used to identify the macromolecular event as an isomerization, and studies of sulfhydryl content to implicate either reversible sulfhydryl oxidation or thiol-disulfide interchange in the isomerization mechanism.