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Biomedical subjects

J Halpert

Publications and source records attributed to J Halpert.

At least 37 records · Page 2Linked to original sources
PubMed1983

Immunochemical detection and quantitation of microsomal cytochrome P-450 and reduced nicotinamide adenine dinucleotide phosphate:cytochrome P-450 reductase in the rat ventral prostate.

Treatment with beta-naphthoflavone (BNF) was found to induce 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities about 500-fold in the microsomal fraction of the rat ventral prostate but had no effect on aminopyrine N-demethylase or reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activities. Phenobarbital (PB) treatment did not alter any of these enzyme activities. Antibodies raised in rabbits against rat liver cytochrome P-450 reductase (P-450 reductase) and against P-450 BNF-B2 and P-450 PB-B2, the major forms of P-450 isolated from liver microsomes of BNF- and PB-treated rats, respectively, were used to characterize the P-450-dependent monooxygenase system in the rat ventral prostate. Anti-P-450 reductase immunoglobulin G inhibited reduced nicotinamide adenine dinucleotide phosphate:cytochrome c reductase activity in prostatic microsomes, and anti-P-450 BNF-B2 but not anti-P-450 PB-B2 immunoglobulin G inhibited the BNF-induced prostatic microsomal 7-ethoxyresorufin O-deethylase and aryl hydrocarbon hydroxylase activities. A highly sensitive immunoblotting method was used to quantitate P-450 BNF-B2, P-450 PB-B2, and P-450 reductase in prostatic microsomes. Using this technique, prostatic P-450 reductase with a molecular weight corresponding to that of purified liver P-450 reductase was detected at a level of 0.02 nmol/mg of microsomal protein. In the liver, the same enzyme amounts to 0.2 nmol/mg of microsomal protein. P-450 BNF-B2 was not detected in prostatic microsomes from control or PB-treated rats, whereas a protein band with a molecular weight corresponding to that of purified liver P-450 BNF-B2 was found in prostatic microsomes from BNF-treated rats at a level of 0.05 nmol P-450 per mg microsomal protein. P-450 PB-B2 was not detected in prostatic microsomes from either control, PB-treated, or BNF-treated animals.

Animals↗
PubMed1983

Suicide inactivation of rat liver cytochrome P-450 by chloramphenicol in vivo and in vitro.

Intraperitoneal administration of chloramphenicol (100 mg/kg) to phenobarbital-treated rats causes 50% inhibition of liver microsomal 7-ethoxycoumarin and 1,1,2,2 tetrachloroethane metabolism but has no effect on the level of cytochrome P-450 detectable as its carbon monoxide complex or on the NADPH-cytochrome c reductase (EC 1.6.2.4) activity. Both the endogenous NADPH oxidase activity and the enzymatic reduction of cytochrome P-450 are inhibited by chloramphenicol treatment, whereas the Km and Ks for ethoxycoumarin and the cumene hydroperoxide- or iodosobenzene-supported deethylation of ethoxycoumarin are unaffected, suggesting that impaired electron transport to cytochrome P-450 may be the cause of the loss of enzymatic activity. Administration of [14C]chloramphenicol (100 mg/kg) leads to the covalent binding of 0.7 nmole of metabolite(s) per nanomole of the major cytochrome P-450 isozyme. Alkaline hydrolysis of a cytochrome P-450 fraction obtained by chromatography of solubilized 14C-labeled microsomes on octylamino-Sepharose releases oxalic acid and chloramphenicol oxamic acid, whereas enzymatic digestion releases N-epsilon-chloramphenicol oxamyl lysine in addition. These data obtained with radiolabeled chloramphenicol suggest that the same metabolic pathways which lead to the inactivation of cytochrome P-450 in vitro are also operative in vivo.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1983

Xylene induces a cytochrome P-450 isozyme in rat liver similar to the major isozyme induced by phenobarbital.

Rats pretreated with xylene or phenobarbital, and then exposed to n-hexane, exhibited a markedly increased peak serum concentration of the neurotoxic metabolite 2,5-hexanedione. In order to elucidate the mechanism underlying this synergistic effect, the major liver microsomal cytochrome P-450 isozymes induced by xylene and phenobarbital, respectively, were purified. In a reconstituted system both isozymes showed a high enzymatic activity with n-hexane as the substrate. Turnover numbers for the formation of 2-hexanol were 24 and 27 for the xylene- and phenobarbital-induced isozyme, respectively. The turnover numbers for 7-ethoxycoumarin, benzo[a]pyrene, and 1,1,2,2-tetrachloroethane were also in the same range for the two cytochrome P-450 preparations. The isozyme induced by xylene had an amino acid composition very similar to that of the phenobarbital-induced isozyme, and the purified proteins had identical electrophoretic mobilities on polyacrylamide gels in the presence of sodium dodecyl sulfate. Furthermore, similar peptide maps were obtained following digestion with alpha-chymotrypsin and papain, and each isozyme yielded a single immunoprecipitin band upon reaction with the immunoglobulin G fraction from rabbits immunized with the phenobarbital-induced enzyme. We conclude that xylene induces a rat liver microsomal cytochrome P-450 isozyme very similar to the major isozyme induced by phenobarbital and that this induction is the probable explanation for the enhanced formation of 2,5-hexanedione from n-hexane in vivo.

Amino Acids↗
PubMed1982

Toxicology of thiono-sulfur compounds.

Thiono-sulfur-containing compounds cause a wide variety of toxic effects in mammals. These toxic effects of thiono-sulfur-containing compounds appear to be at least partially the result of their metabolism to reactive intermediates by the cytochrome P-450-containing monooxygenase enzyme systems. Covalent binding of (atomic) sulfur released in the cytochrome P-450 monooxygenase catalyzed metabolism of certain thiono-sulfur compounds appears to be responsible for the inhibition of monooxygenase activity and the loss of cytochrome P-450 seen on administration of these thiono-sulfur compounds in vivo or incubation with cytochrome P-450 monooxygenase enzymes in vitro. Liver necrosis and perhaps the induction of lung edema and neoplasia as well as other effects of thiono-sulfur-containing compounds are more likely the result of the covalent binding of the electrophilic S-oxides or S-dioxides or carbene derivatives of these S-oxides and S-dioxides to tissue macromolecules. The rationale for implicating metabolites of thiono-sulfur compounds other than atomic sulfur in these effects derives from the experiments with thioacetamide and the fact that atomic sulfur is highly reactive and appears to bind predominantly or exclusively to cytochrome P-450. It is difficult to rationalize why binding to and inhibition of cytochrome P-450 would lead to the production of, for examples, liver necrosis.

Animals↗
PubMed1982

Further studies of the suicide inactivation of purified rat liver cytochrome P-450 by chloramphenicol.

The kinetics and reversibility of the suicide inactivation of rat liver cytochrome P-450 by chloramphenicol have been investigated with the use of a reconstituted monooxygenase system purified from liver microsomes of phenobarbital-treated rats. At a ratio of 1 unit of NADPH-cytochrome P-450 reductase per nanomole of cytochrome P-450 and a chloramphenicol concentration of 1 mM, the t1/2 for the inactivation of cytochrome P-450 is less than 2 min. The inactivated cytochrome regains some of its activity upon incubation at 25 degrees or 37 degrees, and experiments with [14C]chloramphenicol show that this partial reactivation is accompanied by the release of some of the 14C originally bound covalently to the cytochrome P-450. Previous work has shown that the 14C-labeled material spontaneously released from 14C-labeled cytochrome P-450 is in the form of oxalic acid, and that the latter is derived from a hydroxylamine-labile adduct of chloramphenicol and cytochrome P-450 [Biochem. Pharmacol. 30:875-881 (1981)]. In the present investigation the 14C-labeled material released by hydroxylamine was identified as the hydroxamic acid of oxalic acid. Trapping experiments with the amino acid cysteine suggest that the adduct, the spontaneous degradation of which appears to be involved in the reactivation of cytochrome P-450, contains an ester rather than a thioester linkage between cytochrome P-450 and a metabolite of chloramphenicol. However, this metabolite may not be identical with chloramphenicol oxamyl chloride, which was the active metabolite implicated in the formation of the 50% covalently bound material which was stable to hydroxylamine treatment.

Animals↗
PubMed1980

Inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion (diethyl p-nitrophenyl phosphorothionate).

The mechanism of the inactivation of purified rat liver cytochrome P-450 during the metabolism of parathion has been investigated with special reference to the covalent binding to the enzyme of the atomic sulfur (S) released upon oxidation of the compound. In addition to the previously reported loss of cytochrome P-450 detectable as its carbon monoxide complex, a loss of heme detectable as pyridine-hemochromagen, and a loss of enzymatic activity toward benzphetamine and ethoxycoumarin are shown. The loss of heme is 80% of the loss of cytochrome P-450, which in turn is less than the loss of benzphetamine demethylase activity, the turnover number of the modified enzyme being two-thirds that of the native based on the amount of cytochrome P-450 detectable as its carbon monoxide complex. Treatment with performic acid or dithiothreitol removes 75% of the 35S covalently bound to the cytochrome P-450 during parathion metabolism. However, the dithiothreitol treatment does not restore cytochrome P-450 detectable as its carbon monoxide complex or enzymatic activity. Likewise, no protection against the loss of heme, cytochrome P-450, or enzymatic activity is afforded by inclusion of 1 mM dithiothreitol during the incubation with parathion, although the amount of irreversibly bound sulfur, i.e. sulfur not dissociable by dithiothreitol, is significantly reduced. In the absence of dithiothreitol, 4 nmol of 35S become bound to the cytochrome P-450 for each nanomole of P-450 no longer detectable as its carbon monoxide complex. Evidence is presented for the covalent binding of sulfur to at least three other amino acids than cysteine. It is suggested that structural changes resulting from the covalent binding of atomic sulfur to cysteine residues are responsible for at least some of the loss of enzymatic activity observed.

Animals↗
PubMed1979

Isolation and incorporation of rabbit liver epoxide hydrase into phospholipid vesicles.

Methods are described for the incorporation into phospholipid vesicles of epoxide hydrase isolated from liver microsomes of phenobarbital-treated rabbits. Chromatography on a Sephadex G-50 column of epoxide hydrase and egg yolk phosphatidylcholine treated with sodium cholate yielded homogeneous vesicles with a diameter of about 25 nm and containing 80 to 85% of the protein applied. At high substrate concentrations, the vesicles catalyzed the hydration of benzo(a)pyrene-4,5-oxide and styrene-7,8-epoxide at a rate similar to that obtained with the enzyme in a soluble form. However, the kinetics of styrene glycol formation catalyzed by the vesicular or microsomal preparations were complex. Convex Lineweaver-Burk plots and concave Hill plots were obtained, whereas normal Michaelis-Menten kinetics characterized the hydration catalyzed by the enzyme in a soluble form. The results could be explained if reconstitution of the enzyme into the vesicles gives rise to low affinity high capacity sites for the substrate on the enzyme, or alternatively facilitates the interaction of the substrate with such sites already present. It is suggested that reconstituted liposomes containing both the liver microsomal hydroxylase system and epoxide hydrase may prove to be a good model system for evaluating substrate specificity and factors of importance in the formation of toxic and carcinogenic metabolites by these enzymes.

Animals↗
PubMed1979

Amino acid sequence of a postsynaptic neurotoxin from the venom of the Australian tiger snake Notechis scutatus scutatus.

Although 60 percent of the protein in tiger snake (Notechis scutatus scutatus) venom consists of the basic per-synaptically neurotoxic and myotoxic phospholipases notexin and Notechis II-5 and other phospholipase homologs such as Notechis II-1, several post-synaptic "curaremimetic" neurotoxins are present in small amounts. The major one of these is a typical "long" neurotoxin containing 73 amino acids in a single peptide chain cross-linked by five disulfide bridges. The formula weight calculated from the amino acid sequence is 8,051. The LD50 for intravenous injection into mice is 125 micrograms/kg.

Amino Acid Sequence↗