John Westley

Long-lasting depletions of striatal dopamine and loss of dopamine uptake sites following repeated administration of methamphetamine.

Repeated administration of high doses of methamphetamine produced long-term decreases in dopamine (DA) levels and in the number of DA uptake sites in the rat striatum. These two effects were dose-related and did not appear to be due to the continued presence of drug in striatal tissue. Long-lasting depletions induced by methamphetamine were selective for striatal DA neurons since norepinephrine (NE) levels in all of the rat brain regions examined were not changed on a long-term basis by methamphetamine treatments. Supersensitivity of DA receptors did not accompany the loss of striatal DA and its uptake sites.

Rhodanese as a thioredoxin oxidase

A major catalytic difference between the two most common isoforms of bovine liver mitochondrial rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed. Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate. However, only the less negative form in common with the recombinant mammalian rhodanese expressed in E. coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species. These activities are understood in terms of the established persulfide structure (R-S-SH) of the covalently substituted rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R-S-OH) when the enzyme acts as a thioredoxin oxidase. The observations suggest a role for one rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals.

Reduced thioredoxin as a sulfur-acceptor substrate for rhodanese

Mammalian rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) catalyzes the transfer of sulfane sulfur from donors such as S2O2-(3) and organic thiosulfonate anions (RS(O)2S-) to nucleophilic acceptors such as CN- and dithiols. The work reported here used an NADPH-coupled assay with thioredoxin reductase to show that reduced thioredoxin at micromolar concentrations is also an effective sulfur-acceptor substrate for rhodanese under conditions where millimolar concentrations of lipoate or dithiothreitol would be required. At near K(m) concentrations of the other substrate, apparent K(m) values for thioredoxin and methane thiosulfonate were 18.5 +/- 1.8 microM and 20 +/- 4 mM, respectively. The physiological compound alanine thiosulfonate also could serve as a donor substrate. In these systems, after a brief lag, inorganic sulfide accumulated as a final product. A formal mechanism in accord with all the results is proposed.

Steady-state kinetics of 3-mercaptopyruvate sulfurtransferase from bovine kidney☆

Abstract Mammalian 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2), purified to apparent homogeneity by a new procedure, was studied by steady-state kinetic methods. The enzyme-catalyzed transfer of a sulfur atom from 3-mercaptopyruvate either to 2-mercaptoethanol or to a second molecule of 3-mercaptopyruvate was found to proceed by a sequential formal mechanism. An overall mechanism incorporating both of these transfers was shown to be capable of generating all of the initial velocity and product inhibition behavior observed.

Biological sulfane sulfur

A voltammetric method for determining cyanide-reactive sulfane sulfur in biological materials is described. Samples are incubated with a sulfurtransferase, a thiolic cofactor, and cyanide. Thiocyanate formed and/or residual cyanide may then be determined electrochemically with either a silver rotating disk electrode or a dropping mercury electrode in differential pulse mode to provide estimates of sulfane sulfur content. The thiocyanate-based procedure is preferable, particularly when samples contain either serum albumin or inorganic sulfide.

Enzyme Inhibition in Open Systems SUPERIORITY OF UNCOMPETITIVE AGENTS

Investigations of the open system behavior of reversible dead-end inhibitors were carried out by means of computer simulations and experimental studies. The results from both approaches indicate that substrate-competitive inhibition may often be an inappropriate basis for design of potential therapeutic agents. The use of uncompetitive (also called anticompetitive) inhibitors in this role is likely to be far more effective. Chemical analogs of pathogen-specific enzymic reaction products rather than analogs of substrates provide a promising basis for the systematic design of such uncompetitive inhibitors.

Sulfate activation and transport in mammals: system components and mechanisms

Extensive studies on the mammalian sulfate-activating enzymes and PAPS translocase have enhanced our understanding of the overall pathway of sulfate activation and utilization. Isolation of the PAPS-synthesizing activities from rat chondrosarcoma and preparation of stable non-hydrolyzable analogs of APS and PAPS have facilitated the kinetic characterization of mammalian ATP sulfurylase and APS kinase. These studies provided the basis for further experimental work showing that APS, the labile intermediate product, is channeled directly between the sulfurylase and kinase active sites. The defect in the brachymorphic mutant mouse lies in this channeling mechanism, thus interfering with efficient PAPS production. The rat chondrosarcoma ATP sulfurylase and APS kinase activities, in fact, reside in a single bifunctional cytoplasmic protein, which has now been cloned and expressed. The mechanism by which PAPS reaches its sites of utilization in the Golgi lumen has also been elucidated: The PAPS translocase is a 230-kDa integral Golgi membrane protein which functions as an antiport.

Votammetric determination of cyanide and thiocyanate in small biological samples

Practical methods for the quantitative measurement of cyanide and thiocyanate content in small biological samples are presented. The basis of the procedures is electrochemical determination of CN- with a silver rotating disk electrode at 90 mV, relative to the Ag/AgCl reference electrode, or with a dropping mercury electrode in differential pulse mode at -240 mV. Thiocyanate is oxidized to CN- with permanganate under controlled conditions prior to its analysis. Endogenous cyanide and cyanide from SCN- oxidation are recovered, separately, by volatilization and trapping. Emphasis is placed on careful handling of specimens prior to analysis. Precautions include a provision for sequestering cyanide in storage, the use of an antioxidant, and an avoidance of the spontaneous sulfide formation from proteins that occurs in even mildly alkaline solutions.

The effect of pH on the kinetic parameters and mechanism of beef liver monoamine oxidase

The effects of pH on the oxidation of benzylamine from the pH range 6.0–11.0 have been investigated. The p K m RNH 2 and the −log V m versus pH plots disclosed inflection points at about pH 7.3 and about pH 10.5. The p K m O 2 and the−log V m versus pH plots showed an inflection point at pH 7.1–7.3. No significant changes in these kinetic parameters were observed in the pH range of about 7.3 to at least pH 10. When the benzylamine and oxygen concentrations were varied simultaneously at fixed ratios, the double reciprocal plots in the acid range below pH 7.3 and above pH 10 disclosed that a ping pong mechanism is operative over the entire pH range. The product inhibition pattern at the acidic and alkaline pH ranges was investigated. A theoretical formal mechanism is presented to account for the observed results. From the changes in the values of the kinetic constants over the pH range 6–11.5 and with temperature, it is suggested that an amino acid side chain containing a group with a p K 1 of 7.0–7.3 is involved in amino binding and that another group with a p K 2 value of about 10.3–10.4 is involved in the cleavage of the amine.

Active site peptides of rhodanese.

Abstract The active site cysteinyl peptide isolated from a tryptic digest of rhodanese (thiosulfate:cyanide sulfurtransferase, EC 2.8.1.1) by column chromatography has been found to contain 15 amino acid residues, of which 7 are hydrophobic. The tryptophyl peptides in such digests also appear to consist predominantly of residues which are hydrophobic.