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Title:Nitrite Mode of Action: The Inhibition of Yeast Pyruvate Decarboxylase and Clostridial Pyruvate:ferredoxin Oxidoreductase by Nitric Oxide and Menadione
Author(s):McMindes, Matthew Kirk
Doctoral Committee Chair(s):Siedler, Arthur J.
Department / Program:Food Science
Discipline:Food Science
Degree Granting Institution:University of Illinois at Urbana-Champaign
Degree:Ph.D.
Genre:Dissertation
Subject(s):Agriculture, Food Science and Technology
Abstract:Nitrite mode of action as an anticlostridial agent was investigated by studying nitric oxide (NO) inhibition of thiamin dependent pyruvate decarboxylases. Two enzymatic decarboxylation systems were studied: pyruvate decarboxylate (PD) from brewer's yeast which catalyzes the non-oxidative decarboxylation of pyruvate and pyruvate:ferredoxin oxidoreductase (PFO) from Clostridium perfringens (CP; ATCC no. 3624A) which catalyzes the oxidative decarboxylation of pyruvate in clostridia. Menadione (MD), which was previously observed to inhibit pyruvate dehyrogenase from pigeon breast muscle in a similar manner to NO, was studied as a possible nitrite alternative.
PD was inhibited by NO (50% inhibition @ 0.5mM) under anaerobic but not under aerobic conditions; prior inhibition was not reversed by aerobic conditions. Sodium nitrite (0.5 mM) was not inhibitory. Inhibition of PD by NO was markedly enhanced in the presence of ascorbate. Inhibition of PD by NO appeared to be non-competitive.
A purified preparation of PFO from CP was characterized with respect to molecular weight (255,000 daltons), subunit composition (dimer 127,000 daltons each), temperature optimum (45$\sp\circ$C), pH optimum (pH 7.5), Michaelis constant (Km pyruvate = 1.0 mM), and NADH oxidase activity (positive). The effects of NO on PFO resulted in non-competitive inhibition (Ki apparent = 0.015 mM). NADH oxidase activity of PFO was also inhibited by NO. Nitrate was not inhibitory under anaerobic testing conditions at concentrations of 1.0 mM.
MD was found to inactivate PD (Ki apparent = 0.065 mM). Inactivation was dependent on the presence of substrate and increased under anaerobic conditions implying a paracatalytic mechanism of inhibition. Structure activity relationships were analyzed for other naphthoquinones, phenolic antioxidants, and known sulfhydryl groups reagents. All the naphthoquinones (NQ) tested were inhibitory at the 0.05 mM concentration (vitamin K$\sb4$, 13.7%, 1,2-NQ 42%, 1,4-NQ 58%, 1,2-NQ-4-sulfonic acid 33%, 1,4-NQ-2-sulfonic acid 28%) except vitamin K$\sb1$ and 2,3-dimethyl-1,4-NQ. Incubation of PD with sulfhydryl inhibitors (5$\prime,5\prime$ dithiobis-(2-nitrobenzoic acid), N-ethylmaleimide, or iodoacetate) produced slight inhibition at the 0.5 mM concentration (12.7%, 6.9%, 5.1%) whereas p-chloromercuribenzoic acid inhibited 56.5% at 0.005 mM. Phenolic antioxidants were not significantly inhibitory at 0.1 mM concentration.
Issue Date:1988
Type:Text
Description:136 p.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1988.
URI:http://hdl.handle.net/2142/70101
Other Identifier(s):(UMI)AAI8815389
Date Available in IDEALS:2014-12-15
Date Deposited:1988


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