An enzyme-linked immunosorbent assay for the detection of Shiga toxins (Premier EHEC assay; Meridian Diagnostics, Inc.) was compared to conventional sorbitol-MacConkey culture for the recovery of enterohemorrhagic Escherichia coli. A total of 74 enteric pathogens, including 8 E. coli O157:H7 isolates, were recovered from 974 stool specimens. Two of these specimens were not tested by Premier assaying due to insufficient sample and are not considered in the data analysis. The Premier EHEC assay detected the 6 evaluable specimens which were culture positive for E. coli O157:H7 and identified an additional 10 specimens as containing Shiga toxin. Seven isolates were recovered from these 10 specimens by an immunoblot assay and were confirmed as toxin producers by a cytotoxin assay. Of these seven, four isolates were serotype O157:H7, one was O26:NM, one was O6:H ؊ , and one was O untypeable:H untypeable. Three specimens contained Shiga toxin by both EHEC immunoassaying and cytotoxin testing; however, no cytotoxin-producing E. coli could be recovered. The sorbitol-MacConkey method had a sensitivity and a specificity of 60 and 100%, respectively, while the Premier EHEC assay had a sensitivity and a specificity of 100 and 99.7%, respectively, for E. coli O157:H7 only. The Premier EHEC assay also detected an additional 20% Shiga toxin-producing E. coli (STEC) that were non-O157:H7. Thus, the Premier EHEC assay is a sensitive and specific method for the detection of all STEC isolates. Routine use would improve the detection of E. coli O157:H7 and allow for determination of the true incidence of STEC other than O157:H7. The presence of blood in the stool and/or the ages of the patients were poor predictors of the presence of STEC. Criteria need to be determined which would allow for the cost-effective incorporation of this assay into the routine screen for enteric pathogens in high-risk individuals, especially children.

3-Chloropropionyl coenzyme A (3-chloropropionyl-CoA) irreversibly inhibits avian liver 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). Enzyme inactivation follows pseudo-first-order kinetics and is retarded in the presence of substrates, suggesting that covalent labeling occurs at the active site. A typical rate saturation effect is observed when inactivation kinetics are measured as a function of 3-chloropropionyl-CoA concentration. These data indicate a Ki = 15 microM for the inhibitor and a limiting kinact = 0.31 min-1. [1-14C]-3-Chloropropionyl-CoA binds covalently to enzyme with a stoichiometry (0.7 per site) similar to that measured for acetylation of enzyme by acetyl-CoA. While the acetylated enzyme formed upon incubation of HMG-CoA synthase with acetyl-CoA is labile to performic acid oxidation, the adduct formed upon 3-chloropropionyl-CoA inactivation is stable to such treatment. Therefore, such an adduct cannot solely involve a thio ester linkage. Exhaustive Pronase digestion of [14C]-3-chloropropionyl-CoA-labeled enzyme produces a radioactive compound which cochromatographs with authentic carboxyethylcysteine using reverse-phase/ion-pairing high-pressure liquid chromatography and both silica and cellulose thin-layer chromatography systems. This suggests that enzyme inactivation is due to alkylation of an active-site cysteine residue.

Spinach leaf ribulosebisphosphate carboxylase forms a quaternary complex with CO2, carboxyarabinitol bisphosphate, and Cr2+ or Co2+. Oxidation of the cation in these complexes produces a protein--cation adduct which is sufficiently stable to be chromatographically isolated after enzyme denaturation. While stoichiometric levels of slowly exchanging cation can be specifically trapped after addition of protein denaturants as well as a vast molar excess of Mg2+, neither CO2 nor carboxyarabinitol bisphosphate remains bound to denatured protein under the conditions employed in these experiments. These observations demonstrate direct innersphere liganding of protein to the exchange-inert cation, which appears to bind at the site normally occupied by the physiologically active cation. Dimeric ribulosebisphosphate carboxylase from Rhodospirillum rubrum also forms a quaternary complex containing stoichiometric amounts of enzyme protomer, CO2, Co2+, and carboxyarabinitol bisphosphate. Lack of a small subunit in the R. rubrum enzyme does not impair binding of the components of the quaternary complex in a nonexchangeable mode. Substantial amounts of protein--cation adduct are recovered upon oxidation and denaturation of the R. rubrum complex, supporting the prediction that the large subunits of the octameric plant enzyme should be the sites of cation binding. The first direct proof for such a hypothesis has been generated by separation of protein subunits derived from a spinach quaternary complex and by the demonstration that the bound cation is associated with the large subunit.

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