High Cleavage Activity Research Articles

Cleavage of the cap binding protein complex polypeptide p220 is not effected by the second poliovirus protease 2A

Poliovirus protein 2A contains a short amino acid sequence that also occurs in the putative active site of the known viral proteinase, 3C, previously shown to be responsible for glutamine/glycine cleavages in the poliovirus polyprotein precursor. Experimental evidence indicates that 2A is a second viral proteinase that mediates the cleavage of two tyrosine/glycine cleavages in the generation of virus-specific proteins. Since poliovirus inhibition of host cell protein synthesis correlates with the specific cleavage of the 220,000-Da component of the cap binding protein complex, we have tested whether viral protein 2A contains the p220 cleavage activity. The results show that 2A does not copurify with p220 cleavage activity, partially purified fractions containing high p220 cleavage activity contain no detectable 2A sequences in the form of either mature or precursor protein, and anti-2A serum or IgG does not inhibit p220 cleavage in vitro.

Recovery and activation of hydroxymethylglutaryl coenzyme A reductase from rat small intestine.

We describe a method for estimating the total activity of 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase (EC 1.1.1.34) in the small intestine of rats. An homogenate of the whole small intestine is prepared rapidly and assayed directly to maximize the yield of enzyme and to minimize opportunity for uncontrolled change in activity. Fresh homogenate inhibits the expression of reductase in hepatic microsomes, has high HMG-CoA cleavage activity, and forms NADPH-independent metabolites which contaminate mevalonolactone isolated by thin-layer chromatography. When homogenate is preincubated, these interfering factors are decreased and reductase activity is increased. Part of this increase can be inhibited by F-. After freezing and preincubation, total reductase activity recovered from homogenates of small intestine from 300-g male rats at the middle of their dark period is 40 nmol mevalonate per min compared to 70 from hepatic microsomes. If F- is added at the time of homogenization, an activity of 11 nmol per min is recovered from each organ. Reductase in intestinal homogenate has an apparent Km for S-HMG-CoA of 4 microM.

Adaptive alterations in Lactobacillus casei: II. Variation in the levels of aldolase in cells grown on glucose and ribose

Abstract The growth of homofermentative Laclobacillus casei on ribose caused reduction in aldol cleavage of fructose-1,6-diphosphate (FDP) while enhancing activities for condensation of triose phosphates and of fructose-1,6-diphosphatase. The purified preparation of aldolase from L. casei was not inhibited by EDTA and was not affected by bivalent metal ions (Fe2+ and Zn2+). The inactivation of the enzyme by sodium borohydride only in presence of dihydroxyacetone phosphate (DHAP) suggested formation of a Schiff-base intermediate. The enzyme could be resolved into three active components on DEAE-cellulose chromatography. These fractions showed distinct differences in properties with respect to cleavage of FDP and condensation of triose phosphates. The fractions having high cleavage activity gave low condensation reaction. This component was also active for transaldolase reaction. The other two fractions gave higher rate of condensation and lower cleavage values. The profiles obtained for aldolase prepared from ribose-grown cells of L. casei indicated that there was drastic reduction in the component having high cleavage activity. The results suggest that the equilibrium of the reaction toward cleavage or condensation was determined by isoenzymic composition of aldolase which appears to be adaptively altered by metabolic regulatory controls operative in glucose or ribose-grown cells of L. casei.