Modified activity of Aeromonas aminopeptidase: metal ion substitutions and role of substrates

Aeromonas aminopeptidase contains two nonidentical metal binding sites that have been shown by both spectroscopy and kinetics to be capable of interacting with one another [Prescott, J.M., Wagner, F.W., Holmquist, B., & Vallee, B.L. (1985) Biochemistry 24, 5350-5356]. The effects of metal ion substitutions on the susceptibility of the p-nitroanilides of L-alanine, L-valine, and L-leucine--substrates that are hydrolyzed at widely differing rates by native Aeromonas aminopeptidase--were studied by determining values of kcat and Km for the 16 metalloenzymes that result from all possible combinations of Zn2+, Co2+, Ni2+, and Cu2+ in each of the two sites. The different combinations of metal ions and substrates yield a broad range in kinetic values; kcat varies by more than 1800-fold, Km by 3000-fold, and kcat/Km ratios by more than 10,000. L-Leucine-p-nitroanilide is by far the most susceptible of the three substrates, and the hyperactivation previously observed with aminopeptidase containing either Ni2+ or Cu2+ in the first binding site and Zn2+ in the second site occurs only with the two poorer substrates, L-alanine-p-nitroanilide and L-valine-p-nitroanilide. Although the enzyme with Zn2+ in both sites hydrolyzes the substrates with N-terminal alanine and valine poorly, it is extremely effective toward L-leucine-p-nitroanilide. Neither metal binding site can be identified as controlling either Km or kcat; both parameters are influenced by the identity of the metal ions, by the site each occupies, and, most strongly, by the substrate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation in Saccharomyces cerevisiae Conferring Streptomycin and Cold Sensitivity by Affecting Ribosome Formation and Function

A cold-sensitive, streptomycin-sensitive mutant of Saccharomyces cerevisiae accumulates a 28S ribonucleoprotein particle when grown at low temperature. This particle contains 17S ribosomal ribonculeic acid which is degraded when exposed to ribonuclease. The particle does not serve as a precursor to 60 and 40S ribosomal subunits nor is it turned over when growth is allowed to resume at the permissive temperature; rather it is only diluted by growth. That streptomycin sensitivity (allelic with cold sensitivity) is ribosomal is evidenced by the inhibition of protein synthesis in vitro by streptomycin and the binding of labeled streptomycin to the mutant but not the parental 40S ribosomal subunit.     

Characterization of genetic markers in the 5′ flanking region of the apo A1 gene

Genetic variation of apoA1/C3/A4 is associated with hyperlipidaemia and coronary heart disease. We report the polymerase chain reaction (PCR) conditions for determining three polymorphic sites in the 5 flanking region of apoA1 using DNA prepared from small aliquots of whole blood. These polymorphisms identify six haplotypes that will be of value in genetic studies.     

Age-related changes in the composition and structure of human articular-cartilage proteoglycans.

1. Analysis of the purified proteoglycans extracted from normal human articular cartilage with 4M-guanidinium chloride showed that there was an age-related increase in their content of protein and keratan sulphate. 2. The hydrodynamic size of the dissociated proteoglycans also decreased with advancing age, but there was little change in the proportion that could aggregate. 3. Results suggested that some extracts of aged-human cartilage had an increased content of hyaluronic acid compared with specimens from younger patients. 4. Dissociated proteoglycans, from cartilage of all age groups, bind to hyaluronic acid and form aggregates in direct proportion to the hyaluronic acid concentration. 5. Electrophoretic heterogeneity of the dissociated proteoglycans was demonstrated on polyacrylamide/agarose gels. The number of proteoglycan species observed was also dependent on the age of the patient.