Studies on B. subtilis Ribonuclease. II. Molecular Weight and Physical Homogeneity

Studies on Bacillus subtilis Ribonuclease. III. Purification and Amino Acid Composition*

“…The new medium will also facilitate studies, included in our long-range plans, on the incorporation of radioactive isotopes and amino acid analogs into the enzyme. The amount of enzyme in the original culture fluid is only about 25% of that obtained in the neopeptone-starch medium (Rushizky et al, 1963;Hartley et al, 1963). However, the recovery of the final purified preparation was 26% as contrasted with 3 % obtained from the previous medium.…”

“…A later report (Nishimura, 1960) that this enzyme was specific for the secondary phosphate esters of purine riboside 3'-phosphates in ribonucleic acid (RNA), stimulated detailed chromatographic analysis of the products of enzyme action upon TMV-RNA (Whitfield and Witzel, 1963), and, in this laboratory, upon yeast RNA (Rushizky et al, 1963). Both groups found that although certain preferences were shown, both pyrimidine and purine linkages were hydrolyzed, and therefore the specificity was not as sharp as originally indicated. During the course of an investigation of the physical homogeneity of this enzyme (Hartley et al, 1963), evidence was obtained that its molecular weight was significantly lower than that of pancreatic ribonuclease (10,700 as opposed to 13,700). Since it is small, stable, lacks disulfide bonds, and is amenable to genetic manipulation, a detailed study of the chemical structure, enzymatic activity, and physical chemistry of this enzyme should be of interest.…”

“…Bacterial strains, enzyme activity units, ribonuclease assay, spectrophotometric measurements, oligonucleotide mapping, and scanning of disc electrophoresis gels have been described in previous papers from this laboratory (Rushizky et al, 1963;Hartley et al, 1963). All chemicals were reagent grade.…”

“…The percent nitrogen and per cent weight recoveries were 101.6 and 102.4%, respectively. Ninety-four residues per mole are calculated for molecular weight of 10,700 (Hartley et al, 1963).…”

Escherichia coli lactose repressor Isolation of two different homogeneous headpieces and the existence of a hinge region between residues 50 and 60 in the repressor molecule

“…Digestion with carboxypeptidases A and B was as in [7]. The amino-terminal residues were determined by the dansyl-procedure [9].…”