Evidence for proteolytic degradation of histidinol phosphate phosphatase specified by nonsense mutants of the hisB gene of Salmonella typhimurium.
The gene products from hisB nonsense mutants having histidinol phosphate phosphatase activity were isolated from Salmonella typhimurium. The enzyme from strain TR691 (hisB278 hisT1529 aroD5) was isolated in the presence of diisopropylfluorophosphate. Three electrophoretically separable forms were demonstrated, and all were shown to have a mol wt of approximately 38,000 and to consist of a single polypeptide chain. Previously, two forms of the phosphatase enzyme from this strain were isolated without diisopropylfluorophosphate and shown to have a different subunit composition. Strain TA387 (hisB2133 his 01242) was shown to have two electrophoretically separable phosphatases with a mol wt of about 52,000 and consisted of 17,000- to 19,000-mol wt polypeptide chains as evidenced by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The results could be explained by proteolytic cleavage of the primary gene product which can partially be prevented by the protease inhibitor. Strain TA387 phosphatase lost all activity in 8 M urea but could be renatured by dialysis. Gel filtration showed that it also regained its original molecular weight. The values of K(m) of histidinol phosphate and the competition inhibition constant for histidinol were determined. The addition of MnCl(2) to the assay was shown to shift the optimal pH value to a lower pH value.
D000605:Amino Alcohols; D002474:Cell-Free System; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005796:Genes; D006639:Histidine; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007531:Isoflurophate / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008970:Molecular Weight; D009153:Mutagens; D009154:Mutation; D009604:Nitrosoguanidines; D010455:Peptides / Q000032:analysis; D010744:Phosphoric Monoester Hydrolases / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*; D011489:Protein Denaturation; D012486:Salmonella typhimurium / Q000201:enzymology*; D014508:Urea / Q000494:pharmacology
D000605:Amino Alcohols; D009153:Mutagens; D009604:Nitrosoguanidines; D010455:Peptides; D007531:Isoflurophate; D008345:Manganese; D006639:Histidine; D014508:Urea; D010744:Phosphoric Monoester Hydrolases