Abstract
Abstract A distinct change occurs in the molecular size of the enzyme, histidinol phosphate phosphatase, specified by nonsense mutants with lesions near the middle of hisB. The hisB gene of Salmonella typhimurium controls two different activities, imidazoleglycerolphosphate dehydratase and histidinol phosphate phosphatase, both of which reside in a single protein. The phosphatase activity from mutants with lesions distal to this transition point appears in or near the void volume of Sephadex G-200, whereas that from mutants with lesions proximal to the transition point produce a polypeptide with little or no activity. One mutant with its lesion near this transition point was chosen for further study. The phosphatase enzyme was purified from strain TR691, a non-complementing UGA hisB278 mutant. Two forms of the enzyme were found by preparative polyacrylamide gel electrophoresis. One form had a molecular weight of 38,000 and was a dimer of 18,000 molecular weight polypeptide chains as shown by electrophoresis in sodium dodecyl sulfate. The other form of the enzyme had a molecular weight of 47,000 and was a dimer of dissimilar chains with molecular weights of 38,000 and 11,500. The pH optima for catalytic activity of both forms was 7.0 in the absence of MnCl2 and 6.5 in the presence of 1.0 mm MnCl2. The Km for histidinol phosphate was 0.83 mm and was unchanged by the addition of Mn2+, although the velocity at the pH optimum increased about 30%. No difference in the Ki between the two forms could be detected towards the competitive inhibitor, histidinol. The mutant enzymes had no imidazoleglycerolphosphate dehydratase activity and did not cleave p-nitrophenylphosphate. It is possible that the 38,000 molecular weight form is produced by limited proteolysis of the 47,000 molecular weight form. Preliminary experiments using trypsin and subtilisin NOVO did not show this conversion; however, the 11,500 molecular weight component appeared to be very sensitive to this treatment.
Highlights
Nonsense mutants with their lesions in the operator proxirnal region specify a polypeptide with littlo or no histidinol phosphate phosphatase activity; strains with nonsense lesions in the distal region specify a protein which has a high molecular weight similar to the native enzyme
Purification of Two Forms of Histidinol Phcsphate Phmphatase-The specific activity of histidinol phosphate phosphatase of TR691 crude extract varied between 0.6 and 5.9 pmoles of histidinol phosphate hydrolyzed per hour per mg of protein after dialysis
No activity towards 0.01 M p-nitrophenylphosphate was found within the peak of histidinol phosphate phosphatase activity
Summary
A distinct change occurs in the molecular size of the enzyme, histidinol phosphate phosphatase, specified by nonsense mutants with lesions near the middle of hisB. Recent evidence obtained in this laboratory has demonstrated a transition point near the middle of h&-B that defines a subregion within the gene [8] Nonsense mutants with their lesions in the operator proxirnal region specify a polypeptide with littlo or no histidinol phosphate phosphatase activity; strains with nonsense lesions in the distal region specify a protein which has a high molecular weight similar to the native enzyme. Preparative Gel Electrophoresis-The active fractions in the low molecular weight range from Sephadex G-75 chromatography were pooled and concentrated to about 10 ml by ultrafiltration using a UM-2 membrane; PM-10 membranes were not used because they allow subst)antial quantities of the dilute enzyme to escape.
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