Nitrate reductase activity in heme-deficient mutants of Staphylococcus aureus

The cytoplasmic nitrate reductase in heme mutant H-14 of Staphylococcus aureus was partially purified by steps which included ammonium sulfate fractionation and chromatography on Bio-Gel A 1.5m and ion-exchange columns. The active fractions from the ion-exchange columns showed two forms of the enzyme upon electrophoresis in nondenaturing gels of polyacrylamide; these corresponded to proteins of R f 0.16 and 0.28. Each form contained a predominant polypeptide of molecular weight 140,000, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The R f 0.16 form contained another major polypeptide of molecular weight 57,000, but the R f 0.28 form contained several other polypeptides. The sedimentation properties of the enzyme were examined after partial purification on Bio-Gel A 1.5m. In sucrose gradients containing Triton X-100 the enzyme sedimented as a homogeneous peak with an estimated molecular weight of 225,000; without detergent a heterogeneous profile was observed of molecular weight greater than 250,000. Treatment of the enzyme with trypsin increased the specific activity, and the enzyme sedimented as a homogeneous peak in sucrose gradients without Triton X-100, with an estimated molecular weight of 202,000. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicated that trypsin treatment converted the polypeptide of molecular weight 140,000 to a polypeptide of molecular weight 112,000. We conclude that the cytoplasmic nitrate reductase of S. aureus has a large subunit of molecular weight 140,000, which can be modified by trypsin to a polypeptide of molecular weight 112,000 without loss of catalytic activity.

mentioning

confidence: 99%

Partial Purification and Some Properties of the Staphylococcus aureus Cytoplasmic Nitrate Reductase

Burke

Lascelles

1979

“…Reconstruction of G3P-nitrate reductase activity in mutant H-14. hem mutants of S. aureus, including H-14, fonn apo-cytochrome when grown without hemin; the addition of hemin in vitro to depleted membranes restores apparently normal b-type cytochrome (2). To determine whether G3P-nitrate reductase activity could be reconstituted, heme-depleted membranes with G3P dehydrogenase and nitrate reductase activities were prepared from mutant H-14 after a two-step induction procedure (Table 4).…”

Section: Resultsmentioning

confidence: 99%

“…This suggests that apo-cytochrome is formed by the mutant, which can readily combine in vitro with the heme prosthetic group to reconstitute functional cytochrome. The reconstructed system is also capable of coupling the L-lactate dehydrogenase with nitrate reductase (2).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

sn-Glycerol-3-phosphate dehydrogenase and its interaction with nitrate reductase in wild-type and hem mutant strains of Staphylococcus aureus

Lascelles¹

1978

Self Cite

Staphylococcus aureus has membrane-associated sn-glycerol-3-phosphate dehydrogenase activity that is strongly activated by detergents. The enzyme can be measured spectrophotometrically in intact cells in assay systems containing lauryldimethylamine oxide (Ammonyx LO). The dehydrogenase activity was located exclusively in the membrane fraction of cells grown with glycerol under aerobic conditions or under anaerobic conditions with the addition of nitrate; there was no evidence of multiple forms. Development of sn-glycerol-3-phosphate dehydrogenase activity was studied with suspensions of cells grown previously under semianaerobic conditions with glucose and nitrate. The wild-type strain rapidly formed the enzyme when incubated with glycerol under aerobic conditions or under semianaerobic conditions in the presence of nitrate. Under similar conditions, suspensions of hem mutant H-14 required the addition of hemin. Induction of the enzyme was strongly repressed by glucose with both organisms. A procedure was established to obtain cells of mutant H-14 with sn-glycerol-3-phosphate dehydrogenase and nitrate reductase activities, but which could not link the systems unless supplemented with hemin. The coupled activity could also be reconstructed in vitro by the addition of hemin to the depleted membranes.

“…Previous studies have shown that in hemerequiring strains of B. melaninogenicus (15), B. ruminicola (5,19), B. fragilis (13), S. aureus (3), and Haemophilus parainfluenzae (18), hemin is utilized for the synthesis of cytochrome of type b or c. We, therefore, investigated whether or not C. pyogenes contains cytochrome(s) and whether the synthesis of such a cytochrome is dependent on the presence of hemin in the medium. Cells were grown aerobically on medium of tryptose broth plus minerals with and without hemin, harvested by centrifugation, and washed three times in ten volumes of potassium phosphate buffer, pH 7.0.…”

mentioning

confidence: 99%

Hemin-dependent growth stimulation and cytochrome synthesis in Corynebacterium pyogenes

Reddy¹,

Cornell²,

M³

1977