No abstract

Sperm cytosolic pH, determined by the spectral properties of intracellular carboxyfluorescein, is decreased rapidly by the diffusion and subsequent dissociation of the uncharged weak acids pyruvic, lactic, or hydroxybutyric and is increased by diffusion and subsequent intracellular protonation of the weak base NH3. Metabolic and kinetic activity increases dramatically when intracellular pH is elevated above 6.8-6.9 by addition of 50 mM NH4CI to sperm suspended in a 120 mM NaCl medium. Respiratory stimulation is not observed upon comparable additions of 50 mM Li' or K+ or when the pH of the medium is increased from 6.5 to 8.2. However, increases of the external pH to 7.8-8.2 in medium employing 120 mM KCI result in increased metabolic and kinetic activity, comparable to the maximal stimulation induced by the phosphodiesterase inhibitor caffeine. An increase in cytosolic pH from 6.3-6.6 to 6.8 occurs concomitant with the respiratory stimulation induced by KCI in alkaline media. No change in cytosolic pH follows addition of caffeine. Cyclic AMP-dependent protein kinase activity ratios, determined in cellular extracts, are increased by caffeine treatment but are not elevated by 120 mM KCI, by alkaline pH, or by their combination. These observations indicate that cytosolic pH plays a role in the regulation of motility and metabolism ofmammalian sperm that is not mediated by cyclic AMP but that may be under control of a plasma membrane voltage-dependent proton channel. However, H+ fluxes across vesicles prepared from sperm membranes are unaffected by variation in the magnitude of the transvesicular K+ concentration gradient.Processes mediated by the intracellular concentrations ofcyclic AMP (1,2) and Ca2+ (3,4) play important roles in regulating function in both invertebrate and mammalian spermatozoa (5, 6). Initial observations of the consequences of treating intact sperm with specific ionophores (7) and of the pH dependence of the activity of isolated sperm components in vitro (8) indicated that intracellular pH also may control exocytotic or contractile events in these cells. In addition a Na+/H+ exchange mechanism has been implicated in the initiation of rat sperm motility (9) and in the stimulation of sperm motility and respiration that is induced by the "speract" peptide released from sea urchin eggs (10).These contentions were supported by estimations of intracellular pH before (10-12) and after (11) the exocytotic acrosome reaction of sea urchin (11) or hamster (12) sperm and by apparent changes in internal pH during Na+-dependent initiation of sea urchin (13) and rat (9) sperm motility. However, the observed net proton release from sperm suspensions (10) or the changes in the pH of cell homogenates (9) or in the distribution of fluorescent amines (11-13) are difficult to relate to true quantitative alterations in intracellular pH.Recently the spectral properties ofcarboxyfluorescein, generated intracellularly from its permeant diacetate derivative, were used to determine cytosolic pH of ascites tumor...

We have purified a minor extracellular serine protease from a strain of Bacillus subtilis bearing null mutations in five extracellular protease genes: apr, npr, epr, bpr, and mpr

The gene for a novel extracellular metalioprotease was cloned, and its nucleotide sequence was determined. The gene (mpr) encodes a primary product of 313 amino acids that has little similarity to other known Bacillus proteases. The amnmo acid sequence of the mature protease was preceded by a signal sequence of approximately 34 amino acids and a pro sequence of 58 amino acids. Four cysteine residues were found in the deduced amino acid sequence of the mature protein, indicating the possible presence of disulfide bonds. The mpr gene mapped in the cysA-arol region of the chromosome and was not required for growth or sporulation.The gram-positive, spore-forming bacterium Bacillus subtilis produces and secretes proteases, esterases, and other types of exoenzymes at the end of the exponential phase of growth (15). The principal extracellular proteolytic enzymes, alkaline (subtilisin) and neutral (metallo-) proteases, are encoded by the apr and npr genes, respectively (11,24,26,29). In addition, the genes for two minor extracellular proteases, epr (21) and bpr (encoding bacillopeptidase F; A. Sloma, G. A. Rufo, Jr., C. F. Rudolph, B. J. Sullivan, K. A. Theriault, and J. Pero, submitted for publication) have been identified. Strains bearing null mutations in these four protease genes and the major intracellular protease gene isp-i (12) still produce extracellular protease activity. The majority of the residual protease activity can be attributed to a novel cysteine-containing metalloprotease (Mpr) that has been purified to apparent homogeneity (17). Here we report the cloning of the gene encoding this cysteine-containing protease. MATERIALS AND METHODSBacterial strains and plasmids. B. subtilis strains are listed in Table 1. Plasmid pBs81/6 is a derivative of pBD64 (8). Plasmids pBR322 and pUC19 were used for cloning into Escherichia coli DH5 cells obtained from Bethesda Research Laboratories, Inc., Gaithersburg, Md. The cat gene was obtained from plasmid pMI1101 (30), and the ble gene was obtained from pUB110 (20). B. subtilis strains were grown on tryptose blood agar base or minimal glucose medium and were made competent by the procedure of Anagnostopoulos and Spizizen (1). Selection for phleomycin resistance was carried out on tryptose blood agar base by the overlayer method after a 1.5-h delay at 37°C to allow for expression.The final concentration of phleomycin was 2 jig/ml. Plasmid DNA from B. subtilis and E. coli was prepared by the alkaline lysis method of Birnboim and Doly (3). Plasmid DNA transformation in B. subtilis was performed as described by Gryczan et al. Amino acid sequence determination. The N-terminal amino acid sequence of purified Mpr was determined by automated sequential Edman degradation with subsequent identification and quantification of phenylthiohydantoin-labeled amino acids by reverse-phase high-performance liquid chromatography. Additional amino acid sequences were obtained from internal fragments of Mpr generated by trypsin digestion. The purified enzyme was incubated with trypsin, and t...

We have purified a minor extracellular serine protease from Bacillus subtilis. Characterization of this enzyme indicated that it was most likely the previously reported enzyme bacillopeptidase F. The amino-terminal sequence of the purified protein was determined, and a "guess-mer" oligonucleotide hybridization probe was constructed on the basis of that sequence. This probe was used to identify and clone the structural gene (bpr) for bacillopeptidase F. The deduced amino acid sequence for the mature protein (496 amino acids) was preceded by a putative signal sequence of 30 residues and a putative propeptide region of 164 amino acids. The bpr gene mapped near pyrD on the chromosome and was not required for growth or sporulation.