Characterization of soybean trypsin inhibitor sensitive protease from unfertilized sea urchin eggs

A serine protease from sea urchin eggs has been isolated by affinity chromatography on soybean trypsin inhibitor-agarose. Benzamidine hydrochloride was included to minimize autodegradation. We present data on the properties of the protease with respect to molecular weight and its interaction with trypsin inhibitors and substrates. The molecular weight of the enzyme is 47 000 by gel filtration under nonreducing conditions and 35 000 by electrophoresis in the presence of sodium dodecyl sulfate and dithiothreitol. The pH optimum and Km with N alpha-benzoyl-L-arginine ethyl ester (BAEE) are 8.0 and 75 microM, respectively. The specific activity is comparable to that of bovine pancreatic trypsin. Proteolytic activity was measured by beta-casein hydrolysis. The caseinolytic activity is completely inhibited by 1 mumol of soybean trypsin inhibitor (SBTI) per micromole of enzyme. BAEE esterase activity is inhibited competitively by SBTI (Ki = 1.6 nM), lima bean trypsin inhibitor (150 nM), chicken ovomucoid (100 nM), and leupeptin (130 nM). Bowman-Birk inhibitor, benzamidine hydrochloride, and antipain are also inhibitors of the purified enzyme. Inhibition by phenylmethanesulfonyl fluoride and N alpha-p-tosyl-L-lysine chloromethyl ketone indicates the presence of serine and histidine residues in the active center, respectively. The chymotrypsin inhibitor L-1-(tosylamido)-2-phenylethyl chloromethyl ketone is ineffective. The protease is susceptible to autodegradation which can result in the appearance of a minor 23-kilodalton component. The egg protease appears to be similar in many respects to trypsins and trypsin-like enzymes isolated from a wide variety of sources, including sea urchin and mammalian sperm.     

Proteolytic system of the sperm of Apis mellifera drones

During many insemination interventions semen coagulates already within the insemination needle, which considerably lengthens the duration of inseminating a single queen bee. Considering this, the authors decided to determine the type and activity of proteases and their inhibitors in normal and coagulated sperm. The samples were collected from mature and old drones. The sperm proteins were isolated in 1% Triton X-100. The samples containing isolated proteins were tested as follows: protein concentration assay by the Lowry method; proteolytic activity in relation to various substrates (gelatine, haemoglobin, ovoalbumin, albumin, cytochrome C, casein) by the modified Anson method; proteolytic activity in relation to diagnostic inhibitors of proteolytic enzymes (pepstatin A, PMSF, iodoacetamide, o-phenantrolin), using the Lee & Lin method; acidic, neutral and basic protease activity by means of the modified Anson method; electrophoretic analysis of proteins in a polyacrylamide gel for protease detection with the Laemmli method; the activity of aspartic and serine protease inhibitors by the Lee and Lin method; electrophoretic analysis of proteins in a polyacrylamide gel for protease inhibitor activity detection by means of the modified Felicioli method. The mixing of non-coagulated semen from different drones increased protein concentration. The activities of proteases were decreased in normal sperm samples as compared with a corresponding rise in the sperm mixture from many drones. The non-coagulated sperm samples were found to contain aspartic and serine proteases. Additionally, thiolic and metallic proteases were also found in the coagulated sperm samples. There was a rise in protease inhibitor activity at pH 3.0 and 12.0, and a fall at pH 7.0 after mixing the sperm samples collected from numerous drones. Oscillation in these activities stemmed from sperm coagulation.     

Presence of a trypsin-like protease in starfish sperm acrosome.

Marthasterias glacialis sperm cells were treated with ionophore A23187, centrifuged, and the supernatants were assayed for esterase activity. With N-benzoyl-L-arginine ethyl ester-HCl (BAEE) as substrate, a net activity was determined which was not detectable when N-acetyl-L-tyrosine ethyl ester (ATEE) was used. The BAEE trypsin-like activity was inhibited by soybean trypsin inhibitor (SBTI), N-alpha-p-tosyl-L-lysine chloromethyl ketone-HCl (TLCK), and phenyl methyl sulfonyl fluoride (PMSF), but not by L-1-tosylamido-2-phenylethyl chloromethyl ketone (TPCK). The presence of proteolytic activity in acrosomal exudates was further demonstrated by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic zymography (gelatin-SDS-PAGE). The presence of several bands of low proteolytic activity and of one band of high proteolytic activity, which also has the lower molecular weight, together with the fact that all are inhibited by benzamidine, suggests the existence of a trypsin-like proteinase system. The effect of the acrosomal exudate on the oocyte jelly coat was investigated by SDS-PAGE analysis. All jelly proteins appeared to be digested by the acrosomal enzymes. Furthermore, if SBTI is added shortly after insemination, the sperm fail to fertilize the oocytes. These results indicate that the starfish sperm acrosomal vesicle contains a trypsin-like protease which may be involved in sperm penetration through the oocyte jelly coat.     

The effect of dibutyryl cyclic AMP and butyrate on F9 teratocarcinoma cellular retinoic acid-binding protein activity

F9 teratocarcinoma cells contain a cellular retinoic acid-binding protein (CRABP) that may mediate the retinoic acid-induced differentiation of this cell line. Specific [3H]retinoic acid binding to CRABP in F9 stem cell cytosol is protein-dependent, reaches equilibrium within 4 h at 4 degrees C, and yields 643 +/- 105 fmol of [3H]retinoic acid per mg of protein with an apparent dissociation constant of 9.2 +/- 1.1 nM. When F9 stem cells are grown in the presence of either dibutyryl cyclic AMP or sodium butyrate, CRABP activity is stimulated 2-4-fold. The effect of these drugs on CRABP activity is both time and concentration-dependent, resulting in an increase in the number of binding sites for [3H]retinoic acid with no change in their affinity. The new [3H]retinoic acid-binding sites have a sedimentation coefficient of 2 S and are not displaced by excess retinol. When F9 stem cells are grown in the presence of cyclic 8-bromo-AMP or cholera toxin, no increase in CRABP activity is observed. We conclude that the stimulation of CRABP activity by dibutyryl cyclic AMP may result from the action of butyrate. In addition, the stimulation of retinoic acid-induced F9 cell differentiation by cyclic AMP analogs (Strickland, S., Smith, K.K., and Marotti, K.R. (1980) Cell 21, 347-355) and the inhibition of this differentiation by butyrate (Levine R. A., Campisi, J., Wang, S.-Y., and Gudas, L. J. (1984) Dev. Biol. 105, 443-450) are not correlated with increases or decreases, respectively, in the level of CRABP activity.     

Identification of serine esterases in tissue homogenates

Serine esterases react with [3H]diisopropylphosphofluoridate ([3H]DFP) to produce radioactive adducts that can be resolved by denaturing slab gel electrophoresis. To identify an esterase or its catalytic subunit, a potential substrate was included in the reaction mixture with the expectation that it would suppress the enzyme's reaction with [3H]DFP. The nature of the enzyme could be inferred from the character of the substrates that suppress labeling. The validity of this analytical method was tested with two serine proteases, trypsin and alpha-chymotrypsin, and two serine esterases, acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE), and several of their natural or model substrates or inhibitors. Application of the method to complex biological systems was tested with chicken embryo brain microsomes. Trypsin labeling with [3H]DFP was suppressed by alpha-N-benzoyl-l-arginine ethyl ester (BAEE) and poly-l-lysine but not by benzoyl-l-tyrosine ethyl ester (BTEE). [3H]DFP labeling of chymotrypsin was suppressed by both BAEE and BTEE. Labeling of AChE and BuChE was suppressed by their natural and some related substrates and inhibitors. [3H]DFP reacted with brain microsomes to produce nine distinct radioactive bands. When the relevant substrates and inhibitors of AChE were included in the reaction mixtures, labeling of only the 95-kDa band was suppressed, implicating it as AChE. Labeling of the 85- and 79-kDa bands was inhibited by butyrylcholine, suggesting that these proteins have BuChE activity.     

Biochemical and functional properties of serine esterases in acidic cytoplasmic granules of cytotoxic T lymphocytes

Percoll gradient fractions of homogenates of murine cloned cytotoxic T lymphocytes (CTL) were analyzed for the trypsin-like enzyme alpha-N-benzyloxy-carbonyl-L-lysinethiobenzyl ester (BLT) esterase recently described in CTL homogenates. Enzymatic activity was found in three areas of the gradient: the dense cytolysin containing granules; a light granule fraction; and a variable amount in the soluble fraction at the top of the gradient. Gel filtration columns showed a major peak of BLT esterase activity eluted at the position of a 60-kDa protein, and an additional, minor BLT esterase peak eluting at about 27 kDa. The separated enzymes were both significantly inhibited by the serine protease inhibitors diisopropylfluorophosphate and phenylmethyl sulfonyl fluoride (PMSF), indicating they are both serine proteases, but showed different patterns of inhibition by a series of inhibitors, suggesting the larger enzyme is not a simple dimer of the smaller. pH activity profiles of both CTL BLT esterases showed an optimum at about pH 8. PMSF inactivation of BLT esterase in detergent extracts of CTL diminished sharply as the pH was dropped below 7. Agents which raise the pH of acidic intracellular compartments were found to markedly enhance the PMSF inactivation of BLT esterase in intact CTL, showing that the granules have a low internal pH. Similarly, [3H]diisopropylfluorophosphate labeling of intact CTL gave four protein bands on non-reduced gels, of which two were labeled threefold more effectively in the presence of chloroquine. In parallel studies of inactivation of CTL lytic activity, PMSF pretreatment caused a 50% reduction of the lytic activity under conditions where greater than 90% of the BLT esterase activity was inactivated. Addition of agents raising the intragranular pH dramatically enhanced the BLT esterase inactivation but did not concomitantly reduce CTL lytic activity. These results indicate that inactivation of lytic function by PMSF is unlikely to be due to its reaction with protease in acidic granules, and suggest that the activity of these enzymes may not be required for cytotoxicity.     

Papain kinetics in the presence of a water-miscible organic solvent

The effects of various concentrations of a water-miscible organic solvent [a 7:3 (v/v) mixture of N, N dimethylformamide and dimethylsulfoxide] on the kinetics of papain have been investigated. The parameters k(cat) and K(m) for the amidase and esterase activity of papain using N-alpha-benzoyl-DL-arginine-p-nitroanilide (BAPNA) and N-alpha-benzoyl-L-arginine ethyl ester (BAEE) as substrates were determined. For both types of activity, k(cat) initially increased (up to about 15% solvent), and then decreased with increasing concentrations of organic solvent. In contrast, K(m) increased sharply with the organic solvent concentration. Active site titration at 0 and 50% solvent indicated no change in the amount of active enzyme. Fluorometric measurements of the emission spectrum of papain did not indicate any major conformational changes with increasing concentrations of organic solvent.     

Role of serine esterase in hydrogen peroxide-mediated activation of phospholipase A2 in rabbit pulmonary arterial smooth muscle cells.

Exposure of rabbit pulmonary arterial smooth muscle cells to hydrogen peroxide cause dose-dependent stimulation of [14C] arachidonic acid (AA) release and enhancement of the cell membrane-associated phospholipase A2 activity as well as of the cell membrane-bound serine esterase activity tested against synthetic substrate p-tosyl-L-arginine methyl ester. While pretreatment of cells with serine protease inhibitors, viz. phenyl methyl sulphonyl fluoride, diisopropyl fluorophosphate and alpha-1-proteinase inhibitor, and antioxidant vitamin E prevents H2O2 stimulation of AA release and the cell membrane-bound serine esterase and PLA2 activities, that with actinomycin D and cycloheximide is devoid of any effect on H2O2 caused stimulation of AA release and the smooth muscle cell membrane associated serine esterase and PLA2 activities. Treatment of the smooth muscle cell membrane suspension with the serine protease trypsin markedly stimulates PLA2 activity. These results suggest that on exposure to H2O2 the smooth muscle cell membrane-bound serine esterase plays an important role in stimulating the cell membrane associated PLA2 activity thereby resulting in an increase in AA release.     

Reproductive characteristics of Rayini male goats of Kerman province in Iran

An artificial vagina was used to collect semen from 14 native Iranian Rayini goats, at 15-day intervals starting on 1 July and ending on 30 December 2000. Testicular size, semen volume, sperm concentration, percent live sperm, percent normal sperm, and total number of live-normal sperm were significantly higher during the summer months. The average semen volume, percent live sperm and percent abnormal sperm during the sampling period varied between 1 and 1.4 mL, 60 and 78%, and 7 and 13%, respectively. The total number of live and normal sperm in the ejaculate during the sampling period varied from 1000 to 2500 million. Testicular size, semen volume and the total number of live and normal sperm were significantly greater in bucks weighing 55-60 kg as compared with 50-54 kg. Seminal fluid pH values were significantly lower from July to October (pH <6) than the values from November to December (pH >6.1). Lowest level of lactate dehydrogenase in the seminal fluid was recorded in early September (2.2 U/mL) and the highest level in November (2.5 U/mL). Seminal fluid K ion level increased gradually from July (52 mg/dL) to the November (97 mg/dL). Variation of seminal fluid Na ion concentration (71-74 mg/dL) was not significant during the sampling period. The correlation coefficients of total number of live-normal sperm with seminal fluid K level (r=-0.65) and LDH (r=-0.36) were negative (P<0.01). The data indicated that the semen quality and quantity of Rayini bucks were higher during summer and early autumn.     

Purification and characterization of a novel thiol protease involved in processing the enkephalin precursor

Proteolytic processing enzymes are required to convert the enkephalin precursor to active opioid peptides. In this study, a novel 33-kDa thiol protease that cleaves complete precursor in the form of [35S]methionine preproenkephalin was purified from bovine adrenal medullary chromaffin granules. Chromatography on concanavalin A-Sepharose and Sephacryl S-200, chromatofocusing, and chromatography on thiopropyl-Sepharose resulted in an 88,000-fold purification with a recovery of 35% of enzyme activity. The thiol protease is a glycoprotein with a pI of 6.0. It cleaves [35S]methionine preproenkephalin with a pH optimum of 5.5, indicating that it is functional at the intragranular pH of 5.5-6.0. Interestingly, production of trichloroacetic acid-soluble products was optimal at pH 4.0, suggesting that processing of initial precursor and intermediates may require slightly different pH conditions. The protease requires dithiothreitol for activity and is inhibited by the thiol protease inhibitors iodoacetate, p-hydroxymercuribenzoate, mercuric chloride, and cystatin. These properties distinguish it from other thiol proteases (cathepsins B, H, L, N, and S), indicating that a unique thiol protease has been identified. The enzyme converted [35S]cysteine preproenkephalin (possessing [35S]cysteine residues specifically within the precursor's NH2-terminal segment) to 22.1-, 21.6-, 17.7-, 17.3-, and 15.0-kDa intermediates that contain the precursor's NH2-terminal segment; proenkephalin in vivo is converted to similar intermediates. The enzyme cleaves peptide F at Lys-Arg and Lys-Lys dibasic amino acid sites to generate methionine enkephalin and intermediates. The appropriate vesicular localization, pH optimum, proteolytic products, and cleavage site specificity suggest that this thiol protease may be involved in enkephalin precursor processing. Most interestingly, [35S]methionine beta-preprotachykinin, a precursor of substance P, is minimally cleaved, suggesting that the thiol protease may possess some selectivity for the enkephalin precursor.     

Correlation of increased intraacrosomal pH with the hamster sperm acrosome reaction

This study describes investigations of the importance of intraacrosomal pH in the hamster sperm acrosome reaction (AR). Washed cauda epididymal sperm were capacitated in vitro in a medium containing 2 mM Ca2+, 144 mM Na+, and 3 mM K+. Such sperm underwent a significant increase in the number of AR within 10 min after the addition of the Mg2+-ATPase (adenosine triphosphatase) inhibitors DCCD (20 microM) or NBD-Cl (10 microM) or the proton ionophore FCCP (6 micrograms/ml) at 3.5 hr of incubation or after addition of HN4Cl (3 mM) at 4 hr of incubation. Addition of the mitochondrial electron transport inhibitor rotenone (2.5 microM) at 3.5 hr or of NaCl (3 mM) or KCl (3 mM) at 4 hr did not stimulate AR over control levels, suggesting that the stimulation of AR by the other compounds was not directly due to depletion of acrosomal adenosine triphosphate (ATP) or alteration of the acrosomal transmembrane potential. The AR also was not stimulated by either DCCD or FCCP added prior to 3 hr of incubation of sperm, whereas both compounds were increasingly effective at stimulating AR with increasing length of preincubation of sperm before the addition of the test compounds. The intraacrosomal pH of sperm incubated in low [K+] (0.6-0.9 mM) for 3.5 hr rose by at least one pH unit (as measured with the fluorescent dye 9-aminoacridine) within 15-30 min after raising extracellular [K+] to 4.2-4.5 mM. The pH rise occurred even in the presence of the Ca2+-chelator EGTA (2 mM). Either FCCP (8 micrograms/ml) or DCCD (20 microM), but not rotenone (2.5 microM), plus K+ (3.6 mM), raised the intraacrosomal pH of sperm incubated for 3 hr in low [K+] within 10 min after addition. No pH rise occurred in the absence of additional K+. These results demonstrate that the intraacrosomal pH of the hamster sperm becomes more alkaline in a process not requiring high concentrations of external Ca2+, but requiring K+. The results of this and previous studies lead us to suggest here that the intraacrosomal pH rise may be mediated via a change in K+ and H+ permeability of sperm head membranes, which allows K+ influx and H+ efflux, and via inhibition of an acrosomal Mg2+-ATPase proton pump. We propose that the permeability changes and the consequent alkalinization of the acrosomal interior are important steps in late capacitation and/or the mammalian AR.     

Comparison of soluble and immobilized trypsin kinetics: Implications for peptide synthesis

The protease trypsin was immobilized to porous glass in both the presence and absence of acetylated soybean trypsin inhibitor (STI) to determine whether immobilization could alter enzyme activity in favor of aminolysis over hydrolysis. Actiive-site titration with 4-methylumbelliferylguanidinobenzoate (MUGB) showed that only about 10% of immobilized trypsin had catalytic activity. Immobilization in the presence of STI produced a higher yield of active enzyme accessible to the inhibitor but did not increase the total yield of MUGB-active immobilized enzyme. Thus, enzyme inactivation upon immobilization could not be attributed to an inaccessible enzyme orientation, nor did STI prevent inactivation by stabilizing the active-site conformation. Kinetic parameters were determined for soluble and immobilized trypsin for two esters, N-tosyl-L-arginine methyl ester (TAME) and N-benzoyl-L-arginine ethyl ester (BAEE), and two amides, N-benzoyl-L-arginine p-nitroanilide (BAPNA) and N-t-boc-leucylglycylarginine p-nitroanilide (LGRNA). In all cases, immobilization caused a greater decrease in k(cat) for amidase activity than for esterase activity. The ratio [k(cat)/ K(m) (ester)]/[k(cat)/K(m) (amide)] increased slightly or stayed the same (for I.GRNA) or decreased sharply (for BAPNA). Including STI during immobilization had little effect on the active enzyme's intrinsic kinetics. A direct comparison of energy diagrams and free energies of activation for BAEE and BAPNA indicates that immobilization raises the free energy barriers for both amide and ester hydrolysis and lowers the energy barrier for aminolysis. In practice, these effects should lower the amidase activity and increase the aminolysis-hydrolysis ratio, rendering the immobilized enzyme a more efficient catalyst for peptide synthesis. (c) 1993 John Wiley & Sons, Inc.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

In vitro maturation of the potential for movement of carp spermatozoa

Carp semen obtained from isolated fish after hormonal stimulation was highly variable in terms of volume of semen, osmotic pressure of the seminal plasma, and sperm capacity to move. Moreover, this last parameter was unstable when the spermatozoa were kept within the seminal plasma, and the present work was designed to investigate and possibly correct this phenomenon. Sperm potential movement was the major parameter studied and was measured by the percentage of motile cells in a final 3.000-fold dilution in a medium of low osmotic pressure in which sperm movement is known to occur (Morisawa and Suzuki, Science 210:1145-1147, 1980). This was completed with occasional measurements of flagellar beat frequencies and demembranation-reactivation of axonemal movement. The results showed that sperm potential movement was preserved upon dilution of the semen into cold 200 mM KCl medium and that semen of initially "poor" quality or spermatozoa that had lost their capacity to move during storage in the semen recovered gradually their potential movement during incubation at 2 degrees C in the same medium. The K+ dependence for both the conservation and the regeneration of sperm capacity to move showed a minimal requirement of 50 mM KCl in media of high osmotic pressure. Na+ ions had similar properties but not divalent cations. The K+ activation was not pH dependent between pH 9.03 and 6.04. Whatever the functional state of live spermatozoa, demembranation-reactivation occurred in ATP-Mg2+. It is concluded that, with dilution of the semen in appropriate conditions, carp spermatozoa retain or acquire potential movement and therefore are a lower vertebrate spermatozoa model available year-round. In addition, obtaining potentially nonmotile sperm and reversion in vitro might be useful to study the control of in vitro maturation.     

Activation of Xenopus Eggs by an Extract of Cynops Sperm

An extract obtained from Cynops sperm induced the activation of both Cynops and Xenopus eggs with accompanying changes in the potential of the egg membrane that were quite similar to those caused by the Cynops sperm. The activation-inducing properties of the extract were abolished by treatment with proteinase K or by heating (60°C, 15 min) and were associated with a protease activity against peptidyl Arg-MCA substrates. The activation of Xenopus eggs by the extract was inhibited by those substrates, or by protease inhibitors, aprotinin or leupeptin. The protease activity was localized in the acrosomal region of Cynops sperm. The activation of Xenopus eggs by the extract was prevented when the exterior concentration of Ca²⁺ions, [Ca²⁺]₀, was reduced to 1.5 μM, but it was enhanced when [Ca²⁺]₀ was increased to 340 μM. The activation of Xenopus eggs by the extract was not affected by positive clamping when [Ca²⁺]₀ was 340 μM. These results suggest that the sperm extract contains a protease that causes an increase in the influx of Ca²⁺ions that results in voltage-insensitive activation of the egg.     

The influence of various cryoprotectants on semen quality of the rainbow trout (Oncorhynchus mykiss) before and after cryopreservation

The influence of permeating and of non-permeating cryoprotectants on motility, eosin permeability (sperm cells viability test) and leaking lactate dehydrogenase activity of spermatozoa of the rainbow trout ( Oncorhynchus mykiss ) was investigated. Correlations between sperm motility rate, percentage of eosinimpermeable (viable) cells and LDH activities established the three parameters as indicators for damages to rainbow trout spermatozoa. High sperm motility rates, velocities, and numbers of linear swimming sperm cells, high numbers of eosinimpermeable spermatozoa and a low extracellular LDH activity were evaluated as positive quality characteristics of semen. With a buffered physiological saline solution as basic extender we found a mixture of 0.67 mol L (5%) dimethylsulfoxide (DMSO) and 0.13 mol/L (1%) glycerol to be the most effective cryoprotectant, followed by DMSO (0.67 mol/L [5%]-1.85 mol/L [15%]) and glycerol (0.65 mol/L [5%]-1.78 mol/L [15%]) and propanediol (1.24mol L [10%]-1.78 mol/L [15%]). Addition of hen egg yolk (7%, 20%) and of 15 mmol/L (0.5%) sucrose significantly increased the semen quality in comparison to the same extender without these additives. Bovine serum albumin (1.5%, 3%) had no influence on the investigated semen parameters.     

A solid phase assay for the protease of human immunodeficiency virus

A solid phase assay for human immunodeficiency virus (HIV) protease using an immobilized substrate, Affi Gel 10-Gly-Gly-Gly-Gly-Val-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-[3H]Gly-OH has been devised. The Tyr-Pro bond of the substrate was hydrolyzed by the protease, releasing the radiolabeled cleavage product, Pro-Ile-Val-Gln-[3H]Gly-OH, into the supernatant. The pH optimum was found to be 6.0, and a high ionic strength was required for maximal activity. The solid phase assay is usable for convenient monitoring of purification procedures, and rapid screening of inhibitors of HIV protease.     

Purification and Characterization of a Neutral Protease from Saccharomycopsis lipolytica

Saccharomycopsis lipolytica 37-1 produced two inducible extracellular proteases, one under neutral or alkaline growth conditions and the second under acid conditions. Secretion of the neutral protease was repressed in the presence of glycerol or glucose, both of which supported rapid growth of the organism. Ammonium ions also repressed the secretion of the enzyme. The neutral protease activity copurified with esterase activity during ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose, and gel filtration on Sephadex G-150. The molecular weight of the enzyme was estimated to be 42,000 by sucrose density gradient centrifugation and 38,500 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The purified enzyme had a pH optimum of 6.8. Phenylmethylsulfonylfluoride inhibited both protease and esterase activities, indicating the presence of a serine residue in the active center. Protease, but not esterase, activity was sensitive to ethylenediaminetetraacetate and was significantly activated by divalent ions. Dithiothreitol inhibited both protease and esterase activities, indicating the presence of a critical disulfide bridge. The enzyme hydrolyzed casein (K(m) = 25.6 muM) and hemoglobin as well as the nitrophenyl esters of tyrosine (K(m) = 2.4 mM), glycine, tryptophan, and phenylalanine.     

土壤中高产蛋白酶菌株产酶条件及酶学性质

【背景】微生物蛋白酶已经成为工业用蛋白酶的主要来源,筛选具有特殊环境适应性的微生物成为生物酶资源的开发热点。【目的】通过对青藏高原土壤微生物产蛋白酶菌株的筛选、优化及相关特性研究,寻找新的蛋白酶资源,为高原菌种资源利用提供科学依据。【方法】采用形态学和分子生物学对筛选菌株进行菌种鉴定,利用单因素试验和正交试验对菌株进行发酵条件优化及酶学性质的探究。【结果】筛选出一株高产蛋白酶菌株XC2,经鉴定菌株XC2为枯草芽孢杆菌(Bacillus subtilis)。XC2最优产酶条件:可溶性淀粉4.0%,牛肉膏1.0%,K~+0.6%,培养温度34°C、初始pH 7.0、接种量2.0%的条件下200 r/min振荡培养13 h,所产蛋白酶活力最高为638.5 U/mL。XC2所产蛋白酶最适反应温度60°C,最适pH9.0;40-50°C、pH8.0-10.0条件下酶活稳定性较高;Mn~(2+)对酶活力有明显激活作用,而Zn~(2+)、Cu~(2+)、Fe~(2+)、Fe3+对酶活力有明显抑制作用。【结论】枯草芽孢杆菌XC2有较强的产碱性蛋白酶的能力,具有较好的应用前景。