Tosylphenylalanine Chloromethyl Ketone Research Articles

The relevance of gap junctions to stage I tumor promotion in mouse epidermis.

A previous paper reports that the potent tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), has a time-dependent effect on mouse epidermal gap junctions. A single topical application of 1.0 micrograms TPA results in the absence of gap junctions from mouse interfollicular epidermis between 18 and 30 h post-treatment. This paper describes the dose-dependent effect of TPA on mouse epidermis. Observations indicate that only promoting doses of TPA affect the gap junctions. Similarly, while a low dose of the hyperplasiogenic compound mezerein (1.0 microgram) is ineffective, a higher dose (4.0 micrograms) results in a significant reduction in the gap junction number. One and two applications of TPA had identical effects. The potent inhibitor of both stage I and stage II of tumor promotion, Fluocinolone acetonide, used in combination with TPA, completely suppressed the hyperplasiogenic and the gap junction modulating effects of TPA. Retinoic acid, which inhibits only stage II of tumor promotion, did not influence the gap junction eliminating property of TPA. Tosylphenylalanine chloromethyl ketone which is a mild but specific inhibitor of only stage I of tumor promotion counteracted the action of TPA on gap junctions to some extent, which remained present in smaller numbers than in normal tissue at 24 h after the treatment. These results suggest that gap junctions are essential and specifically relevant to stage I tumor promotion.

サケ科魚類せっそう病原因菌Ａｅｒｏｍｏｎａｓ ｓａｌｍｏｎｉｃｉｄａの産生する病因物質について ＩＩＩ Ａｅｒｏｍｏｎａｓ ｓａｌｍｏｎｉｃｉｄａ Ａｒ‐４ （ＥＦＬＤ）の産生するプロテアーゼの特性

In a previous paper, we examined the toxicity of the extracellular protease of A. salmonicida, Ar-4 (EFDL) on yamabe (Oncorhynchus masou f. ishikawai) and goldfish (Carassius auratus). From these results, we considered the protease secreted by this bacterium was a causative agent of furunculosis. In this paper, we observed enzymatic properties of the purified protease. The results obtained were summarized as follows: 1. The molecular weight of purified protease was estimated to be 71, 000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis. 2. The purified protease showed maximal activity at pH 9.4 and 50°C. It was stable over the pH range 5.0 to 10.0 and was completely inactivated by temperature at 56°C. 3. The protease was presumably classified an alkaline serine protease and it showed chymotryptic properties since it was significantly inhibited by diisopropylfluorophosphate (DFP) and tosyl-phenylalanine-chloromethyl ketone (TPCK) and hydrolyzed N-benzoyl-L-tyrosine ethyl ester (BTEE).

The role of protease in streptolysin S formation

Production of streptolysin S by streptococci was found to be inhibited by treatment with protease inhibitors, tosylphenylalanine chloromethyl ketone (TPCK), tosyllysine chloromethyl ketone (TLCK), or phenylmethylsulfonyl fluoride (PMSF), even in the presence of the inducer oligonucleotides. Other protease inhibitors, antipain, leupeptin, or pepstatin were found to have little or no effect. Trypsin reversed the effect of TPCK or TLCK. The reversal was dependent upon the amount of added trypsin and the incubation time at 37 °C, suggesting that a protease activity was involved in the hemolysin formation. The effect of trypsin was not observed if chloramphenicol was also added, suggesting that a precursor of streptolysin S was processed as it was synthesized and released into medium as the active hemolysin, by the concerted action of a protease and inducer oligonucleotides. Experiments with the subcellular fractions of streptococci indicated that the streptolysin precursor was localized in the insoluble fraction and the “processing” protease in the supernatant fraction.

A neutral thiol proteinase in normal and dystrophic human muscle

Human skeletal muscle homogenates were found to contain a neutral proteinase that catalyzes the hydrolysis of benzyloxycarbonyl- l-alanyl- l-alanyl- l-lysine 4-methoxynaphthylamine. The proteinase was inhibited by sulfhydryl blocking reagents, tosylphenylalanine chloromethyl ketone, tosyllysine chloromethyl ketone, and benzamidine but not by phenylmethanesulfonyl fluoride and metal chelating agents. It was not thiol dependent but its sensitivity to thiol blocking reagents suggests that the enzyme probably is a thiol proteinase. When the levels in patients were compared with controls, significant increases in muscle proteinase levels were observed in patients with major forms of muscular dystrophies and certain denervating diseases. These results suggest the possibility that this neutral proteinase may be involved in some way in the muscle wasting process in these muscle diseases.

Effect of an extract of nereis virens on mammalian spermatozoa

AbstractEpididymal sperm of the mouse, rat, and guinea pig and ejaculated sperm of rabbits are cleaved at the head‐tail junction by an extract of Nereis virens. Annelids are extracted with water and the extract is purified by ion exchange chromatography. Electron microscopy shows that the extract acts on the filaments connecting the capitulum of the tail with the basal plate lining the nuclear envelope. Following detachment, the basal plate remains with the head. The extract contains proteases as indicated by hydrolysis of tosyl arginine methyl ester (TAME), benzoyl arginine ethyl ester (BAEE), and Azocoll, a general protease substrate. The hydrolysis of TAME is inhibited by tosyl lysine chloromethyl ketone (TLCK), a trypsin inhibitor, but TLCK does not prevent head‐tail separation by the Nereis extract. Similarly tosyl phenylalanine chloromethyl ketone (TPCK), a chymotrypsin inhibitor, and phosphoramidon and leucyltryptophan, both thermolysin and acrolysin inhibitors — singly or in combination — do not prevent hydrolysis of Azocoll. Head‐tail separation activity of the extract was inhibited by dithiothreitol, which reduces disulfide bonds, and phenylmethyl sulfonyl fluoride, an inhibitor of serine proteases. These results indicate that the extract is a mixture of proteases, one being a serine protease similar to trypsin.Digestion of the connecting filaments with the pure proteases, trypsin and Staphylococcus aureus V8 protease, has yielded the following information on the proteins of the filaments. The accessibility of arginine and/or lysine peptide bonds to enzyme action is highest in rat sperm filaments, whereas those in the filaments of mouse, rabbit, and guinea pig sperm are less accessible than in the rat. Another possibility is that the total content of arginine and/or lysine varies between the species. The most dramatic difference is the enzymatic action on glutamyl peptide bonds of the filaments, the order being: mouse 〉 rat 〉 rabbit, with guinea pig sperm filaments completely resistant over the time course of the experiment.

Studies on the mechanisms involved in multistage carcinogenesis in mouse skin.

AbstractSkin tumors can be effectively induced in mice by the repetitive application of a carcinogen. The relative order of sensitivity to complete carcinogenesis is Sencar > CD‐1 > C57BL/6 ≥ BALB/c ≥ ICR/Ha Swiss > C3H. Skin tumors in mice can also be induced by the sequential application of a sub‐threshold dose of a carcinogen (initiation phase) followed by repetitive treatment with a weak or noncarcinogenic tumor promoter (promotion phase). The relative order of sensitivity to initiation‐promotion is Sencar > > CD‐1 > ICR/Ha Swiss ≥ Balb/c > C57BL/6 ≥ C3H ≥ DBA/2. The initiation phase requires only a single application of a carcinogen and is essentially an irreversible step, which probably involves a somatic cell mutation as is evidenced by a good correlation between the carcinogenicity of many chemical carcinogens and their mutagenic activities; the promotion stage, however, is initially reversible, later becoming irreversible. For strains and stocks of mice which respond to initiation‐promotion, there is a good correlation between the tumor‐initiating activities of polycyclic aromatic hydrocarbons (PAH) and their abilities to bind covalently to DNA. Potent inhibitors and stimulators of PAH tumor initiation appear to effect the level of the PAH diol epoxide bound to specific DNA adducts. However, when the binding of a given PAH to DNA is compared in various stocks and strains of mice, there is no correlation, since in those mice which are able to metabolize PAH, the amounts of carcinogen bound to DNA are similar.The phorbol ester tumor promoters have been shown to have several cellular and biochemical effects on the skin. Of all the observed phorbol ester related effects on the skin, the induction of epidermal cell proliferation, polyamines, prostagladins, and dark basal keratinocytes as well as other embryonic conditions appear to correlate the best with promotion. Mezerein, a weak promoter, was found to induce many cellular and biochemical changes similar to 12‐O‐tetradecanoylphorbol‐13 acetate (TPA), especially epidermal hyperplasia and polyamines; however, it was not a potent inducer of dark cells. We recently found that promotion could be divided into at least two stages. The first stage (I) can be accomplished by limited treatment with TPA or the nonpromoting agents, 4‐O‐methyl TPA and the calcium ionophore A23187, and the second stage (II) by repetitive applications of mezerein. The dark basal cells appear to be important in the first stage of promotion, since TPA, 4‐0‐methyl TPA, and A23187 are potent inducers of dark cells. Fluocinolone acetonide (FA) was found to be a potent inhibitor of stage I and II. Retinoic acid (RA) was ineffective in Stage I but was a potent inhibitor of Stage II promotion, whereas tosyl phenylalanine chloromethylketone (TPCK) specifically inhibited Stage I. In addition, FA and TPCK effectively counteracted the appearance of dark basal keratinocytes but had very little effect on polyamines, whereas RA had no effect on dark cells but is a potent inhibitor of TPA‐induced ornithine decarboxylase activity and subsequent putrescine formation. These results provide additional evidence for the importance of dark basal keratinocytes (primitive stem cells) in Stage I of promotion and indicate that most of the other cellular and biochemical responses normally associated with promotion (such as polyamines) are actually associated with Stage II of promotion.Although C57BL/6 mice are relatively resistant to initiation‐promotion by PAH initiation and phorbol ester promotion, they are fairly sensitive to complete carcinogenesis by PAH. This suggests that the C57BL/6 mice are resistant to phorbol ester tumor promotion. Preliminary experiments suggest that C57BL/6 and Sencar mice respond qualitatively but not quantitatively to a single treatment with TPA.

Selective antigen-stimulated release of proteolytic activity from rat mast cells.

Stimulation of rat mast cell suspension from actively sensitized rats with antigen in vitro produced a parallel release of histamine and enzyme, probably proteolytic activity, which releases p-nitrophenol from an L-tyrosine-p-nitrophenyl ester derivative (TPNE). The histamine and enzyme release correlated with respect to their dependence on antigen concentration, reaction time and inhibition by 2,4-DNP and papaverine. In contrast, more than 50% of total histamine but nearly no enzyme was released by the ionophore A 23.187 and C 48/80 (each less than or equal to 1 microgram/ml). The enzyme was apparently secreted predominantly in a particular form. It was approximately 50% inactivated by heating for 1 h at 56 degree C or by incubation for 3 h at 37 degrees C with the chymotrypsin inactivator tosyl-phenylalanine chloromethylketone (TPCK; 2.5 X 10-4 M) or for 5 min at 37 degrees C with benzyl sulphonyl fluoride (2.5 X10-4 M), which reacts with SH groups. Heating for 3 min at 100 degrees C destroyed it completely. On the basis of these properties we suggest that the antigen released enzyme is the known granulabound chymase from rat mast cells. TPNE was not only a cleavable substrate for the enzymatic activity in the 800 g cell supernatant following antigen stimulation, but also a strong inhibitor of the histamine release on administration before antigen (IC50 approximately 10-6 M). It appears that the same enzyme activity acts initially intracellularly as activator of the histamine secretion and then is subsequently released along with histamine as a further mediator. Extracellularly this enzyme may act as a modulator of inflammatory reactions in type I allergy both locally and systemically.

Association of a protease with polytene chromosomes of Drosophila melanogaster.

Incubation of Drosophila salivary glands with radioactive diisopropyl fluorophosphate results in the uniform labeling of polytene chromosomes. Extensive labeling is seen only when chromosome squashes are prepared by a formaldehyde fixation procedure and not by standard acetic acid techniques. The labeling is inhibited in the presence of tosylphenylalanine chloromethyl ketone and phenylmethane sulfonylfluoride but not by tosyllysine chloromethyl ketone, suggesting that a chymotrypsin-like serine protease is associated with the chromosomes. Protease inhibitors show no apparent effect on heat-shock specific puffing.

Age-related changes of liver tyrosine aminotransferase in senescent rats.

Tyrosine aminotransferase was induced in adult and senescent rat liver and its properties studied. We show the appearance of a 'cross-reacting material' for induced tyrosine aminotransferase of old rats compared to basal enzyme; this cross-reacting material can be provoked in adult rats after injection of cycloheximide, and suppressed in adult and old rats after injection of a serine protease inhibitor (tosylphenylalanine chloromethylketone). Other properties of induced tyrosine aminotransferase (thermostability, Km for tyrosine, isoelectrofocusing) are identical except for the proportion of the three forms and their sensitivity to trypsin in the absence of pyridoxal phosphate, which is increased in senescent animals. The suppression of cross-reacting material clearly indicates that it is not due to errors on old rat liver DNA but rather to post-translational modifications. This demonstrates also the role of serine proteases in tyrosine aminotransferase degradation. We suggest that induced enzyme of senescent rats would undergo a conformational change, possibly due to a release of pyridoxal phosphate from the enzymic molecules, which would thus become more susceptible to proteolytic attack than those of adult rats.

Multiple molecular forms of human pancreas alanine aminopeptidase

Human pancreas is the source of an alanine aminopeptidase (HPAA) that is unique to pancreas and is readily distinguishable from the liver, kidney, and duodenal alanine aminopeptidases. Each of these three aminopeptidases appears in small quantities in blood and therefore may constitute tissue/organ specific marker enzymes. In this study alanine aminopeptidase from pancreas has been purified. Pancreas alanine aminopeptidase was resolved upon purification with ion exchange chromatography into three isoenzymes. Gel filtration chromatography of these isoenzymes indicates that their molecular weights are near 235 000 daltons. The isoenzymes contain a firmly bound divalent cation that could be removed with EDTA only at temperatures above 40°C (at which the enzymes were stable) and below 52°C (at which thermal denaturation begins to take place). Treatment with EDTA at 4°C yields fully active enzymes, which, however may be stimulated approximately 200% by the addition of Co 2+ at 10 −4 mol/1. This cobalt stimulation is easily reversed by dialysis of the stimulated isoenzymes against deionized water. The pancreas alanine aminopeptidases were not inhibited by tosyl-phenylalanine chloromethylketone or by tosylleucine chloromethylketone, whereas phenylalanine chloromethylketone was inhibitory. The K m values for methionyl-, arginyl-, leucyl-, alanyl-, and isoleucylβ-naphthylamide for each isoenzyme are statistically identical and the average values are 0.36, 0.60, 0.69, 1.25, and 1.29 × 10 −4 mol/1, respectively. The k cat values are 2.50, 1.58, 0.82, 0.38, and 0.19 × 10 4 sec −1, respectively.

Studies on mechanism of action of anti-tumor-promoting agents: their specificity in two-stage promotion.

The effects of fluocinolone acetonide (FA), retinoic acid (RA), and tosylphenylalanine chloromethyl ketone (TPCK) on two-stage promotion after 7,12-dimethylbenz[a]-anthracene (DMBA) initiation in female Sencar mice were investigated. The two-stage promotion protocol was achieved by twice weekly applications of 2 microgram of 12-O-tetradecanoylphorbol 13-acetate (TPA) for 2 weeks (stage I) followed by twice weekly applications of mezerein for 18 weeks (stage II). Separately stage I and II do not cause any tumors to develop after DMBA initiation. FA was found to be a potent inhibitor of stages I and II but to a greater degree for stage I than for stage II. RA was ineffective in stage I but was a potent inhibitor of stage II; TPCK specifically inhibited stage I but not stage II. FA and TPCK effectively counteract the appearance of the dark basal keratinocytes, whereas RA has no effect. These results provide additional evidence for the importance of dark basal keratinocytes in stage I of promotion and indicate that most of the other biochemical and morphological responses normally associated with promotion (such as polyamines) are actually associated with stage II of promotion.

Biochemical studies of the hatching process in sea urchin embryos. I. Effects of protease inhibitors

AbstractEffects of 12 protease inhibitors both on the hatching process and on the activity of the hatching enzyme of the sea urchins, Hemicentrotus pulcherrimus and Strongylocentrotus intermedius, were studied. Synthetic inhibitors, N‐tosyl‐L‐phenylalanyl‐chloromethane (often called tosylphenylalanine chloromethylketone; TPCK), N‐α‐tosyl‐L‐lysyl‐chloromethane (often called tosyllysine chloromethylketone; TLCK), p‐nitrophenyl p'‐guanidinobenzoate (NPGB), diphenylcarbamyl chloride (DPCC) and phenylmethane sulfonyl fluoride (PMSF), caused cytolysis of embryos, whereas the microbial inhibitors, chymostatin, leupeptin, antipain, pepstatin, elastatinal and phosphoramidon, and soybean trypsin inhibitor did not. Among them, the inhibitors specific against chymotrypsmsy chymostatin and TPCK, delayed the hatching process and inhibited the hatching enzyme. NPGB also inhibited the enzyme but its effect on the hatching process was uncertain because of its general toxicity to embryos. Esterolytic hydrolysis of N‐acetyl‐L‐tyrosine ethyl ester (ATEE) by a crude preparation of the enzyme was found.The evidence presented suggests that the hatching enzyme of sea urchins is similar to chyrmotrypsin.

Purification, characterization and localization of serine protease of Morris hepatoma 8999.

1. A serine protease of hepatoma 8999, isolated in the mitochondrial fraction, was purified and crystallized. The purified enzyme was apparently homogeneous on ultracentrifugal analysis and polyacrylamide disc gel electrophoresis. The ratio of absorbance at 280 nm and 260 nm, A280/A260, was 1.90 and its absorption coefficient, A280 1% was 10.5 cm-1 estimated from dry weight measurements. Its S20, w value was 2.23 S and its molecular weight was estimated to be 24000 +/- 1000. The enzyme contained twice as much lysine, arginine and histidine as chymotrypsinogen did, but had a very similar amino acid composition to serine protease from skeletal muscle. Its isoelectric point was pH 10.6. 2. The substrate specificity of the enzyme was the same as that of chymotrypsin A. Its Km and kcat values for N-acetyl-L-tyrosine ethyl ester, N-acetyl-L-phenylalanine ethyl ester and N-acetyl-L-tryptophan ethyl ester were 0.35 mM and 10.69 s-1, 0.38 mM and 10.7 s-1, and 0.11 mM and 11.8 s-1, respectively. Its activity was completely inhibited by phenylmethylsulfonyl fluoride and partially inhibited with tosylphenylalanine chloromethyl ketone. 3. The enzyme was shown to be located in different granules from the intracellular particules (light and heavy mitochondrial fraction) by sucrose density gradient centrifugation, and it was stained in mast cells of the hepatoma 8999 by the immunofluorescent technique. 4. Serine protease is present in different amounts in various organs of rat and the enzyme from hepatoma 8999 gave a single band that fused completely with those of the enzymes from skeletal muscle, heart, liver and kidney, respectively, on Ouchterlony double-diffusion analysis using antiserum to the crystalline enzyme of hepatoma 8999, but the enzyme from small intestine did not react with the antiserum.

Rat Skin Main Neutral Protease: Purification and Properties

The main neutral protease of rat skin has been purified by extracting the skin first with a dilute buffer, solubilizing the enzyme with 2M KCl, precipitating it by dialysis in dilute buffer, resolubilizing the neutral protease with 2M KCl, chromatographying it on Sephadex G-200 and hydroxyapatite. The purified enzyme is homogenous in sodium dodecylsulfate-gel electrophoresis. It is cationic and, at low ionic strength, forms complexes with heparin, is adsorbed by dextran and polyacrylamide gels, and glass. A molecular weight of 27,500 was obtained with sodium dodecylsulfate-gel electrophoresis and gel chromatography. The specificity of the enzyme resembles that of bovine chymotrypsin; it hydrolyzes proteins and synthetic substrates of chymotrypsin, optimally at pH 8–9. The enzyme is activated by KCl and inhibited by diisopropyl fluorophosphate, phenylmethyl sulfonyifluoride, tosylphenylalanine chloromethylketone, trypsin inhibitors from lima bean and soybean, and by rat serum. The trypsin inhibitors from ovomucoid or bovine lung (Trasylol) did not inhibit the enzyme. Treatment of rats with compound 48/80, a mast cell degranulator, decreases the amount of the neutral protease in skin to 1/50 of the control. This, and the properties of the enzyme, suggest that the main neutral protease of rat skin is identical with the rat mast cell chymase.

Flexibility of myosin rod, light meromyosin, and myosin subfragment-2 in solution.

Myosin rod was prepared by papain proteolysis of myosin. The components of rod, light meromyosin (LMM) and subfragment-2 (S-2), were prepared by proteolysis of myosin and rod, respectively, using trypsin treated with tosylphenylalanine chloromethyl ketone. S-2, thus prepared, was of greater molecular weight than obtained previously, so that the combined molecular weights of LMM and S-2 were equal to that of rod, and S-2 contained virtually all of the region of the rod susceptible to trypsin. Electro-optical measurements were made on the three fragments in 2 mM sodium pyrophosphate, pH 9.3 at 3 degrees, over a large range of protein concentrations. Analysis of the relaxation of birefringence, at low protein concentration where there was no aggregation, showed that LMM (relaxation time 13.1 micros) behaves as a rigid cylinder. Rod (relaxation time 41.2 micros) and S-2 (relaxation time 6.0 micros) had relaxation rates that were too fast for rigid molecules of their dimensions, and therefore are not straight rods. This implies that myosin rod is flexible in the S-2 portion, presumably in the region susceptible to proteolysis. The implications of rod flexibility for the mechanism of muscle contraction are discussed.

Reinvestigation of the phenacyl bromide modification of alpha-chymotrypsin.

The modification of alpha-chymotrysin with phenacyl bromide has been reinvestigated over a wide pH range. Evidence is presented that indicates that the nature of the phenacyl-modified enzymes prepared by this reaction is dependent upon the pH of the reaction medium. The phenacyl alpha-chymotrypsin produced at low pH is most probably the Met-192 phenacylsulfonium salt, as proposed earlier, since it readily undergoes dealkylation using 2-mercaptoethanol. However, the phenacyl-enzyme prepared at neutral pH possesses a much reduced enzymatic activity and does not react with 2-mercaptoethanol to regenerate native alpha-chymotrypsin. In addition, incubation of the Met-192 phenacyl sulfonium enzyme at neutral pH causes a smooth irreversible change to the new phenacyl-enzyme as monitored by changes in enzymatic activity, susceptibility to dealkylation using 2-mercaptoethanol, and ultraviolet difference absorption spectral properties. The stoichiometries of both the low and neutral pH modification reactions have been determined, using [carbonyl-14C]phyenacyl bromide, to be 1 phenacyl group/enzyme molecule. In efforts to obtain information about the nature and mechanism of formation of the phenacyl alpha-chymotrypsin produced at neutral pH, alkylation reactions of modified alpha-chymotrypsins produced by His-57 functionalization with tosylphenylalanine chloromethyl ketone and by Met-192 oxidation to the sulfoxide have been investigated. The combined results of these studies have been initially interpreted in terms of a neutral pH, phenacyl bromide modification resulting in formation of a new modified enzyme via the Met-192 sulfonium salt.

Effect of protease inhibitors on protein degradation in rat hepatoma cells I. Effect on general protein degradation

Tosyllysine chloromethyl ketone and tosylphenylalanine chloromethyl ketone in vitro are active-site specific and irreversible inhibitors of trypsin (EC 3.4.21.4) and chymotrypsin (EC. 3.4.21.1) respectively. Using rat hepatoma cells in suspension culture, both inhibitors were found to partially inhibit breakdown of prelabelled cell proteins ot amino acids, the effect being greastest in the absence of serum. Protein synthesis in rat hepatoma cells, reticulocytes and reticulyte lysates was also irreversibly inhibited by these compounds. Reduction of ATP levels with antimycin a inhibited protein degradation, but neither tosylphenylalanine chloromethyl ketone nor tosyllysine chloromethyl ketone had any effect on ATP concentration in rat hepatoma cells. These results suggest that the degradation of at least some proteins in animal cells may involve the action of serine protease(s).

Effect of protease inhibitors on protein degradation in rat hepatoma cells II. Effects on ornithine decarboxylase and tyrosine aminotransferase

Tyrosine aminotransferase (EC 2.6.1.5) and ornithine decarboxylase (EC 4.1.1.17) are both inducible, pyridoxal phosphate-requiring enzymes, which undergo rapid turnover in cultured rat hepatoma (HTC) cells, as measured by loss of enzyme activity following inhibition of protein synthesis. Under these conditions tosylphenylalanine chloromethyl ketone (Tos-PheCh 2Cl) and tosyllysine chloromethyl ketone (Tos-LysCh 2Cl) both almost completely inhibit the loss of tyrosine aminotransferase activity, implying the involvement of serine proteases in this process. Depletion of intracellular ATP levels to virtually zero also completely inhibits the apparent degradation of tyrosine aminotransferase. In contrast, the decline in ornithine decarboxylase activity following inhibition of protein synthesis is stimulated by both Tos-PheCH 2Cl and Tos-LysCH 2Cl, although with Tos-PheCH 2Cl this stimulation is followed after 45 min by a complete inhibition of apparent degradation. ATP starvation inhibits the loss of ornithine decarboxylase activity by only 50%.

Properties of a proteolytic enzyme from rabbit sperm acrosomes.

Acrosin is a proteolytic enzyme extractable from sperm acrosomes and essential for penetration of the zona pellucida of the ovum by spermatozoa. It has properties in common with both trypsin and plasmin having an optimum pH of 8.0, hydrolyzing benzoyl arginme ethyl ester (BAEE) and tosyl arginine methyl ester (TAME) but not benzoyl tyrosine ethyl ester (BTEE), or acetyl tyrosine ethyl ester (ATEE), being stimulated by calcium ions and stable at pH 3.0. Acrosin differs from pancreatic trypsin in that it hydrolyzes BAEE more rapidly than TAME, is less effectively inhibited by ovomucoid and pancreatic trypsin inhibitor, has a higher molecular weight, undergoes rapid loss of activity even in the presence of calcium and produces fewer peptides on incubation with oxidized nbonuclease. One of two trypsin inhibitors isolated from guinea pig seminal vesicles inhibits acrosin and trypsin but not plasmin, whereas the other inhibits all three enzymes. Acrosin further differs from plasmin in that it has a different molecular weight and is more unstable. Inhibition by diisopropyl fluorophosphate and tosyl lysine chloromethyl ketone shows a similarity to trypsins and lack of inhibition by tosyl phenylalanine chloromethyl ketone indicates that acrosin is not chymotrypsin-like. The enzyme is also inhibited by soybean trypsin inhibitor. Acrosin appears to be a unique proteolytic enzyme and therefore deserves a specific name. Previously we described the purification of an acrosomal trypsin-like enzyme, acrosin, from epididymal sperm with a molecular weight of 55,000 (Polakoski et a!., 1971). The presence of such a proteolytic enzyme in sperm was first reported by Yamane (1935a, b) and shown to be trypsin-like by Buruiana (1956). Hartree and Srivastava (1965), and Srivastava, Adams, and Hartree (1965) showed that acrosomal extracts prepared from ram sperm removed the zona pellucida from rabbit ova in vitro. Stambaugh and Buckley (1968, 1969) confirmed these results using similar extracts prepared from epididymal rabbit sperm. These authors showed that the protease demonstrated to be localized in acrosomal extracts by Srivastava ci a!. (1965) was trypsin-like in nature and responsible for the removal of the zona. It is most likely the same enzyme studied by Buruiana (1956). In vitro fertilization was inhibited by soybean trypsin inhibitor (Stambaugh ci a!., 1969). Fertilization in vivo was inhibited by preincubation of capacitated sperm with pancreatic tryp

The effect of epsilon amino caproic acid and other inhibitors of proteolysis upon the response of human peripheral blood lymphocytes to phytohemagglutinin

Previous work has suggested that intracellular proteolysis may play a role in lymphocyte stimulation. An inhibitor of proteolysis, epsilon amino caproic acid (EACA) was studied for its effect on the lymphocyte response to phytohemagglutinin (PHA). EACA was found to inhibit several parameters of lymphocyte stimulation (e.g. DNA, RNA, and protein synthesis as well as alterations in morphology) This inhibition was not due to diminished cellular viability and did not permanently impair the capacity of the lymphocyte to subsequently respond to PHA. Additionally, there was no evidence that this inhibition was due to other possible effects of EACA, such as alterations in Na(+) - K(+) transport, competitive amino acid deprivation or interference with PHA binding. Moreover, the inhibitors of proteolysis, tosyl arginine methyl ester (TAME), tosyl lysine chloromethyl ketone (TLCK), and tosyl phenyl-alanine chloromethyl ketone (TPCK), were also shown to inhibit lymphocyte stimulation.EACA was most effective when added during the first 24 hr of stimulation. Therefore, these experiments support the hypothesis that proteolysis is an essential step in the early phase of lymphocyte activation.