Partial purification and characterization of a serine endopeptidase from rat liver plasma membranes

A serine endopeptidase was partially purified from rat liver plasma membranes by using a four-step procedure: solubilization with N-lauroylsarcosine; Ultrogel AcA-34 chromatography; CM Affi-Gel blue chromatography; agarose-soybean trypsin inhibitor chromatography. This enzyme was found to hydrolyze casein and various chromogenic peptide substrates; highest activity occurred with H-D-Val-Leu-Arg-p-nitroanilide, reported to be a specific substrate for human glandular kallikreins. The enzyme was heat-sensitive, showed a pH optimum between 8.0 and 9.0 and was inhibited by D-Phe-L-Phe-L-Arg-CH2Cl, aprotinin, diisopropyl fluorophosphate (DFP), soybean trypsin inhibitor, phenylmethylsulphonyl fluoride, leupeptin, antipain and dithiothreitol. This liver plasma membrane proteinase has an apparent molecular weight of about 30 000 as determined by Ultrogel AcA-34 chromatography and by autoradiography of [3H]DFP-labelled protein electrophoresis.     

Trypsin inactivation by intact Hymenolepis diminuta (Cestoda): some characteristics of the inactivated enzyme

In the presence of intact Hymenolepis diminuta, trypsin was inactivated; intact worms had no apparent effect on subtilisin, pepsin, or papain. Inactivation of trypsin was demonstrable using azoalbumin as a substrate, but the inactivated enzyme retained full catalytic activity against benzoyl-DL-arginine-p-nitroanilide, p-tosyl-L-arginine methyl ester (low molecular weight synthetic trypsin substrates) and p-nitro-p-guanidinobenzoate (an active site titrant). Inactivation was not reversible under conditions of heating, freezing and thawing, or prolonged dialysis of the enzyme. Analyses of inactivated 3H-trypsin by cationic and SDS-polyacrylamide gel electrophoresis, and gel chromatography failed to indicate the presence of a high molecular weight trypsin inhibitor associated with the inactivated enzyme; no low molecular weight, dissociable inhibitor was demonstrable following thermal denaturation of the inactivated enzyme. Analyses of trypsin after incubation in the presence of pulse-labeled worms also failed to demonstrate the presence of any inhibitor of worm origin associated with the inactivated enzyme. The data suggest that inactivation is the result of a small structural or conformational change in the enzyme molecule, a change which partially (rather than totally) inactivates the enzyme towards protein substrates.     

Phenylmethylsulfonyl fluoride stimulates proteolysis of nuclear proteins from chick liver.

The degradation of H1 histone and high mobility group (HMG) nonhistone proteins was stimulated when the homogenate from chick liver was incubated in the presence of phenylmethylsulfonyl fluoride (PMSF). Two proteinase inhibitors, elastatinal and chymostatin, significantly inhibited the PMSF-stimulated degradation of H1 histone and HMG proteins. On the contrary, other proteinase inhibitors like leupeptin, pepstatin, trypsin inhibitor, antipain, o-phenanthroline and EDTA had no effect on the degradation of the nuclear proteins. These results warn the researcher to be cautious while using PMSF for preparation of nuclear proteins such as H1 histone and HMG proteins.     

Purification and partial characterization of an exocellular proteinase from Trichophyton rubrum

An exocellular proteinase produced by Trichophyton rubrum in a glucose-peptone broth was purified from lyophilized and dialysed culture filtrate of the dermatophyte by Sephadex G-100 gel filtration and preparative polyacrylamide gel electrophoresis. The purified enzyme was a homogeneous protein of molecular weight 34700 and it could hydrolyse azoalbumin, casein, bovine serum albumin, alpha-N-benzoyl-L-arginine ethyl ester and p-toluenesulfonyl-L-arginine methyl ester but not N-benzoyl-L-tyrosine ethyl ester, alpha-N-benzoyl-DL-arginine-p-nitroanilide and keratin. The enzyme showed an alkaline pH optimum and was not activated by divalent metal ions but inhibited strongly by phenylmethylsulfonylfluoride. Thus the enzyme was identified as an alkaline serine proteinase.     

Purification and characterization of benzoyl-L-tyrosine ethyl ester hydrolase from the yolk sac membrane of chicken egg

An enzyme which hydrolyzes benzoyl-L-tyrosine ethyl ester (BTEE) was purified from yolk sac membranes of day-18 chick embryos. The purified BTEE hydrolase has a molecular weight of 110,000, being composed of 70,000 and 40,000 subunits, and preferred synthetic substrates for chymotrypsin to those for trypsin. The optimum pH and temperature of this enzyme were 6.5-7.0 and 40 degrees C, respectively. The Km value for BTEE of the enzyme was 16 mM at pH 6.5 and 30 degrees C. The enzyme was inhibited markedly by some chymotrypsin inhibitors but scarcely inhibited by trypsin inhibitors. Magnesium ion acted as potent activator, depending on the enzyme purity and its concentration, whereas p-chloromercuribenzoate and zinc ion inactivated the activity markedly. The BTEE hydrolase was found to hydrolyze proteins such as casein and hemoglobin. These data indicated that the enzyme is a proteinase similar to chymotrypsin. This proteinase could act on yolk proteins, suggesting that it plays an important role in the metabolism of yolk at the yolk sac membrane layer.     

A trypsin-like proteinase localized in cortical granules isolated from unfertilized sea urchin eggs by zonal centrifugation. Role of the enzyme in fertilization

A trypsin-like proteinase was localized within a single subcellular compartment of unfertilized Strongylocentrotus purpuratus eggs, the cortical granules. Homogenates of eggs were fractionated by rate-zonal centrifugation. Enzymatic markers were used to determine the distribution of mitochondria (cytochrome oxidase), yolk platelets (acid nitrophenyl phosphatase), and cortical granules (β-1, 3-glucanase) in the sucrose density gradient. A bimodal distribution pattern was obtained for aryl esterase activity (substrate: β-naphthyl acetate), with one peak in the microsomal and the other in the cortical granule fractions. The cortical granule enzyme was characterized as a trypsin-like proteinase, since it also hydrolyzed another typical tryptic substrate α-N-benzoyl-l-arginine ethyl ester and was completely inactivated by soybean trypsin inhibitor (SBTI). The aryl esterase activity in the microsomal fractions was not inhibited by SBTI, while 50% of the total aryl esterase activity in the original egg homogenate was inactivated by SBTI. The identity of the enzyme(s) responsible for the aryl esterase activity associated with the microsomal particles is unknown at present.The cortical granule proteinase functions in the elevation of the fertilization membrane and establishment of the block to polyspermy at fertilization. Arbacia punctulata eggs inseminated in the presence of trypsin inhibitors, SBTI or tosyl lysine chloromethyl ketone (TLCK), failed to elevate normal fertilization membranes and became heavily polyspermic.On the basis of these results and observations made by other investigators with a wide variety of biological systems, it is proposed that trypsin-like proteinases function in the discharge of secretory granules from all types of cells.     

The nonhistone chromosomal protein, H2A-specific protease, is selectively associated with nucleosomes containing histone H1

We have determined the distribution of the nucleosomal bound nonhistone chromosomal protein, H2A-specific protease, in calf thymus and liver chromatin. The protease was unevenly distributed in chromatin with domains containing histone H1 being selectively complexed with the enzyme. Moreover, the protease had a preference for the less compact chromatin domains enriched in the H1 subtypes H1a and -c. We have demonstrated that ubiquitinated H2A is a substrate of the H2A-specific protease and that the enzyme is a serine protease which can be inactivated with protease inhibitors only after it is released from the nucleosome. Possible functions of the protease in modulating chromatin structure are discussed.     

Substrate activation of rat trypsins 1 and 2

Qualitative differences in the active center of rat trypsins 1 and 2 resulted in different ratios of K_cat for N¹-tosyl-l-arginine methyl ester vs K_cat for N¹-benzoyl-l-arginine ethyl ester. These ratios were 2.5 for trypsin 1 and 1.2 for trypsin 2.Substrate activation with N¹-tosyl-l-arginine methyl ester enhanced the catalytic rate constant of rat trypsin 1 2.5-fold and that of rat trypsin 2 only 1.5-fold. The increase in the catalytic rate constant found with N¹-benzoyl-l-arginine ethyl ester was the same (1.5-fold) for both trypsins. Consequently, at 20 mm substrate concentration, trypsin 1 catalyzed the esterolysis of N¹-tosyl-l-arginine methyl ester 4.5 times faster than that of N¹-benzoyl-l-arginine ethyl ester, while trypsin 2 was only 1.3 times more efficient with the first substrate.Furthermore, the activation of both rat enzymes by N-acetyl-l-tyrosine ethyl ester was even more effective than that obtained with the two cationic esters; the maximum rates of hydrolysis of this neutral substrate by trypsins 1 and 2 were enhanced 120- and 50-fold, respectively, by high concentrations of N-acetyl-l-tyrosine ethyl ester.     

Guanidoacetate methyltransferase from rat liver: Purification,properties, and evidence for the involvement of sulfhydryl groups for activity

Guanidoacetate methyltransferase (EC 2.1.1.2) has been purified about 800-fold from rat liver. The purified preparation shows a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the enzyme is estimated to be 25,000 and 26,000 by Sephadex gel molecular-exclusion chromatography and by electrophoresis in polyacrylamide gradient gel, respectively. The sodium dodecyl sulfate-denatured enzyme also has a molecular weight of 26,000; thus, the enzyme is a monomeric protein. Guanidoacetate methyltransferase as isolated is catalytically inactive, but is readily reactivated by incubation with a thiol. The reactivated enzyme, which contains 3 mol of sulfhydryl groups/mol of enzyme, is again inactivated by oxidized glutathione. This inactivation is accompanied by the disappearance of two sulfhydryl residues. The relationship between the loss of enzyme activity and the number of residues disappeared indicates that the integrity of these sulfhydryl residues is critical for activity. The oxidized enzyme fails to bind the substrate S-adenosylmethionine as evidenced by the equilibrium dialysis study. Alkylation of the nonoxidizable sulfhydryl by N-ethylmaleimide shows that this residue is also essential for activity. UV absorption, fluorescence, and CD spectra show no difference between the reduced and oxidized enzymes, but the former is more susceptible to proteolytic attack by trypsin. The enzyme has an isoelectric pH of 5.3, and is most active at pH 9.0. From the CD spectrum, an α helix content of 15% is calculated. The K_m values for guanidoacetate and S-adenosylmethionine are 97.5 and 6.73 μm, respectively, at pH 8.0 and 37 °C.     

Protease-activated protein kinase in rat liver plasma membrane

Upon limited proteolysis with trypsin, a cAMP and Ca2+-independent protein kinase was produced from rat liver plasma membrane. This enzyme showed a multifunctional capacity and phosphorylated calf thymus histone and rat liver ribosomal proteins. The molecular weight was estimated to be 5.0 X 10(4). When plasma membrane was treated with a buffer containing Triton X-100, a proenzyme with a molecular weight of 8.4 X 10(4) was extracted. By tryptic digestion, the proenzyme was converted to an active protein kinase which was similar to the enzyme obtained by the direct digestion of membrane. However, this proenzyme phosphorylated H1 histone in the presence of Ca2+ and phospholipid without proteolytic digestion. These results indicate the existence of a protease-activated protein kinase in rat liver plasma membrane and the proenzyme seems to be same as protein kinase C.     

Properties of the genome in normal and SV-40 transformed WI-38 human diploid fibroblasts. III. Turnover of nonhisone chromosomal proteins and their phosphate groups.

The turnover of nonhistone chromosomal proteins and their phosphate groups was compared in normal and in SV-40 virus transformed WI-38 human diploid fibroblasts. Cells were pulse labelled with tryptophan-³H and ³²P for 30 minutes and the specific activities of tryptophan-³H and ³²P in the various molecular weight classes of nonhistone chromosomal proteins were determined during the first four hours following termination of labelling. While a rapid turnover of high molecular weight nonhistone polypeptides (142, 000 to 200, 000 Daltons) is evident after one hour in SV_40 transformed cells, the specific activities of these nonhistone chromosomal polypeptides are not significantly decreased in normal cells. In contrast, a rapid turnover of low molecular weight (30, 000 to 51, 000 Daltons) nonhistone chromosomal proteins occurs during the first hour in normal WI-38 cells with no corresponding decrease in the specific activities of these proteins in SV-40 transformed cells. There is no apparent net turnover of phosphate groups on nonhistone chromosomal proteins in either normal or SV-40 transformed cells four hours following pulse labelling. Rather, during the first four hours significant fluctuations are observed in the ³²P specific activities of defined molecular weight fractions. Taken together with previous reports of differences in the composition, synthesis and phosphorylation of nonhistone chromosomal proteins in normal and SV-40 transformed human diploid cells the present results further indicate the complex nature of the alterations in these proteins which accompany viral transformation.     

In vivo andin vitro methylation of lysine residues ofEuglena gracilis histone H1

We have earlier identified and purified two protein-lysine N-methyltransferases (Protein methylase III) fromEuglena gracilis [J. Biol. Chem.,260, 7114 (1985)]. The enzymes were highly specific toward histone H1 (lysine-rich), and the enzymatic products were identified as ε-N-mono-, di- and trimethyllysines. These earlier studies, however, were carried out with rat liver histone H1 as thein vitro substrate. Presently, histone H1 has been purified fromEuglena gracilis through Bio-Rex 70 and Bio-Gel P-100 column chromatography. TheEuglena histone H1 showed a single band on SDS-polyacrylamide gel electrophoresis and behaved like other histone H1 of higher animals, whereas it had a much higherR _f value than the other histones H1 in acid/urea gel electrophoresis. When theEuglena histone H1 was [methyl-³H]-labeledin vitro by a homologous enzyme (one of the twoEuglena protein methylase III) and analyzed on two-dimensional gel electrophoresis, three distinctive subtypes of histone H1 were shown to be radiolabeled, whereas five subtypes of rat liver histone H1 were found to be labeled. Finally, by the combined use of a strong cation exchange and reversed-phase Resolve C₁₈ columns on HPLC, we demonstrated thatEuglena histone H1 contains approximately 9 mol% of ε-N-methyllysines (1.40, 1.66, and 5.62 mol% for ε-N-mono-, di- and trimethyllysines, respectively). This is the first demonstration of the natural occurrence of ε-N-methyllysines in histone H1.     

Proteinase Enzyme System of Lactic Streptococci II. Role of Membrane Proteinase in Cellular Function

The effect of several environmental conditions on the structure and activity of a membrane-associated proteinase from Streptococcus lactis was investigated. The activity of the enzyme varied with pH. Before storage at 3 C, maximal activity occurred at pH 6.0, but was minimal at this pH after storage. At all pH values tested, the enzyme was inactivated after storage. After storage at 3 C, the enzyme showed gross structural alterations with a concomitant loss of activity. Gel filtration and sedimentation velocity data indicated that inactivation of the enzyme was the result of aggregation to higher molecular weight forms. p-Hydroxymercuribenzoate prevented inactivation of the enzyme during storage by preventing aggregation. Activity was correlated with disaggregation of polymer forms of the enzyme to an active monomer. The storage-inactivated enzyme could be reactivated by treatment of the enzyme with cysteine, glutathione, or ferrous ion. Glutathione enabled stored cells to produce acid at their original rate when subcultured in milk. This was attributed to the effect of glutathione on the membrane proteinase. The data suggested that the biological activity of stored cells may be dependent upon the activity of the membrane proteinase.     

Neutral elastolytic proteinase from canine leucocytes. Purification and characterization.

1. A neutral proteinase (EC 3.4.-.-) with elastolytic activity was isolated from canine bloodstream leucocytes, and purified to apparent homogeneity by a two-step procedure consisting of DEAE-Sephadex chromatography and molecular sieving on Sephadex G-75. 2. The molecular weight of the enzyme was 23 500, and the absorbance (A1%1cm) at 282 nm was 6.1. Amino acid analysis showed high content of glycine, aspartic acid, and valine, and low proportion of methionine, lysine and histidine as well as the absence of tyrosine in the enzyme molecule. 3. The proteinase was active against several protein substrates as well as towards N-t-butyloxycarbonyl-L-alanine p-nitrophenyl ester, N-acetyl-L-alanyl-tyrosine ethyl ester. 4. The enzyme was inactivated by diisopropylfluorophosphate, N-acetyl-L-alanyl-L-alanyl-L-alanine chloromethyl ketone, and N-p-tosyl-L-phenylalanine chloromethyl ketone. Inhibition by some natural proteinase inhibitors was also noted.     

The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With ³²P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.     

Purification and some properties of two proteinases from Crotalus adamanteus venom that inactivate human alpha 1-proteinase inhibitor.

Two proteinases (proteinases I and II) have been purified from Crotalus adamanteus venom to the stage of electrophoretic homogeneity and proteinase II has been crystallized. The proteinase differ slightly in molecular weight and amino acid composition. Both are metalloenzymes requiring Zn2+ or Ca2+, or both; neither requires thiol compounds for activation. The proteinases are free of esterolytic activity against benzoly-L-arginine ethyl ester and benzoyl--tyrosine ethyl ester. Proteinase II cleaves the oxidized B chain of insulin at the bonds Phe1-Val2, His5-Leu6, His10-Leu11, Ala14-Leu15, Leu15-Tyr16, and Tyr-16-Leu17. Digestion of polylsine and polyarginine by proteinase II liberates products ranging from dodecapeptides to hexapeptides. Proteinases I and II catalytically inactive human plasma alpha 1-proteinase inhibitor (54,000 daltons). Electrophoretic analysis of the reaction of proteinase II with alpha 1-proteinase inhibitor reveals that an inactivated inhibitor species of 50,000 daltons is formed, and a peptide of 4,000 daltons is released. The gradual disappearance of the native inhibitor results in the corresponding loss of inhibitory activity against trypsin and chymotrypsin.     

Effect of the conditions of isolation and incubation of the nuclei on digestion of DNA chromatin by calcium- and magnesium-dependent endonuclease. Change in the spectrum of chromosomal proteins and possible role of DNA-protein interactions]

Digesting of chromosomal DNA of interphase rat liver nuclei by Ca, Mg-dependent endonuclease in situ in the presence of chelating agents results in the appearance of the soluble DNP--up to 30% of the total DNA. In addition, 50% of the chromatin is solubilised after mild ultrasonication. In the absence of the chelating agents the degree of fragmentation is considerably increased. The process is accompanied by a loss of some histone and nonhistone chromosomal proteins; the nonhistone proteins are lost selectively. The preliminary removal of the nuclear membrane and significant part of the proteins by tritone X-100 promotes the chromatin degradation and the appearance of low molecular weight fragments. The DNA-fragments of solubilised chromatin are similar to the DNA-fragments of residual chromatin, but in the presence of the chelating agents the latter does not contain monomeric fragments.     

Effect of thyrotropin on the phosphorylation of thyroid chromosomal proteins.

Thyrotropin (TSH) stimulated the phosphorylation of histone H1 in calf thyroid slices but had no effect on other classes of histones. Phosphorylation of total phenol-soluble nonhistone chromosomal proteins was not affected by incubation with TSH. However, when these phenol-soluble nonhistone chromosomal proteins were analysed by two-dimensional gels involving isoelectrofocusing and dodecyl sulfate-polyacrylamide gel electrophoresis, TSH was shown to stimulate the phosphorylation of two specific groups of phosphoproteins with molecular weights between 35,000 and 45,000 and isoelectric points at pH values of 5.4-6.0. This increase in phosphorylation with TSH stimulation was confirmed by quantitative analysis of one-dimensional isoelectrofocusing gels.     

Changes in mammary-gland acetyl-coenzyme A carboxylase associated with lactogenic differentiation

The process leading to the rise of acetyl-CoA carboxylase activity in rat mammary tissue after the onset of lactation was investigated. The kinetics of change in enzyme activity and enzyme immunotitratable with antibody against avian liver acetyl-CoA carboxylase were determined during the course of lactogenic differentiation. The antibody inactivates and specifically precipitates acetyl-CoA carboxylase from rat mammary tissue as well as that from chicken liver cytosol. Characterization of the immunoprecipitate of the mammary tissue carboxylase by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis reveals a single biotin-containing polypeptide of about 230000mol.wt. This molecular weight is approximately twice that reported for the avian liver enzyme. However, chicken liver cytosol prepared in the presence of trypsin inhibitor and subjected to immunoprecipitation gives rise to a biotin-containing subunit of 230000mol.wt. as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis; omission of proteinase inhibitor leads to a subunit(s) approximately one-half this size. Throughout gestation both carboxylase activity and amounts of immunotitratable enzyme remained low; however, after parturition both parameters rose concomitantly to values 30-40 times the initial values. Therefore the elevated concentration of acetyl-CoA carboxylase appears to result from an increased rate of synthesis of enzyme relative to degradation rather than to activation of a pre-existing form of the enzyme.     

Purine nucleoside phosphorylases purified from rat liver and Novikoff hepatoma cells.

Purine nucleoside phosphorylase (PNP) was purified from rat hepatoma cells and normal liver tissue utilizing the techniques of ammonium sulfate fractionation, heat treatment, ion-exchange and molecular exclusion chromatography, and polyacrylamide gel electrophoresis. Homogeneity was established by disc gel electrophoresis in the presence and absence of sodium dodecyl sulfate. Purified rat hepatoma and liver PNPs appeared to be identical with respect to subunit and native molecular weight, substrate specificity, heat stability, kinetics and antigenic identity. A native molecular weight of 84,000 was determined by gel filtration. A subunit molecular weight of 29,000 was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A single isoelectric point was observed at pH 5.8, and the pH optimum was 7.5. Inosine, guanosine, xanthosine, and 6-mercaptopurine riboside were substrates for the enzymes. The apparent K_m for both inosine and guanosine was about 1.0 × 10^?4m and for phosphate was 4.2 × 10^?4m. Hepatoma and liver PNP showed complete cross-reactivity using antiserum prepared against the liver enzyme.