Purification and characterization of a neutral protease from rat-liver cytosolic fraction

Rat liver cytosol contains a neutral protease which degrades acetylated hemoglobin and some urea-denatured proteins maximally at pH 7.5. The enzyme was purified to homogeneity by conventional chromatographic techniques. It appears to be a metalloprotease since it is inhibited by EDTA and o-phenanthroline, the metal-depleted enzyme can be reactivated by Co2+, Zn2+, Mn2+, or Mg2+, and it is not inhibited by reagents specific for carboxyl, seryl, or thiol proteases. The enzyme has an apparent molecular weight of 200,000 as determined on Sephacryl S-200 column chromatography, and electrophoresis in sodium dodecyl sulfate showed 3 protein bands corresponding to the molecular weights of 110,000, 74,000, and 40,000.     

Age-associated changes in the estrogen receptor-active proteases of female rabbit liver

Female rabbit liver cytosol contains a receptor-modifying activity that converts the 250,000 estrogen receptor of liver and uterine cytosol to a 37,000 form. There is an age-dependent increase in this receptor-active protease and in the general protease activity of rabbit liver cytosol, measured with [14C]casein. Sephacryl S-200 chromatography of liver cytosol shows that in the young animal (5 weeks old) the major receptor-modifying activity elutes near the void volume, while in the older animal (13 weeks old) activities having lower molecular weights are present. The general protease activity elution profile is similar to the receptor-active protease profile for the 5-week-old rabbit but not the 13-week-old rabbit. The liver cytosol of the older animal has a high molecular weight protease active toward [14C]casein but not toward the estrogen receptor. The changes in the estrogen receptor forms and the receptor-modifying activity profiles of liver cytosol that occur during development in the rabbit suggest that receptor-modifying activity may initially be associated with the estrogen receptor to form a high molecular weight complex.     

Studies on a 2,3-diaminopropionate: ammonia-lyase from a Pseudomonad.

2,3-Diaminopropionate:ammonia-lyase, an induced enzyme in a Pseudomonas isolate, has been purified 40-fold and found to be homogeneous by disc gel electrophoresis and by ultracentrifugation. Some of its properties have been studied. The optimum pH and temperature for activity are 8 and 40 degrees C, respectively. The enzyme shows a high degree of substrate specificity, acting only on 2,3-diaminopropionate; the D-isomer is only one-eighth as effective as the L-form. L-Homoserine and DL-cystathionine are not substrates, and 3-cyanolalanine does not inhibit its activity. It is a pyridoxal phosphate enzyme which requires free enzyme sulphhydryls for activity. The Km values for L-2,3-diaminopropionate and pyridoxal phosphate are 1mM and 25 muM, respectively. The molecular weight of the enzyme is about 80 000 as determined by gel filtration. On treatment with 0.5M urea or guanidine by hydrochloride, the enzyme dissociates into inactive subunits with an approximate molecular weight of 45 000. One mole of the active enzyme binds one mole of pyridoxal phosphate. The bacterial enzyme seems to be quite different in many of its properties from the rat liver enzyme which also exhibits the substrate specificity of cystathionine gamma-lyase.     

Purification and characterization of hemoglobin-hydrolyzing acidic thiol protease induced by leupeptin in rat liver

Intraperitoneal administration of leupeptin to rats induced a hemoglobin-hydrolyzing protease which was most active at pH 3.5 and was insensitive to pepstatin in various tissues such as the liver, kidney, and muscle, as observed previously in adult rat hepatocytes in primary culture (Tanaka, K., Ikegaki, N., and Ichihara, A. (1979) Biochem. Biophys. Res. Commun. 91, 102-107). The induced acidic protease was purified about 600-fold in 30% yield from rat liver by conventional chromatographic techniques. The purified enzyme appeared homogeneous by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate and was a monomeric protein of Mr = 20,000. The enzyme appeared to be a glycoprotein because its induction was blocked by the addition of tunicamycin to cultures of hepatocytes and because the induced protease was absorbed on concanavalin A-Sepharose and eluted with methylglucoside. It seemed to be present in lysosomes and was fairly stable at various pH values and temperatures. It showed endopeptidase activity on various protein substrates, but scarcely hydrolyzed N-substituted derivatives of arginine. It did not hydrolyze esters, showed no aminopeptidase or carboxypeptidase activity, and did not inactivate glucose-6-phosphate dehydrogenase or aldolase. The enzyme appeared to be a thiol protease, since it was strongly inhibited by sulfhydryl-reactive compounds and N-( [N-(1-3-trans-carboxyoxiran-2-carbonyl)-L-leucyl]-agmatine and was not inhibited by reagents specific for carboxyl-, serine-, or metalloproteases. This induced protease could be separated from cathepsins B, D, and H by chromatography. The enzyme was similar to cathepsin L in chromatographic behavior, Mr and pI, but differed from the latter in stability and in its inability to inactivate some enzymes. These results suggest that it differs from any known proteases found previously in rat liver.     

Structure of tetanus toxin. Demonstration and separation of a specific enzyme converting intracellular tetanus toxin to the extracellular form.

Protease activity has been demonstrated in culture supernatants of Clostridium tetani at various stages of fermentation. Gel chromatography of the concentrated filtrates revealed the presence of three enzymatically active fractions eluting at separate positions off the column. The smallest protease was found to "nick" the single chain intracellular tetanus toxin, producing the extracellular, two-chain structure of the molecule. As little as 3 ng of active protease were sufficient to cleave 50 microgram of intracellular tetanus toxin, suggesting that this enzyme is responsible for the observed structural change of the toxin molecule during its release into the culture medium. By comparison, the second protease, eluting at an intermediate position, exhibited only marginal activity towards intracellular toxin. The third, largest, enzyme was not active under the conditions of the assay. However, the latter protease effectively hydrolyzed low molecular weight histidyl peptides, and it is concluded that this enzyme is similar to the one described by Miller, P.A. Gray, C.T., and Eaton, M.D. (1960) J. Bacteriol. 79, 95-102. The properties of the partially purified enzymes, including their differential behavior towards a number of protease inhibitors, are reported.     

Properties of mouse α-galactosidase

α-Galactosidase has been examined in various murine tissues using the substrate 4-methylumbelliferyl-α-galactoside. Mouse liver appears to contain a single major form of the enzyme, as judged by chromatography and electrophoresis. The enzyme was purified 467-fold with a yield of about 40% by a method involving chromatography on Concanavalin A-Sepharose. It has maximal activity at pH 4.2, a K_m value of 1.4 mM, an energy of activation of 16 400 cal/mol, and a molecular weight of 150 000 at pH 5.2. It is inhibited at high concentrations of myoinositol and appears to contain N-acetylneuraminic acid. In these characteristics it resembles human α-galactosidase A.The enzyme from various tissues differs in electrophoretic mobility. After treatment with neuraminidase, however, the enzyme from all tissues comigrates as a single band of activity. By this criterion the α-galactosidase of liver is most heavily sialylated and that from kidney the least. As estimated by gel filtration, the enzyme from liver and kidney exists as species of molecular weight 320 000, 150 000 and 70 000, depending upon pH and ionic strength. This appears to be the result of aggregation of the enzyme, since the forms are interconvertible and under some conditions a single molecular weight species is observed. The liver enzyme is primarily lysosomal, while the kidney enzyme is distributed approximately equally between lysosomal and microsomal fractions.     

The release of collagenase as an inactive proenzyme by bone explants in culture

1. A latent collagenase, activated only by limited proteolysis, was found in culture media of mouse bone explants. It could be activated by trypsin or, less efficiently, by chymo-trypsin. Skin explants also released latent collagenase. 2. Bone collagenase attacks native collagen at about neutral pH when it is in solution, in reconstituted fibrils or in insoluble fibres, producing two fragments representing 75 and 25% of the molecule. It requires calcium and is inhibited by EDTA, cysteine or serum. 3. Latent collagenase is not activated by trypsin-activated collagenase but by a distinct unidentified thermolabile agent present in a latent trypsin-activatable state in the culture media, or by purified liver lysosomes between pH5.5 and pH7.4. Trypsin activation decreases the molecular weight of latent collagenase from 105000 to 84000 as determined by gel filtration. 5. The latency of collagenase is unlikely to be due to an enzyme-inhibitor complex. Although some culture media contain a collagenase inhibitor, its presence is not constant and its molecular weight (at least 120000) is not compatible with the decrease in molecular weight accompanying activation; also combinations of collagenase with inhibitor are not reactivated by trypsin. Moreover, the latency remains after gel filtration, or treatment by high dilution, exposure to pH values between 2.5 and 10, or high ionic strength, urea or detergent. 6. It is proposed that latent collagenase represents an inactive precursor of the enzyme, a ;procollagenase', and that the extracellular activity of collagenase is controlled by another protease that activates procollagenase by a limited proteolysis of its molecule.     

Multiple forms of Ca-activated protease from rat brain and muscle

Three Ca-dependent proteases have been identified in rat brain and skeletal muscle using ion exchange, gel filtration, and substrate affinity chromatography. A high degree of homology exists among three enzymes from different sources. Both the high molecular weight protease (154,000) and lower molecular weight protease (96,000) show high affinity for calcium while the third protease (76,000) had low affinity for calcium. Transformation among the three enzymes was calcium-induced and the process was unidirectional, generating a lower molecular weight form with decreased affinity for calcium. The protease with low affinity for calcium was susceptible to calcium-induced inactivation by autocatalysis. Immunologically the three proteases were equivalent, if not identical, and the brain and muscle proteases cross-react. All three proteases degraded neurofilament proteins; however, the protease with low affinity for calcium had 3 to 6 times higher specific activity. It is suggested that the high molecular weight enzyme (154,000) may be the native form of the Ca-dependent protease present in vivo.     

Purification of a liver alkaline protease which degrades oxidatively modified glutamine synthetase. Characterization as a high molecular weight cysteine proteinase

A nonlysosomal alkaline protease which degrades the oxidatively modified form of Escherichia coli glutamine synthetase has been purified to apparent homogeneity from rat and mouse liver acetone powders. Its molecular weight was determined to be 300,000 by Sephacryl S-300 gel filtration but results of further studies using high pressure liquid chromatography gel filtration suggest a value of 650,000. Examination of the subunit structure by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple bands of molecular weights between 22,000 and 34,000. The alkaline protease was inhibited by thiol reagents. Phenylmethylsulfonyl fluoride, aprotinin, leupeptin, antipain, and chymostatin partially inhibited the protease. The inhibition by phenylmethylsulfonyl fluoride was prevented by dithiothreitol, and alpha 1-antitrypsin and soybean trypsin inhibitor did not inhibit. No inhibition was observed with metalloprotease inhibitors. The alkaline protease is active over a broad range of pH with optimum activity for the degradation of oxidized glutamine synthetase around pH 9.0. Its activity is not stimulated by MgATP. A study of the products of insulin B chain degradation demonstrated major cleavage sites at Gln13-Ala14, Leu15-Tyr16, Cys(SO3H)19-Gly20, Gln4-His5, and Leu17-Val18. Based on its endopeptidase activity and its inhibitor specificity, the alkaline protease should be classified as a cysteine proteinase. It appears to be distinct from previously described proteinases and is likely involved in nonlysosomal mechanisms of intracellular protein turnover.     

Purification and properties of the thermostable acid protease of Penicillium duponti.

An acid protease produced by the thermophilic fungus Penicillium duponti K 1014 has been purified by consecutive ion-exchange and gel permeation chromatography, and crystallized from aqueous acetone solution. The purified endopeptidase gave a symmetrical schlieren peak by sedimentation velocity, and was found to be homogeneous upon disc gel electrophoresis at pH 9.5. The enzyme was most active at pH 2.5 against milk casein and showed high thermostability. An isoelectric point of 3.81 was found by isoelectric focusing. A minimum molecular weight of 41 590 was calculated from the amino acid composition, adopting an arginine content of one residue per mole of enzyme. This minimum molecular weight is in good agreement with the value of 41 000 previously found by gel permeation (Hashimoto, H., Iwaasa, T., and Yokotsuka, T. (1973), Appl. Microbiol. 25, 578). Besides the thermostability, the purified P. duponti protease differs from other well-characterized acid proteases in that it contains carbohydrate, 4.33% expressed as glucose. The enzyme was not affected by p-bromophenacyl bromide, but was completely inactivated by alpha-diazo-p-bromoacetophenone, diazoacetyl-DL-norleucine methyl ester, and diazoacetylglycine ethyl ester, in the presence of Cu2+. The complete inactivation of the protease by diazoacetyl-DL-norleucine methyl ester resulted in the specific incorporation of 1 mol of norleucine/mol of enzyme. On the basis of similar behavior of other acid proteases toward this inactivator, the results suggest the presence at the active site of an unusually reactive carboxyl group, involved in the catalytic function. The naturally occurring pepsin inhibitor of Streptomyces naniwaensis [Murao, S., and Satoi, S. (1970), Agric. Biol. Chem. 34, 1265] inhibited also the protease, at a threefold molar excess with respect to the enzyme.     

Purification and properties of a thiol protease from rat liver nuclei

A thiol protease was purified about 800-fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G-100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H-labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30-40% of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2-4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.     

Purification and characterization of a novel protease from the latex of Pedilanthus tithymaloids

A novel protease was purified to homogeneity from the latex of Pedilanthus tithymaloids by a simple purification procedure involving ammonium sulfate precipitation and cation-exchange chromatography. The molecular weight of the protease was estimated to be approximately 63.1 kDa and the extinction coefficient (epsilon(1%)(280nm)) was 28.4. The enzyme hydrolyzes denatured natural substrates like casein, azoalbumin and azocasein with a high specific activity but little activity towards synthetic substrates. The pH and temperature optima were pH 8.0-9.5 and 65-70 degrees C, respectively. The proteolytic activity of the enzyme was inhibited by different protease-specific inhibitors (e.g., thiol, serine, metallo, etc.) up to a certain extent but not completely by any class of inhibitors. The enzyme was relatively stable towards pH change, temperature, denaturants and organic solvents. The enzyme consists of five disulfide bridges compared to three observed in most plant cysteine proteases. Overall, the striking features of this protease are its high molecular weight, high cysteine content and only partial inhibition of activity by different classes of protease inhibitors contrary to known proteases from other plant sources. The enzyme is named as pedilanthin as per the protease nomenclature.     

Purification and Some Properties of a Protease from Streptomyces limosus

Streptomyces limosus was selected because it secreted a novel protease that catalyzed the synthetic reaction forming Pro-Pro-Pro from Pro-Pro. The protease was purified to an electrophoretically homogeneous state and an activity of more than about 20,000-fold that of the culture broth. The molecular mass of the enzyme was estimated to be 50 kDa by SDS-polyacrylamide gel electrophoresis. The enzyme was most active in alkaline pH for the synthetic reaction producing Pro-Pro-Pro from Pro-Pro, although for the hydrolytic reaction forming proline it was most active in neutral pH. The enzyme was inhibited by 1,2-epoxy-3-(p-nitrophenoxy)propane (EPNP) and diazoacetyl-DL-norleucine methyl ester (DAN). It can be considered that this enzyme belongs to the class of aspartic proteases. The substrate specificity indicates that this enzyme has a strong affinity for proline as a N-terminal amino acid of peptides.     

尖孢镰刀菌M1耐热蛋白酶纯化及酶学性质

【背景】前期工作中,从北大仓白酒大曲分离到一株真菌,经形态学和分子生物学方法,将其鉴定为尖孢镰刀菌(Fusarium oxysporim)M1,研究发现该菌能产中性蛋白酶。中性蛋白酶是应用于工业化生产的重要酶制剂。由于其作用条件温和、催化速率较高,被广泛应用于食品、医药、皮革、饲料、化工和废弃物处理行业。【目的】为了使该菌蛋白酶应用于相关工业生产,需要对该蛋白酶进行纯化和酶学特性研究。【方法】采用硫酸铵分级分离、疏水和离子交换层析对该菌蛋白酶进行纯化,通过SDS-PAGE测定酶的纯度和分子量,并研究其热稳定性和酸碱适应性。【结果】经各步层析,蛋白酶纯化倍数达26.1,得率为7.9%;经测定纯酶的分子量为62 kD;该酶最适温度为40℃,最适pH为7.0,属于中性蛋白酶;该酶对酸较敏感,对碱有较强的耐受性;耐热性较强,但酶活性不受乙二胺四乙酸二钠盐抑制。【结论】由于该中性蛋白酶具有较好的耐热性,因此,可作为工业生产上潜在的生物催化剂。     

Multiplicity of molecular forms and thermostability of Torula thermophila UzPT-1 protease]

Alkaline protease preparations with different ratio of molecular forms are isolated from cultural medium of thermophilic fungi Torula thermophila UzPT-1 by means of protein fractionation with (NJ/)2SO4 and gel filtration through Sephadex G-75. The enzyme preparations differ in their thermostability in water at 60 degrees C. High molecular weight oligomeric enzyme forms dissociate in water (at 2-4 degrees C) forming dimeric and monomeric forms. Disaggregation is accompanied by the change in the thermostability of the enzyme preparations. It is concluded that protease thermostability depends on the ratio of dimeric and monomeric forms of the preparation, and it is associated with the conformational state of the enzyme molecules, and it is associated with the conformational state of the enzyme molecules. Oligomeric forms do not dissociate in 1% sodium dodecylsulphate and in 6 M urea. Ca2+ produces dissociation of high molecular weight enzyme forms and the conformational transition into the thermostable state.     

In vitro processing of the human alkyl-dihydroxyacetonephosphate synthase precursor.

Alkyl-dihydroxyacetonephosphate synthase, a peroxisomal enzyme involved in the biosynthesis of ether phospholipids, is synthesized with a cleavable N-terminal presequence containing the peroxisomal targeting signal type 2. The human alkyl-dihydroxyacetonephosphate synthase precursor produced in vitro or expressed in Escherichia coli could be processed to a lower molecular weight protein by incubation at 37 degrees C with a guinea pig liver fraction, enriched in mitochondria, lysosomes, and peroxisomes. This lower molecular weight protein was identified as the mature human alkyl-dihydroxyacetonephosphate synthase by radiosequencing, indicating that the processing protease is present in this organellar fraction. Characterization of the processing protease indicated that it is a cysteine protease with a pH optimum of 6.5. Furthermore, it was demonstrated that exogenously added pre-alkyl-dihydroxyacetonephosphate synthase was imported and processed in purified peroxisomes in vitro. Processing of alkyl-dihydroxyacetonephosphate synthase did not increase the activity of the enzyme. This indicates that the presence of the presequence does not affect the activity of the enzyme.     

Chymotryptic-like hydrolysis of luliberin (LH-RF) by an adenohypophyseal enzyme of high molecular weight

An enzyme that hydrolyzes the fluorogenic chymotrypsin substrate glutaryl-Gly-Gly-Phe-β-naphthylamide has been partially purified from extracts of bovine anterior pituitaries. Like chymotrypsin, this enzyme hydrolyzes the neuropeptide Luliberin (LH-RF, <Glu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH₂) at the carboxyl-side of Trp and Tyr, but it differs from the pancreatic protease by its high molecular weight, insensitivity towards OH-reactive agents and other enzymechemical parameters. It seems, however, to be identical to the “cation-sensitive neutral endopeptidase”. In the course of this study evidence has also been obtained that LH-RF is not degraded by the cystinyl-arylamidase.     

The purification of protease IV of E.coli and the demonstration that it is an endoproteolytic enzyme

A strong proteolytic activity is unmasked and solubilized when $\text{E. coli}$ outer membrane fragments are preincubated with 0.083% sodium dodecyl sulfate. This proteolytic activity cleaves αS1 casein into the same degradation products as protease IV, a recently described protease of $\text{E.coli}$ located in the outer membrane (Ph. Régnier, preceding paper), it is concluded that sodium dodecyl sulfate solubilizes the same protease. Protease IV has been purified 11,200 fold, probably to homogenetiy, by sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by elution of the protein from gel slices. The purified enzyme is fully active, its molecular weight, determined from its migration in denaturating gels is 23,500. αS1 casein is cleaved by protease IV into two large polypeptides which are not further degraded and some small peptides of about 5,000 daltons. The production of discrete polypeptide species suggests that protease IV is an endoproteolytic enzyme.     

A high molecular weight protease in the cytosol of rat liver. I. Purification, enzymological properties, and tissue distribution

Rat liver cytosol has low hydrolytic activity against [3H]methylcasein at neutrality, but activity increases greatly on addition of various compounds such as poly-L-lysine, N-ethylmaleimide, and sodium dodecyl sulfate, suggesting that it contains latent proteolytic activity. The latent enzyme was found to be stabilized in the presence of 20% glycerol and to be activated by addition of poly-L-lysine. The latent enzyme was purified from a crude extract of rat liver to apparent homogeneity in the presence of 20% glycerol by conventional chromatographic techniques. The purified enzyme showed endoproteolytic activity toward various proteins when it was activated by the compounds listed above. It preferentially degraded N-substituted tripeptide substrates with a basic amino acid at the carboxyl terminus, as well as peptides containing neutral hydrophobic amino acids. It did not require activation for these peptidase activities, in contrast to its activity toward large proteins. Interestingly, a proteinase and a trypsin-like and a chymotrypsin-like peptidase activity could not be separated by customary chromatographic methods but were distinguishable by their sensitivities to various inhibitors, activators, and covalent modifiers, suggesting that the enzyme has three distinct active sites within a single protein. The enzyme seems to be a seryl endopeptidase showing maximal activity at neutral and weakly alkaline pH values. Thus, the enzyme is a unique protease with latent multifunctional catalytic sites. The distribution of the protease in soluble extracts of various rat tissues and cells was examined quantitatively by an enzyme immunoassay. The enzyme level was highest in liver and also in spleen, stomach, lung, small intestine, and kidney, but was low in heart, diaphragm, skeletal muscle, brain, and skin. The concentrations of enzyme in some established cell lines including hepatoma and rat kidney cells were comparable to that in normal liver hepatocytes. The enzyme was found mainly in the cytosol fraction, although a small amount was associated with microsomal membranes, suggesting that it is an extralysosomal protease. Immunohistochemical staining of the liver and skeletal muscles showed that the protease is distributed diffusely in panlobular hepatocytes with slight centrilobar predominance and is present in Kupffer cells, vascular endothelial cells, and bile duct epithelial cells in the liver and also diffusely in the intermyofibrillar spaces and vascular endothelial cells in skeletal muscle. The quantitative data obtained in the present study indicate the presence of the protease in the cytosol fraction of all rat tissues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and characterization of two leaf polypeptide inhibitors of leaf protease from alfalfa (Medicago sativa)

Two polypeptides with antiproteolytic activities have been isolated from alfalfa leaves. Polypeptide I resembles the previously described plant protease inhibitors in both structural and functional features; it has a molecular weight of 15,000, a random coil secondary structure, and inhibits exogenous protease as well as alfalfa leaf protease. Polypeptide II is a novel type of plant inhibitor with a molecular weight of 6300 and a highly organized structure with a high (40-50%) alpha-helix content. It only inhibits endogenous protease with a molar stoichiometry polypeptide/enzyme protein of 1.