Identification and primary characterization of specific proteases in the digestive juice of Archachatina ventricosa

The profile of sedimentation on a 4-20% (w/v) linear sucrose gradient of the digestive juice of the mollusk Archachatina ventricosa revealed the presence of at least four specific proteases. A first peak, corresponding to a sedimentation coefficient of 3.9 S, contained two endoproteases that could be assayed, one with Leu-pNA and the other with Met-pNA. Their activity was maximal at pH 8.0 and increased in the presence of Ca(2+) ions. Both enzymes were inhibited by the chelating agent 1,10-phenanthroline but their thermal inactivation kinetics were different. A second protease peak was observed at 6.8 S and corresponded to a metallo-endoprotease that hydrolyzed with a maximal activity at pH 8.0 only the amide bonds of peptide substrates having a threonine residue at the P1' position. A last protease peak identified at 9.0 S contained a protease that preferentially acted on tripeptides, such as Val-Pro-Leu (diprotin B) and Thr-Val-Leu, releasing the C-terminal residue. Unlike the proteases identified in the two other peaks, its activity was maximal at acid pH (5.0) and was inhibited by the serine protease inhibitors. Together these results show the potential of A. ventricosa as a source of specific proteases.     

Extracellular acid protease of Aspergillus oryzae grown on liquid media: multiple forms due to association with heterogeneous polysaccharides.

The acid protease (EC 2.4.23.6) that is produced extracellularly when Aspergillus oryzae is grown on liquid media has been isolated and characterized. The enzyme was purified by precipitation with tannic acid, chromatography on Duolite A-2, and gel filtration on Sephadex G-100. The last step yielded four active components, with varying molecular weights ranging from 42,000 to 60,000. Two of them, designated E1 and E1a, with molecular weights of 60,000 and 55,000, respectively, were heterogeneous on isoelectric focusing, both giving at least three enzyme species with different isoelectric points, whereas the other two, E1b and E2, with molecular weights of 49,000 and 42,000, respectively, were essentially homogeneous. These four enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They had essentially the same amino acid composition and immunologically cross-reacted with each other. These catalytic, chemical, and immunological properties are similar to those of acid protease A1 and A2 from A. oryzae grown on solid bran media. Unlike acid protease from solid bran culture, which contains both carbohydrate-containing and the carbohydrate-free species, all of the four enzymes, E1, E1a, E1b, and E2, contained carbohydrate, ranging from 18.9 to 43% and comprising three hexoses, glucose, galactose, and mannose. The carbohydrate portions were polysaccharide in nature and heterogeneous with respect to both molecular weight and sugar composition, and at least a part of the carbohydrate was present in the form of homopolysaccharides such as galactan and mannan. These findings indicate that polysaccharide chains with different molecular weights and with different chemical compositions are apparently responsible for the microheterogeneity of acid protease.     

Purification and some properties of the extracellular acid proteases from Mucor renninus

A complex of proteases was fractionated into three enzymes by chromatography of a crude enzyme preparation obtained from culture fluid of the fungus Mucor renninus on biospecific polystyrene adsorbent. Electrophoretically homogeneous proteases I-III were obtained by subsequent rechromatography on biospecific adsorbent and gel filtration on Sephadex G-75. Optimal proteolytic activities occurred at pH 4.25; 3.5 and 2.5 for enzymes I, II and III, respectively. Milk-clotting activity was exhibited only by protease II. All three proteases hydrolysed haemoglobin, Na caseinate and bovine serum albumin. Enzyme I hydrolysed Na caseinate the most effectively, while haemoglobin was the most effective substrate for proteases II and III. Trypsinogen was activated only by protease I. All three enzymes have a molecular weight ～35 000 as determined by gel chromatography on Sephadex G-75 column and by sodium dodecylsulphate disc electrophoresis. Isoelectric points, pH-stability range, amino acid composition, carbohydrate content were determined for each enzyme and the influence of metal ions (Ca²⁺, Mg²⁺, Cu²⁺, Co²⁺) on proteolytic activities of these enzymes studied.     

Purification and characterization of the two molecular forms of membrane acid protease from Aspergillus oryzae.

Two forms (M1 and M2) of the membrane-bound acid protease of Aspergillus oryzae have been purified by extraction with Triton X-100, washing with cold acetone, and repeated gel filtration on Bio-Gel A-15 m in the presence and absence of Triton X-100. The purified membrane enzymes, M1 and M2, moved as a single band in acrylamide gel electrophoresis and had apparent molecular weights of 150 000 and 60 000, respectively, as estimated by sodium dodecyl sulfate/acrylamide gel electrophoresis. These two membrane enzymes activated bovine pancreatic trypsinogen and had the same pH optima in the acid pH range. They immunologically cross-reacted with each other and with an extracellular acid protease from A. oryzae, and contained carbohydrate, ranging from 52.5 to 80.5% and comprising three hexoses, glucose, galactose, and mannose. While these catalytic, chemical and immunological properties are similar to those of the extracellular acid protease from A. oryzae, both membrane enzyme differed in their hydrophobic properties from external enzymes. Thus they are activated by the detergent Triton X-100 and some polar lipids.     

Monitoring of the production of monoclonal antibodies by hybridomas. Part II: Characterization and purification of acid proteases present in cell culture supernatant.

An acid proteolytic activity has been found in cell culture supernatants from long-term cultivations of hybridoma cells in hollow fibre bioreactors using serum free medium. The proteolytic activity has now been further characterized and the main results were: (1) the proteolytic activity showed a maximum around pH 3 and declined essentially to zero at pH 8; (2) the activity was specifically inhibited by pepstatin A; (3) the acid proteases consisted of two sets of closely spaced bands with apparent molecular weights of 40-45K and 90-105K, respectively; (4) the protease bands (40-45K and 90-105K) were reactive with anti-human cathepsin D; (5) the IEP values of the acid proteases ranged from pH 4.55-6.5. Furthermore, IgG incubation with the acid proteases isolated from hybridoma cells yielded fragments similar to those found in serum-free hollow fibre cell culture supernatants. These results indicated that the IgG fragments are the result of degradation by cathepsin D like proteases released after cell death or cell lysis.     

竹叶青蛇毒丝氨酸蛋白酶的分子克隆和序列比较

利用逆转录酶与聚合酶链反应相结合的RT—PCR法,扩增出5个竹叶青(Trimeresurus stejnegeri)蛇毒丝氨酸蛋白酶的cDNAs;将扩增的cDNA片段克隆入pGEM-T载体中,筛选得到它们的基因,分别命名为TSSP-1、TSSP-2、TSSP-3、TSSP-4和TSSP-5。经末端终止法测定核苷酸序列,推导出5个丝氨酸蛋白酶的全序列;结合纯化的蛋白酶N-末端序列测定结果,推导TSSP-2、-3和-4分别编码凝血酶样酶stejnobin、纤溶酶stejnefibrase 1和2。5个丝氨酸蛋白酶分别含有1～6个N-型糖基结合位点,表明它们的计算分子量与纯化蛋白表观分子量之间的差异是由糖含量的不同造成,而其氨基酸序列相似度在60％～90％。TSSP-1和-2编码的成熟蛋白酶由236个氨基酸残基组成,TSSP-3、-4和-5的则由234个氨基酸残基组成。TSSP-1编码的蛋白酶在组成丝氨酸蛋白酶三联体催化活性中心产生了His^41-Arg^41的天然突变,这与其他自然界已发现的丝氨酸蛋白酶明显不同。     

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 of several proteolytic enzymes by tosyl- and carbobenzoxy-triethylene-tetramine-sepharoses.

Tosyl-triethylenetetramine-Sepharose (Tos-T-Sepharose) and carbenzoxytriethylenetetramine-Sepharose (Z-T-Sepharose) were found to be adsorbents utilizable in the purification of several microbial and animal proteases. The former Sepharose derivative adsorbed alpha-chymotrypsin, trypsin, subtilisin, thermolysin and neutral subtilopeptidase at neutral pH range, and acid proteases such as pepsin and Rhizopus niveus protease at pH 3.5-6.5. alpha-Chymotrypsin and trypsin were eluted with 0.1 N acetic acid and Rhizopus protease with 0.5 N acetic acid, thermolysin with 1 M guanidine-HCl or 33% ethyleneglycol, whilst pepsin was recovered by elution with 2 M guanidine-HCl at pH 3.5. The binding of neutral subtilopeptidase and subtilisin to this adsorbent was comparatively weak and both the enzymes were recovered by elution with 0.5 M NaCl at neutral pH. On the other hand, Z-T-Sepharose was found to bind tightly to these proteolytic enzymes except neutral subtilopeptidase. Trypsin and alpha-chymotrypsin were released from the adsorbent column with 1 M p-toluenesulfonate, and subtilisin with 1 M guanidine-HCl or 33% ethyleneglycol at neutral pH region. By these chromatographic procedures, the specific activities of these proteolytic enzymes increased effectively. Comparison of the binding abilities of acetyl-, benzoyl-, tosyl- and carbobenzoxy-T-Sepharoses to these enzymes suggests that hydrophobicity of tosyl and carbobenzoxy groups plays an important role in the enzyme-adsorbent interaction.     

Extracellular acid and alkaline proteases from Candida olea

Candida olea 148 secreted a single acid protease when cultured at acidic pH. In unbuffered medium, the culture pH eventually became alkaline and a single alkaline protease was produced. This was the only proteolytic enzyme produced when the organism was grown in buffered medium at alkaline pH. Both proteolytic enzymes were purified to homogeneity (as assessed by SDS-PAGE). The Mr of the acid protease was 30900, the isoelectric point 4.5; optimum activity against haemoglobin was at 42 degrees C and pH 3.3. This enzyme was inactivated at temperatures above 46 degrees C and was inhibited by either pepstatin and diazoacetyl-norleucine methyl ester but was insensitive to inhibition by either 1,2-epoxy-3-(p-nitrophenoxy)-propane or compounds known to inhibit serine, thiol or metallo proteases. The acid protease contained 11% carbohydrate. The alkaline protease had an Mr of 23400 and isoelectric point of 5.4. The activity of this enzyme using azocoll as substrate above 42 degrees C and was inhibited by phenylmethyl-sulphonyl fluoride and irreversible inactivated by EDTA. The enzyme was also partially inhibited by DTT but was insensitive to either pepstatin or p-chloromercuribenzoic acid.     

THE LYSOSOME PERIPHERY: BIOCHEMICAL AND ELECTROKINETIC PROPERTIES OF THE TRITOSOME SURFACE

Normal rat liver lysosomes were isolated by the technique of loading with Triton WR-1339. Purity of the preparation was monitored with marker enzymes; a high enrichment in acid hydrolases was obtained in the tritosome fraction. In 0.0145 M NaCl, 4.5% sorbitol, 0.6 mM NaHCO₃, pH 7.2 at 25°C the tritosomes had an electrophoretic mobility of -1.77 ± 0.02 µm/s/V/cm, a zeta potential of 23.2 mV, a surface charge of 1970 esu/cm², and 33,000 electrons per particle surface assuming a tritosome diameter of 5 x 10^-7 m. Treatment of the tritosomes with 50 µg neuraminidase/mg tritosome protein lowered the electrophoretic mobility of the tritosome to -1.23 ± 0.02 µm/s/V/cm under the same conditions and caused the release of 2.01 µg sialic acid/mg tritosome protein. Treatment of the tritosomes with hyaluronidase did not affect their electrophoretic mobility, while trypsin treatment elevated the net negative electrophoretic mobility of the tritosomes. Tritosome electrophoretic mobilities indicated a homogeneous tritosome population and varied greatly with ionic strength of the suspending media. pH vs. electrophoretic mobility curves indicated the tritosome periphery to contain an acid-dissociable group which likely represents the carboxyl group of N-acetylneuraminic acid; this was not conclusively proven, however, since the tritosomes lysed below a pH of 4 in the present system. Total tritosome carbohydrate (anthrone-positive material as glucose equivalents) was 0.19 mg/mg tritosome protein while total sialic acid was 3.8 µg (11.4 nmol)/mg tritosome protein. A tritosome "membrane" fraction was prepared by osmotic shock, homogenization, and sedimentation. Approximately 25% of the total tritosome protein was present in this fraction. Analysis by gas-liquid chromatography and amino acid analyzer showed the following carbohydrate composition of the tritosome membrane fraction (in microgram per milligram tritosome membrane protein): N-acetylneuraminic acid, 14.8 ± 3; glucosamine, 24 ± 3; galactosamine, 10 ± 2; glucose, 21 ± 2; galactose, 26 ± 2; mannose, 31 ± 5; fucose, 7 ± 1; xylose, 0; and arabinose, 0. The results indicate that the tritosome periphery is characterized by external terminal sialic acid residues and an extensive complement of glycoconjugates. Essentially all the tritosome N-acetylneuraminic acid is located in the membrane and about 53% of it is neuraminidase susceptible.     

Properties of Two Homologous Alkaline Proteases from Streptomyces rectus

Some physicochemical properties of two thermostable proteases from Streptomyces rectus are described. The enzymes were judged to be identical with respect to molecular weight, inactivation with serine protease inhibitors, and in primary structure by peptide analysis. Amino acid analysis indicated the enzymes had identical compositions except for their amide content. The molecular weights of the enzymes were judged to be 28,000 by sedimentation equilibrium, 26,200 by sedimentation diffusion, and 29,100 from amino acid analysis. Titration of the proteases with diisopropylfluorophosphate and phenylmethane sulfonylfuoride indicate equivalent weights of 28,500 and 32,800 g, respectively, for the proteins. The pentapeptide around the serine residue reacting with diisopropylfluorophosphate was isolated and had the composition: Asx(1), Gly(1), Thr(1), Ser(1), Met(1).     

Studies on extracellular proteases of Streptococcus sanguis. Purification and characterization of a human IgA1 specific protease.

Extracellular caseinolytic activity was found in the culture fluid of Streptococcus sanguis ATCC 10556 grown in a dialyzed culture medium. This activity was due to multiple proteases that differed in their elution from hydroxyapatite, sensitivity to enzyme inhibitors, specificity and optimum pH. IgA protease, which splits human immunoglobulin A1 into intact Fc and Fab could be effectively separated from these relatively non-specific proteases and purified to apparent homogeneity in 20% yield by a five-step procedure. Although the bulk of the dextran sucrase activity was separated from the IgA protease, a small amount of sucrase activity remained with the final IgA protease preparation. In polyacrylamide gel electrophoresis at pH 9.5 both activities were located in the single protein band detected in this preparation. A quantitative method for the assay of IgA protease was developed, based on radial immunodiffusion to quantitate the Fab produced. This was used to follow the specific activity and yield during purification, and to characterize some of the catalytic properties of the enzyme. At an enzyme/substrate ratio of 1: 400 (w/w) the protease could effect 50% proteolysis of IgA in overnight incubation at 37 degrees C. The optimum activity was at pH 8.0, and 50% inhibition was achieved at 4 . 10(-4) M o-phenanthroline or 8 . 10(-4) M ethylene diamine tetraacetate. Concentrations of diisopropyl phosphofluoridate, phenylmethyl-sulfonyl fluoride, iodoacetate and p-chloromercuribenzoate up to 10(-2) M were without effect on the IgA protease activity. Full reactivation of the chelator inhibited enzyme could be achieved by the addition of Mg2+, Mn2+ or Ca2+.     

Protease nexin. Properties and a modified purification procedure

The present paper describes chemical and functional properties of protease nexin, a serine protease inhibitor released from cultured human fibroblasts. It is shown that protease nexin is actually synthesized by fibroblasts and represents about 1% of their secreted protein. Analysis of the amino acid composition of purified protease nexin indicates that it is evolutionarily related to antithrombin III and heparin cofactor II. Protease nexin contains approximately 6% carbohydrate, with 2.3% amino sugar, 1.1% neutral sugar, and 3.0% sialic acid. The Mr calculated from equilibrium sedimentation analysis is 43,000. Protease nexin is a broad specificity inhibitor of trypsin-like serine proteases. It reacts rapidly with trypsin (kassoc = 4.2 +/- 0.4 X 10(6) M-1 s-1), thrombin (kassoc = 6.0 +/- 1.3 X 10(5) M-1 s-1), urokinase (kassoc = 1.5 +/- 0.1 X 10(5) M-1 s-1), and plasmin (kassoc = 1.3 +/- 0.1 X 10(5) M-1 s-1), and slowly inhibits Factor Xa and the gamma subunit of nerve growth factor but does not inhibit chymotrypsin-like proteases or leukocyte elastase. In the presence of heparin, protease nexin inhibits thrombin at a nearly diffusion-controlled rate. Two heparin affinity classes of protease nexin can be detected. The present characterization pertains to the fraction of protease nexin having the higher affinity for heparin. The low affinity material, which is the minor fraction, is lost during purification.     

Gastric proteases of the Greenland cod Gadus ogac. II. Structural properties

Three gastric proteases were isolated from the stomach mucosa of the Greenland cod (Gadus ogac). The cod proteases were all less stable to heating and protease 1 retained less activity at 5 degrees C when the pH was greater than 5 in comparison with porcine pepsin. The activities of cod proteases 1 and 2, with hemoglobin as the substrate, were doubled in the presence of 25 mM NaCl, while cod protease 3 and porcine pepsin were not stimulated by the salt. The cod proteases did not cross-react with antibodies raised against porcine pepsin. However, some cross-reactivity was noted with antibodies raised against proteases from psychotrophic pseudomonads. The molecular weights of all the cod proteases were in the range of 36,000-38,000. The amino acid compositions of the cod proteases as compared by the Metzger difference index differed from the mammalian gastric proteases by about the same extent that pepsin, gastricsin, and chymosin differ from each other. Of the cod enzymes, protease 1 differed from mammalian gastric proteases, while cod proteases 3 was more like chymosin with respect to amino acid composition. Cod protease 1 had the lowest hydrophobicity index and chymosin had the highest. The hydrophobicity indices of cod proteases 2 and 3 were intermediate between that of porcine pepsin and bovine chymosin. It is suggested that the Greenland cod proteases represent less differentiated forms of gastric proteases than the mammalian pepsins, gastricsins, and chymosins.     

Purification and Characterization of an Extracellular Protease from Penicillium chrysogenum Pg222 Active against Meat Proteins

An extracellular protease from Penicillium chrysogenum (Pg222) isolated from dry-cured ham has been purified. The purification procedure involved several steps: ammonium sulfate precipitation, ion-exchange chromatography, filtration, and separation by high-performance liquid chromatography. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis and gel filtration, the purified fraction showed a molecular mass of about 35 kDa. The hydrolytic properties of the purified enzyme (EPg222) on extracted pork myofibrillar proteins under several conditions were evaluated by SDS-PAGE. EPg222 showed activity in the range of 10 to 60°C in temperature, 0 to 3 M NaCl, and pH 5 to 7, with maximum activity at pH 6, 45°C, and 0.25 M NaCl. Under these conditions the enzyme was most active against tropomyosin, actin, and myosin. EPg222 showed collagenolytic activity but did not hydrolyze myoglobin. EPg222 showed higher activity than other proteolytic enzymes like papain, trypsin, and Aspergillus oryzae protease. The N-terminal amino acid sequence was determined and was found to be Glu-Asn-Pro-Leu-Gln-Pro-Asn-Ala-Pro-Ser-Trp. This partial amino acid sequence revealed a 55% homology with serine proteases from Penicillium citrinum. The activity of this novel protease may be of interest in ripening and generating the flavor of dry-cured meat products.     

Isolation and properties of four alpha-amylase isozymes from human submandibular saliva

Four isoamylases have been isolated from human submandibular secretions by gel filtration and isoelectric focusing. The isozymes (1A, 1B, 2A, 2B) were each purified about 8-fold and each yielded one major band on disc gel electrophoresis. In all cases the major protein band contained more than 95% of the protein and amylase activity recovered. The isoenzymes, in order of their relative positions on the polyacrylamide gels (from the anodal end), their isoelectric points, and percentage distribution in the submandibular secretion are as follows: isozyme 2A, pH 5.9, 9%; isozyme 1A, pH 5.9, 18%; isozyme 2B, pH 6.4, 63%; isozyme 1B, pH 6.4, 10%. Amino acid analyses showed that the protein compositions of the four isoamylases were essentially the same. Possible differences were noted in aspartic acid, serine, glutamic acid, and proline contents. Molecular weights, determined by SDS disc gel electrophoresis, were 57,000 for 1A and 1B, and 54,000 for 2A and 2B. This molecular weight difference is attributed mainly to the presence of bound carbohydrate on isozymes 1A and 1B. Gas Chromatographic analysis was used for determining the carbohydrate compositions. Molar ratios of sugars were similar for both glycoprotein amylases (moles sugar/mole enzyme): glucosamine, 3; mannose, 3; galactose, 2; fucose, 3. Isoamylase 1A, which had more carbohydrate than 1B, also contained about 2 moles of N-acetylneuraminic acid. Sialic acid was not detected in isozyme 1B.     

Comparison of the carbohydrate and amino acid composition of normal and S-variant α-1-antitrypsin

α-1-Antitrypsin has been isolated and purified from the serum of an individual with the Pi S phenotype whose serum contains only 50–60% as much α-1-antitrypsin as normal M-type serum. The preparation was homogeneous by the criteria of sodium dodecyl sulfate polyacrylamide gel electrophoresis and sedimentation equilibrium ultracentrifugation. When analyzed in the ultracentrifuge, the S-type α-1-antitrypsin exhibited a molecular weight of 47500 which was essentially the same as that of the M-type (47300) and the Z-type (47500) α-1-antitrypsin. The S-type α-1-antitrypsin contains 15.2% carbohydrate consisting of 16.4 residues/mol of N-acetylglucosamine, 7.8 residues/mol of mannose. 6.7 residues/mol of galactose and 7.1 residues/mol of sialic acid which is essentially the same as the carbohydrate composition of the M-type α-1-antitrypsin. In addition, M- and S-type α-1-antitrypsin have very similar amino acid compositions.     

Properties of Pseudomonas AM1 primary-amine dehydrogenase immobilized on agarose.

1. The primary-amine dehydrogenase of Pseudomonas AM1 (primary amine:(acceptor) oxidoreductase (deaminating), EC 1.4.99.-) was purified by an improved method and covalently attached to cyanogen bromide-activated Sepharose 4B. The immobilized enzyme showed very little change in its sensitivity to heat and to inhibition by semicarbazide as compared with the soluble enzyme, but had enhanced stability at 0 degrees C. The pH optimum of the immobilized enzyme remained unchanged at pH 7.4. 2. A new type of spectrophotometric assay is described in which sedimentation of the immobilized enzyme in the cuvette is prevented by increasing the viscosity by the presence of 10% (w/w) polyethylene glycol (M1 20 000). Detailed kinetic analysis using this assay showed only insignificant differences in the Km values for n-butylamine and phenazine methosulphate between the soluble and Agarose-bound enzymes. The results are compared with those for other oxidoreductase enzymes immobilized on Sepharose.     

Purification and characterization of high Ca2+-requiring neutral proteases from porcine and bovine brains

Porcine and bovine brain high Ca2+-requiring neutral proteases were purified to homogeneity by the same isolation procedures, and their properties were compared. A high degree of similarity existed between the two proteases. The purification procedures included ion-exchange chromatography on DEAE-cellulose, hydrophobic chromatography on phenyl-Sepharose CL-4B, second DEAE-cellulose chromatography, second phenyl-Sepharose CL-4B chromatography, and gel filtration on Ultrogel AcA 34. Both purified enzymes were composed of Mr 75,000 and 29,000 subunits, as shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Both enzymes required 250 microM Ca2+ for half-maximal activity and 700 microM Ca2+ for maximal activity. Sr2+ and Ba2+, but not Mg2+ or Mn2+, also activated both enzymes but not as effectively as Ca2+. Both enzymes displayed maximum activity at pH 7.5-8.0. Leupeptin, antipain, and trans-epoxysuccinyl-L-leucylagmatine inhibited both enzymes. Neurofilament triplet proteins and microtubule-associated proteins were extensively hydrolyzed by both proteases, but tubulin and actin were not hydrolyzed. The amino acid compositions of the two proteases were very similar. Antisera against bovine brain protease cross-reacted with porcine brain protease when examined by immunoelectrotransfer blot techniques.     

Optimization of Protease Extraction from Horse Mango (Mangifera foetida Lour) Kernels by a Response Surface Methodology

Protease is one of the most important industrial enzymes with a multitude of applications in both food and non-food sectors. Although most commercial proteases are microbial proteases, the potential of non-conventional protease sources, especially plants, should not be overlooked. In this study, horse mango (Mangifera foetida Lour) fruit, known to produce latex with a blistering effect upon contact with human skin, was chosen as a source of protease, and the effect of the extraction process on its protease activity evaluated. The crude enzyme was extracted from the kernels and extraction was optimized by a response surface methodology (RSM) using a central composite rotatable design (CCRD). The variables studied were pH (x(1)), CaCl(2) (x(2)), Triton X-100 (x(3)), and 1,4-dithryeitol (x(4)). The results obtained indicate that the quadratic model is significant for all the variables tested. Based on the RSM model generated, optimal extraction conditions were obtained at pH 6.0, 8.16 mM CaCl(2), 5.0% Triton X-100, and 10.0 mM DTT, and the estimated response was 95.5% (w/w). Verification test results showed that the difference between the calculated and the experimental protease activity value was only 2%. Based on the t-value, the effects of the variables arranged in ascending order of strength were CaCl(2)< pH < DTT < Triton X-100.