Purification and Characterization of Extracellular Amylolytic Enzymes from the Yeast Filobasidium capsuligenum

The extracellular amylolytic system of Filobasidium capsuligenum consisted of an alpha-amylase (1,4-alpha-d-glucan glucanhydrolase, EC 3.2.1.1) and two forms of glucoamylase (1,4-alpha-d-glucan glucohydrolase, EC 3.2.1.3). The enzymes were purified by ammonium sulfate fractionation, repeated ion-exchange chromatography (DEAE-Sephadex A-50), and gel filtration (Sephadex G-25, Sephadex G-100 sf). alpha-Amylase had an optimum pH of 5.6 and an optimum temperature of 50 degrees C but was rapidly inactivated at higher temperature. The molecular weight was estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to be 64,000. An acarbose concentration of 20 mug/ml was required for 50% inhibition of the alpha-amylase. Both glucoamylases are glycoproteins of identical molecular weight (60,000) and produce only glucose by exohydrolysis. The debranching activity of the glucoamylases was evidenced with substrates containing alpha-1,6 linkages. The pH optima were 5.0 to 5.6 for glucoamylase I and 4.8 to 5.3 for glucoamylase II. Glucoamylase I had a higher optimum temperature (55 degrees C) than glucoamylase II (50 degrees C) and was also more resistant to thermal inactivation. Only low acarbose concentrations (<0.1 mug/ml) were required to reduce the activity of the glucoamylases by 50%.     

Comparative studies on glucoamylases from three fungal sources

Five commercial preparations of glucoamylases (three fromAspergillus niger, one each fromAspergillus foetidus andAspergillus candidus) were purified by ultrafiltration, Sepharose-gel filtration and DEAE-sephadex chromatography. Two forms of the enzyme, namely glucoamylase I and glucoamylase II were obtained from the fungi except from one strain ofA. Niger. All the enzymes appeared homogeneous by electrophoresis and ultracentrifugation. The specific activities varied between 85 and 142 units. The pH and temperature optima were between 4 and 5, and 60‡C respectively. The molecular weight as determined by the sodium dodecyl sulphate-polyacrylamide gel electrophoresis ranged from 75,000 to 79,000 for glucoamylase I and 60,000 to 72,000 for glucoamylase II. OnlyA. niger glucoamylases contained phenylalanine at the N-terminal end. The amino acid composition of the enzymes was generally similar. However,A. niger andA. foetidus glucoamylases, in contrast toA. candidus enzymes, contained greater percentage of acidic than of basic amino acids. The enzymes contained 15 to 30% carbohydrate and 49 to 57 residues of monosaccharides per mol.A. niger enzymes contained mannose, glucose, galactose, xylose and glucosamine but theA. candidus enzyme lacked xylose and glucose and only xylose was absent inA, foetidus enzymes. Majority of the carbohydrate moieties were O-glycosidically linked through mannose to the hydroxyl groups of seline and threonine of the polypeptide chain.     

Purification and characterization of a thermostable glucoamylase from Chaetomium thermophilum

Thermostable amylolytic enzymes are currently being investigated to improve industrial processes of starch degradation. A thermostable extracellular glucoamylase (exo-1, 4-alpha-D-glucanohydrolase, E.C.3.2.1.3) from the culture supernatant of a thermophilic fungus Chaetomium thermophilum was purified to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) homogeneity by using ammonium sulfate fraction, DEAE-Sepharose Fast Flow chromatography, and Phenyl-Sepharose Fast Flow chromatography. SDS-PAGE of the purified enzyme showed a single protein band of molecular weight 64 kDa. The glucoamylase exhibited optimum catalytic activity at pH 4.0 and 65 degrees C. It was thermostable at 50 degrees C and 60 degrees C, and retained 50% activity after 60 min at 65 degrees C. The half-life of the enzyme at 70 degrees C was 20 min. N-terminal amino acid sequencing (15 residues) was AVDSYIERETPIAWN. Different metal ions showed different effects on the glucoamylase activity. Ca2+, Mg2+, Na+, and K+ enhanced the enzyme activity, whereas Fe2+, Ag+, and Hg2+ cause obvious inhibition. These properties make it applicable to other biotechnological purposes.     

Purification of Glucoamylase from Lactobacillus amylovorus ATCC 33621

An intracellular glucoamylase (E.C. 3.2.1.3) was purified to homogeneity from Lactobacillus amylovorus on a Fast Protein Liquid Chromatography System (FPLC) with a Mono Q ion-exchanger and two Superose 12 gel filtration columns arranged in series. The enzyme activity was quantified with a specific, chromogenic substrate, p-nitrophenyl-β-maltoside. Preparative gel electrophoresis was then used to further purify active enzyme fractions. Native polyacrylamide gel electrophoresis (Native-PAGE) and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified enzyme showed a single protein band of molecular weight 47 kDa. Glucoamylase activity of the purified protein was confirmed by its ability to degrade starch on a 0.025% starch-polyacrylamide gel stained with I₂/KI. Glucoamylase exhibited optimum catalytic activity at pH 6.0 and 45°C, and the enzyme had an isoelectric point near 4.39. The glucoamylase contained high levels of hydrophilic amino acids, comparable to fungal glucoamylases. Received: 12 July 1996 / Accepted: 10 September 1996     

Purification and characterization of two bifunctional chitinases/lysozymes extracellularly produced by Pseudomonas aeruginosa K-187 in a shrimp and crab shell powder medium.

Two extracellular chitinases (FI and FII) were purified from the culture supernatant of Pseudomonas aeruginosa K-187. The molecular weights of FI and FII were 30,000 and 32,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 60,000 and 30,000, respectively, by gel filtration. The pIs for FI and FII were 5.2 and 4.8, respectively. The optimum pH, optimum temperature, pH stability, and thermal stability of FI were pH 8, 50 degrees C, pH 6 to 9, and 50 degrees C; those of FII were pH 7, 40 degrees C, pH 5 to 10, and 60 degrees C. The activities of both enzymes were activated by Cu2+; strongly inhibited by Mn2+, Mg2+, and Zn2+; and completely inhibited by glutathione, dithiothreitol, and 2-mercaptoethanol. Both chitinases showed lysozyme activity. The purified enzymes had antibacterial and cell lysis activities with many kinds of bacteria. This is the first report of a bifunctional chitinase/lysozyme from a prokaryote.     

Modification of a minor glucoamylase from Aspergillus saitoi with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate

In order to elucidate the structure-function relationship of glucoamylases [EC 3.2.1.3, alpha-D-(1-4)-glucan glucohydrolase] from Aspergillus saitoi, the reaction of a minor component, Gluc M2 with 1-cyclohexyl-3-(2-morpholinyl-(4)-ethyl)carbodiimide metho p-toluenesulfonate (CMC) was studied at pH 4.5. Inactivation of Gluc M2 with [14C]CMC proceeded with the incorporation of about 5 CMC moieties. From the results of analyses of amino acid and sulfhydryl contents of CMC-modified Gluc M2 and the hydroxylamine treatment of the CMC-modified Gluc M2 at pH 7.0, it was concluded that the sites of CMC-modification were carboxylic acids of Gluc M2. In the presence of maltose, when Gluc M2 was treated with [14C]CMC, ca. 4 CMC moieties were incorporated with a simultaneous decrease in activity (30%). The Gluc M2 modified in the presence of maltose was re-modified with CMC after elimination of maltose. The CMC-modified Gluc M2 (70% activity) was inactivated completely with the further incorporation of ca. 2 CMC moieties. The logarithm of the half-life of the inactivation of Gluc M2 by CMC was a linear function of log[CMC] indicating that one carboxyl group among the modified ones was crucial for the inactivation of Gluc M2. From the results of these modification reactions, it was concluded that one or two carboxylic acids in Gluc M2 were crucial for the catalysis of glucoamylase from A. saitoi. Based on the analysis of the pH-profile of CMC inactivation of Gluc M2, the participation of a carboxylic acid having pKa 5.7 in the active site is proposed.     

Purification and properties of two forms of glucoamylase fromAspergillus niger

A. niger produced α-glucosidase, α-amylase and two forms of glucoamylase when grown in a liquid medium containing raw tapioca starch as the carbon source. The glucoamylases, which formed the dominant components of amylolytic activity manifested by the organism, were purified to homogeneity by ammonium sulfate precipitation, ion-exchange and two cycles of gel filtration chromatography. The purified enzymes, designated GA1 and GA2, a raw starch digesting glucoamylase, were found to have molar masses of 74 and 96 kDa and isoelectric points of 3.8 and 3.95, respectively. The enzymes were found to have pH optimum of 4.2 and 4.5 for GA1 and GA2, respectively, and were both stable in a pH range of 3.5–9.0. Both enzymes were thermophilic in nature with temperature optimum of 60 and 65°C, respectively, and were stable for 1 h at temperatures of up to 60°C. The kinetic parametersK _m andV showed that with both enzymes the branched substrates, starch and amylopectin, were more efficiently hydrolyzed compared to amylose. GA2, the more active of the two glucoamylases produced, was approximately six to thirteen times more active towards raw starches compared to GA1.     

嗜热真菌Thermomyces lanuginosus A_(236)热稳定葡萄糖淀粉酶的纯化及其特性

嗜热真菌ThermomyceslanuginosusA_236在液体培养基中50℃下静止培养14天,粗提酶液经硫酸铵分级沉淀、DEAE-Toyopearl离子交换层析、Butyl－Toyopearl疏水层析、SephacrylS100凝胶过滤和FPLCMonoQ离子交换层析,得到了凝胶电泳均质的葡萄糖淀粉酶。酶促反应产物经TLC分析为葡萄糖,证明纯化的酶为葡萄糖淀粉酶（EC3．2．1．3）。SDS－PAGE测定其分子量为72,000,不具亚基,PI为4．0,富含Val和Leu。酶反应最适温度和pH分别为70℃和5．0。在pH5．0条件下,酶在60℃保温1h,仍具有原酶活性。酶活性在70℃和80℃的半衰期分别为20min和6min。Ca2+对酶有激活作用,Fe3+、Al3+、Hg2+等金属离子对酶活力有一定的抑制作用。纯酶碳水化合物含量为12．4％。纯酶可水解可溶性淀粉、直链淀粉、支链淀粉、糊精、糖原、麦芽三糖和麦芽糖,其中可溶性淀粉为最适底物。     

Comparison of the properties of glucoamylases from Rhizopus niveus and Aspergillus niger

Some properties of the glucoamylase from Rhizopus niveus have been determined and compared with the comparable properties of the glucoamylase from Aspergillus niger. The enzymes from these organisms possess the following common properties: quantitative conversion of starch to glucose, molecular weights in the range 95,500 to 97,500, and glycoprotein structures with many oligosaccharide side chains attached to the protein moieties of the enzymes. Differences in the glucoamylases exist in electrophoretic mobility, amino acid composition, nature of carbohydrate units, and types of glycosidic linkages. Lysine, threonine, serine, glutamic acid, tyrosine, and phenylalanine differ in the two glucoamylases by 25 to 50%. Whereas the enzyme from R. niveus contains mannose and glucosamine, in the N-acetyl form, as the carbohydrate constituents, the enzyme from A. niger contains mannose, glucose, and galactose. The carbohydrate chains of the R. niveus enzyme are linked by O-glycosidic and N-glycosidic linkages to the protein, while those of the A. niger enzyme are linked by O-glycosidic linkages only. Antibodies directed against the two glucosamylases have been isolated by affinity chromatography and found to be specific for the carbohydrate units of the glucoamylases. Cross reactions did not occur between the glucoamylases and the purified antibodies.     

Production of multiple forms of glucoamylase in Aspergillus awamori

The biosynthesis of glucoamylases in Aspergillus awamori was studied by in vivo protein labelling and analysis of glucoamylase-specific mRNAs. Two types of glucoamylases with molecular weights of 100,000 and 82,000 were shown to be synthesized de novo. Deglycosylation of the 100,000 molecular weight glucoamylase type resulted in the formation of another glucoamylase form with molecular weight of about 94,000. De novo synthesis of two types of glucoamylases was further confirmed by the existence of two types of glucoamylase-specific mRNAs, as demonstrated by in vitro translation and Northern blot analysis studies.     

Different behavior towards raw starch of three forms of glucoamylase from a Rhizopus sp

Three forms of glucoamylase [EC 3.2.1.3] of a Rhizopus sp., Gluc1 (M.W. 74,000), Gluc2 (M.W. 58,600), and Gluc3 (M.W. 61,400), which have similar pH optima and specific activities towards soluble starch were studied as to their behavior towards raw starch. The pH optima for raw starch digestion were different, that is, 4.5 for Gluc1 and 5.0 for both Gluc2 and Gluc3. All the enzymes digested raw starch almost completely but at quite different rates; Gluc2 and Gluc3, which lack the N-terminal portions of Gluc1, were 22 and 25 times less effective, respectively, for raw starch digestion than Gluc1. Of the three enzymes, only Gluc1 tightly bound to raw starch. Binding of Gluc1 to raw starch occurred pH-dependently with a broad pH optimum of 4.5-5.5, but temperature and ionic strength affected it only slightly and little, respectively. The binding constant of Gluc1 for raw starch at pH 5.0 and 4 degrees C was estimated to be 1.2 X 10(5) M-1. Fragment H (M.W. 16,700), presumably released from the N-terminal part of Gluc1, not only bound to raw starch itself but also inhibited the binding of Gluc1 to raw starch. pap-Gluc (M.W. 57,000) and chymo-Gluc (M.W. 64,000), which are papain- and chymotrypsin-modified Gluc1, respectively, and lack the N-terminal portions of Gluc1, resembled Gluc2 and Gluc3 in raw starch binding as well as digestion. All these results indicate that Gluc1 has a raw starch-binding site, different from the active center, in the N-terminal region. Various substrates and analogs inhibited the binding of Gluc1 to raw starch, presumably due to steric hindrance.     

Detection of chitinase activity after polyacrylamide gel electrophoresis

Commercial Streptomyces griseus and Serratia marcescens chitinases and purified wheat germ W1A and hen egg white lysozymes were subjected to polyacrylamide gel electrophoresis under native conditions at pH 4.3. After electrophoresis, an overlay gel containing 0.01% (W/V) glycol chitin as substrate was incubated in contact with the separation gel. Lytic zones were revealed by uv illumination with a transilluminator after staining for 5 min with 0.01% (W/V) Calcofluor white M2R. As low as 500 ng of purified hen egg lysozyme could be detected after 1 h incubation at 37 degrees C. One band was observed with W1A lysozyme and several bands with the commercial microbial chitinases. The same system was also used with native polyacrylamide gel electrophoresis at pH 8.9. Several bands were detected with the microbial chitinases. The same enzymes were also subjected to denaturing polyacrylamide gel electrophoresis in gradient gels containing 0.01% (W/V) glycol chitin. After electrophoresis, enzymes were renatured in buffered 1% (V/V) purified Triton X-100. Lytic zones were revealed by uv after staining with Calcofluor white M2R as for native gels. The molecular weights of chitinolytic enzymes could thus be directly estimated. In denaturing gels, as low as 10 ng of purified hen egg white lysozyme could be detected after 2 h incubation at 37 degrees C. Estimated molecular weights of St. griseus and Se. marcescens were between 24,000 and 72,000 and between 40,500 and 73,000, respectively. Some microbial chitinases were only resistant to denaturation with sodium dodecyl sulfate while others were resistant to sodium dodecyl sulfate and beta-mercaptoethanol.     

Purification and properties of two endo-1,4-beta-xylanases from Irpex lacteus (Polyporus tulipiferae)

Two different endo-1,4-beta-xylanases [1,4-beta-D-xylan xylanohydrolases, EC 3.2.1.8], named Xylanases I and III, were purified to homogeneity by gel filtration and ion exchange column chromatography from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae). The purified enzymes were found to be homogeneous on polyacrylamide disc electrophoresis and their specific activities toward xylan were increased approximately 28.7 and 19.8 times, respectively. The activities of each enzyme were considerably inhibited by Hg2+, Ag+, and Mn2+. Their molecular weights were estimated to be approximately 38,000 and 62,000 by gel filtration and sodium dodecyl sulfate (SDS)-polyacrylamide electrophoresis, respectively. Their carbohydrate contents were 2.5% and 8.0% as glucose, and their amino acid composition patterns resembled each other, showing high contents of acidic amino acids, serine, threonine, alanine, and glycine. Both enzymes were most active at pH 6.0 but Xylanase I was more stable as to pH. Their optimum temperatures were 60 degrees C and 70 degrees C, respectively. Xylanase I split up to 34.5% of larchwood xylan whereas Xylanase III split only 18.9% of it. The products with the former were mainly xylose (X1), xylobiose (X2), and xylotriose (X3), whereas X2 and X3 were the main products with the latter. Both enzymes did not hydrolyze X2. Xylanase I produced almost equal quantities of X1 and X2 from X3, while Xylanase III did not attack this substrate. Both enzymes showed no activity toward glycans, other than xylan, such as starch, pachyman and Avicel (microcrystalline cellulose), except the almost one twentieth activity of Xylanase III toward sodium carboxymethyl cellulose (CMC).     

Neutral proteinases of human spleen. Purification and criteria for homogeneity of elastase and cathepsin G.

1. Human spleen was found to contain proteinases active against azo-casein at neutral and alkaline pH values. 2. The activity was stimulated by high ionic strength and some detergents. 3. Optimal extraction of the proteinases from the tissue was achieved with 1.0M-NaCl containing 0.1% Brij 35 and 0.1% trisodium EDTA. 4. The proteinases were efficiently adsorbed to insoluble material in the absence of salt in the initial stages of purification. 5. Two distinct proteinases were separated by chromatography on DEAE-cellulose, an elastase and a chymotrypsin-like enzyme designated cathepsin G. 6. Both enzymes were highly purified by further column chromatography. 7. The molecular weights of the enzymes were estimated by gel chromatography and sodium dodecyl sulphate-gel electrophoresis. 8. It was shown by isoelectric focusing and gel electrophoresis that both enzymes are cationic proteins that occur in multiple forms.     

Glucoamylases of a fungus isolated from a rotting cassava tuber

Summary Aspergillus niger H-9 is a fungal strain isolated from a rotting cassava tuber in Thailand. In the present study, the production of the enzymes was carried out as solid state ricebran-soybean fermentation. Two types of glucoamylases were isolated and purified. The purified glucoamylases were found to be homogenous on 7.5% polyacrylamide gel disc electrophoresis. The molecular weights of glucoamylase I and II were 59,400–72,600 and 43,000–52,600 respectively. The Km values of glucoamylase I and glucoamylase II were 12.5 and 6.25 mg glucose/ml when soluble starch was used as substrate. The optimal pH of both enzymes was 4.0–5.0. The optimum temperatures for the activities of glucoamylase I and glucoamylase II were 60 and 70°C respectively. Both enzymes were stable in the pH range 3.0–6.0 and temperature stable below 50°C. Both glucoamylases were active on various kinds of starch and dextrin including raw starch. Glucoamylase II was, however, found to hydrolyse raw starch better than glucoamylase I.

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Resumen Aspergillus niger H-9 es una cepa aislada en Tailandia a partir de tuberculos de cassava afectados de podredumbre. En este trabajo la producción de enzimas tuvo lugar mediante fermentación en un medio sólido compuesto por fibra de arroz y soja. Se aislaron y purificaron dos tipos de glucoamilasas. Al realizar una electroforesis en disco de polyacrilamida al 7.5% se observó que las glucoamilasa purificadas eran homogeneas. Los pesos de las glucoamilasas I y II eran respectivamente 59,400–72,600 y 43,000–52,600. Las Km respectivas fueron 12.5 y 6.25 mg ml^–1 cuando se utilizó almidón soluble como substrato. El pH optimo para ambos enzimas fue 4.0–5.0. Las temperaturas óptimas para la glucoamilasa I y la glucoamilasa II fueron respectivamente de 60 y 70°C respectivamente. Ambos enzimas eran estables en el intérvalo de pH 3.0–6.0 y a temperaturas por debajo de 50°C. Los dos enzimas eran activos fiente a distintos tipos de almidón y dextrina incluyendo almidón bruto. La glucoamilasa II hidrolizó mejor el almidón bruto que la glucoamilasa I.

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Résumé Aspergillus niger H-9 est une souche de moisissure isolée en Thailande à partir de tubercules pourris de manioc. Dans cette étude, la production d'enzymes a été obtenue par fermentation en milieu solide sur son de riz et soja. Deux types de gluco-amylases ont été isolés et purifiés. Les enzymes purifiés sont homogènes en disque-éléctrophorèse sur gel de polyacrylamide. Les poids moléculaires des gluco-amylases I et II sont respectivement de 59,400–72,600 et 43,000–52,000 et leurs Km pour l'amidon soluble de 12.5 et 6.25 mg de glucose/ml. Le pH optimum des deux enzymes est compris entre 4.0 et 5.0. Leurs températures optimales sont respectivement de 60 et 70°C. Les deux enzymes sont stables de pH 3.0–6.0 et aux températures inférieures à 50°C. Les deux gluco-amylases sont actives sur différents types d'amidon et de dextrines, y compris l'amidon cru. Toutefois, la gluco-amylase II hydrolyse l'amidon cru plus activement que la gluco-amylase I.

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Paper presented at the VII International Conference on the Global Impacts of Applied Microbiology, Helsinki, 12–16 August 1986.     

Properties of a purified thermostable glucoamylase from <Emphasis Type="Italic">Aspergillus niveus</Emphasis>

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40°C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0–5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0–9.5. The temperature optimum was 65°C and retained 100% activity after 240 min at 60°C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K _m was calculated as 0.32 mg ml⁻¹. Circular dichroism spectroscopy estimated a secondary structure content of 33% α-helix, 17% β-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.     

Nucleotide sequence of the glucoamylase gene GLU1 in the yeast Saccharomycopsis fibuligera.

The complete nucleotide sequence of the glucoamylase gene GLU1 from the yeast Saccharomycopsis fibuligera has been determined. The GLU1 DNA hybridized to a polyadenylated RNA of 2.1 kilobases. A single open reading frame codes for a 519-amino-acid protein which contains four potential N-glycosylation sites. The putative precursor begins with a hydrophobic segment that presumably acts as a signal sequence for secretion. Glucoamylase was purified from a culture fluid of the yeast Saccharomyces cerevisiae which had been transformed with a plasmid carrying GLU1. The molecular weight of the protein was 57,000 by both gel filtration and acrylamide gel electrophoresis. The protein was glycosylated with asparagine-linked glycosides whose molecular weight was 2,000. The amino-terminal sequence of the protein began from the 28th amino acid residue from the first methionine of the putative precursor. The amino acid composition of the purified protein matched the predicted amino acid composition. These results confirmed that GLU1 encodes glucoamylase. A comparison of the amino acid sequence of glucoamylases from several fungi and yeast shows five highly conserved regions. One homology region is absent from the yeast enzyme and so may not be essential to glucoamylase function.     

Affinity purification and properties of cathepsin-E-like acid proteinase from rat spleen.

A unique acid proteinase different from cathepsin D was purified from rat spleen by a method involving precipitation at pH 3.5, affinity chromatography on pepstatin-Sepharose 4B and concanavalin A-Sepharose 4B, chromatography on Sephadex G-100 and DEAE-Sephacel, and isoelectric focusing. A purification of 4200-fold over the homogenate was achieved and the yield was 11%. The purified enzyme appeared to be homogeneous on electrophoresis in polyacrylamide gels. The isoelectric point of the enzyme was determined to be 4.1-4.4. The enzyme hydrolyzed hemoglobin with a pH optimum of about 3.1. The molecular weight of the enzyme was estimated to be about 90000 by gel filtration on Sephadex G-100. In sodium dodecylsulfate polyacrylamide gel electrophoresis, the purified enzyme showed a single protein band corresponding to a molecular weight of about 45000. The hydrolysis of bovine hemoglobin by the enzyme was much higher than that of serum albumin. Various synthetic and natural inhibitors of the enzyme were tested. The enzyme was inhbited by Zn2+, Fe3+, Pb2+, cyanide, p-chloromercuribenzoate, iodoacetic acid and pepstatin, whereas 2-mercaptoethanol, phenylmethyl-sulfonyl fluoride and leupeptin showed no effect.     

Purification and properties of alkaline ribonuclease from human serum.

1. Five alkaline ribonucleases (EC 3.1.4.22) were purified about 140- to 1900-fold from human serum by phosphocellulose and DEAE-cellulose chromatographies and Sephadex G-75 filtration, with a total recovery of 22%. These were designated as RNAases 1-5. 2. Optimum activities were observed at pH 8.5-8.7 for RNAases 1-4, and at pH 7.5 for RNAase 5. The molecular weights of these enzymes were estimated by gel filtration as 45 000, 32 000, 20 000, 13 000 and 8500, respectively. 3. These RNAases were found to be heat-labile proteins but are markedly stabilized with bovine plasma albumin. The reaction was activated by Na+, K+, Mg2+ and Ca2+, and inhibited by Co2+, Fe2+, Cu2+ and Zn2+. EDTA had little effect on the velocity of the reaction. Spermine caused 2- to 7-fold activation. 4. Among the substrates examined, these RNAases preferentially hydrolyzed pyrimidine bodies and except for RNAase 5 had a higher affinity for poly(C) than poly(U) as substrate. Each enzyme was free from other nucleolytic enzymes and hydrolyzed only RNA.     

A Ca2+-activated protease possibly involved in myofibrillar protein turnover. Partial characterization of the purified enzyme.

The purified Ca2+-activated protease (CAF) isolated from porcine skeletal muscle and capable of removing Z-disks from intact myofibrils is optimally active on either myofibril or casein substrates at pH 7.5 and in the presence of 1 mM Ca2+ and at least 2 mM 2-mercaptoethanol. No CAF activity is detected when 1 mM Mg2+, Mn2+, Ba2+, Co2+, Ni2+, and Fe2+ are added singly. When added with 1 mM Ca2+, Co2+, Cu2+, Ni2+, and Fe2+ inhibit, whereas Mg2+, Mn2+, and Ba2+ have no effect on CAF activity. CAF is irreversibly inhibited by iodoacetate but is unaffected by soybean trypsin inhibitor. S0/20,W=5.90 S, and sedimentation equilibrium molecular weight - 112 000 for purified CAF. Because purified CAF migrates as two polypeptide chains with molecular weights of 80 000 and 30 000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the CAF molecule must consist of one each of these two polypeptide chains. Approximate molecular dimensions of 38 X 220 A can be calculated for CAF from calibrated gel permeation column data or from S0/20,W and the molecular weight. Amino acid composition and physical properties of purified CAF distinguish it from the known catheptic enzymes and from other proteases found in blood or in granulocytes. Purified CAF removes Z-disks the 400-A periodicity associated with troponin in the I band and partly degrades M lines but causes no other ultrastructurally detectable effects when incubated with myofibrils. These results agree with the earlier finding that purified CAF degrades troponin, tropomyosin, and C-protein but has no effect on myosin, actin, or alpha-actinin, and suggest that CAF may have a physiological role in disassembly of intact myofibrils during metabolic turnover of myofibrillar proteins.