The cellular distribution of some rat-kidney glycosidases

1. Free and total activities of beta-glucosidase, beta-galactosidase, N-acetyl-beta-glucosaminidase and beta-glucuronidase have been determined fluorimetrically in five subcellular fractions of rat kidney. 2. The beta-glucosidase activity appeared in the soluble fraction, beta-glucuronidase had the distribution pattern of a lysosomal enzyme, and both beta-galactosidase and N-acetyl-beta-glucosaminidase had bimodal distributions. 3. Two types of beta-galactosidase activity were found: a sedimentable type, having optimum pH3.7, mol.wt. about 80000 and slow electrophoretic mobility at pH7.0 in starch gel; and a soluble type of much faster mobility, having optimum pH5.5-6.5 and mol.wt. about 40000. 4. Evidence is presented that the beta-glucosidase and the soluble type of beta-galactosidase are the same enzyme. 5. Most of the N-acetyl-beta-glucosaminidase activity was in the lysosome-rich fractions, but a significant proportion occurred in the microsomal fraction in a non-latent form. 6. The use of beta-galactosidase and N-acetyl-beta-glucosaminidase as lysosomal marker enzymes is complicated by the possible presence of multiple forms, but this limitation does not apply to beta-glucuronidase in the rat kidney.     

Structure and function of mitochondria in hepatopancreas cells from metabolically depressed snails

Mitochondria in cells isolated from the hepatopancreas of aestivating land snails (Helix aspersa) consume oxygen at 30% of the active control rate. The aim of this study was to investigate whether the lower respiration rate is caused by a decrease in the density of mitochondria or by intrinsic changes in the mitochondria. Mitochondria occupied 2% of cellular volume, and the mitochondrial inner membrane surface density was 17 microm(-1), in cells from active snails. These values were not different in cells from aestivating snails. The mitochondrial protein and mitochondrial phospholipid contents of cells were also similar. There was little difference in the phospholipid fatty acyl composition of mitochondria isolated from metabolically depressed or active snails, except for arachidonic acid, which was 18% higher in mitochondria from aestivating snails. However, the activities of citrate synthase and cytochrome c oxidase in mitochondria isolated from aestivating snails were 68% and 63% of control, respectively. Thus the lower mitochondrial respiration rate in hepatopancreas cells from aestivating snails was not caused by differences in mitochondrial volume or surface density but was associated with intrinsic changes in the mitochondria.     

Morpho-functional modifications of human syncytiotrophoblast plasma membrane during Pregnancy Induced Hypertension

Changes in [³⁵S]methionine protein labeling patterns were examined by following incorporation into the acid precipitate protein fraction of land snails,Otala lactea (Müller) (Pulmonata, Helicidae). Labeled proteins were analyzed by SDS polyacrylamide gel electrophoresis and isoelectric focusing columns. Snails in four different physiological states were compared: active controls, short term aestivating snails (injected and allowed to enter aestivation), long term aestivating snails (aestivated for 14 days, injected, and maintained in the aestivating state), and snails aroused after aestivation (aestivated, injected, and aroused). Protein associated radioactivity was measured over a 7 day time course post injection. Autoradiographic analysis of SDS-polyacrylamide gels showed increases in the radioactivity of four proteins: 91 kDa (hepatopancreas, day 1 in long term aestivating animals), 50 kDa (hepatopancreas, day 2 in short term aestivating snails), 70 kDa and 30 kDa (foot, day 2 in short term aestivating animals). Hepatopancreas and foot from day 1 long term aestivating and day 2 short term aestivating animals were also analyzed by isoelectric focusing columns. Several pH-specific differences were apparent when controls and aestivating animals were analyzed. In particular a peak of radioactivity was observed at pH 5.05 in 1 d long term aestivating hepatopancreas and at pH 4.30 in 2d short term aestivating animals. Several differences were noted in foot with no specific pattern emerging. SDS-polyacrylamide gel electrophoresis analysis of the hepatopancreas peaks showed the appearance of several bands with increased radioactivity, including the 91 kDa and 50 kDa proteins described above. These results suggest thatO. lactea aestivation specific proteins may be involved in the transition to a depressed metabolic state.Abbreviations dpm radioactive disintegrations per minute - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulphate - SRP stress related protein     

Comparison of fermentation reactions in different regions of the human colon

Colonic contents were obtained from two human sudden-death victims within 3 h of death. One of the subjects (1) was methanogenic, the other (2) was a non-CH, producer. Measurements of bacterial fermentation products showed that in both individuals short-chain fatty acids, lactate and ethanol concentrations were highest in the caecum and ascending colon. In contrast, products of protein fermentation, such as ammonia, branched chain fatty acids and phenolic compounds, progressively increased from the right to the left colon, as did the pH of gut contents. In Subject 1, cell population densities of methanogenic bacteria (MB) increased distally through the gut and methanogenic activity was lower in the right (0.78–1–18 μmol CH₄ produced/h/g dry wt contents) than in the left colon (1.34 μmol CH₄ produced/h/g dry wt contents). Methane production rates did not correlate with MB numbers.
Sulphate-reducing bacteria (SRB) were not found and dissimilatory sulphate reduction was not detected in any region of the colon. Methanogenic bacteria did not occur in subject 2, but high numbers of SRB were present throughout the gut ( ca 10⁹/g dry wt contents). Sulphate reduction rates were maximal in the ascending and transverse colons (0.24 and 0.22 μmol ³⁵SO^2–₄ reduced/h/g dry wt contents, respectively). Short-chain fatty acid production by caecal contents was up to eight-fold higher than contents from the sigmoid/rectum. These findings demonstrate significant differences in fermentation reactions in different regions of the large gut.     

Peptide fragmentation suitable for solid-phase microsequencing. Use of N-bromosuccinimide and BNPS-skatole (3-bromo-3-methyl-2-[(2-nitrophenyl)thio]-3H-indole).

A new intracellular beta-glucosidase was isolated from Trichoderma reesei. It was sequentially purified by (NH4)2SO4 precipitation and chromatography and rechromatography on Sephadex G-150. The enzyme has a mol.wt. of 98 000, optimal activity at pH 6.5, pI 4.4 and Km values of 6.7 mM and 3.3 mM for sophorose and cellobiose respectively. Possible functions of the enzyme may be regulation of cellulase induction and/or to serve as a proenzyme.     

Purification and characterization of a broad specificity beta-glucosidase from sheep liver

1. A soluble beta-glucosidase from sheep liver has been isolated and purified 114-fold by conventional enzyme fractionation procedures. The specific activity of the purified enzyme was 5910 mU/mg of protein. 2. The enzyme has a broad specificity and hydrolyzes the p-nitrophenyl derivatives of beta-D-glucose, beta-D-galactose, beta-D-fucose, beta-D-xylose and alpha-L-arabinose. The best Vmax/Km value corresponds to the beta-glucosidase activity. 3. The enzyme has a pH optimum between 4.5-5.5 for all the activities, and mol. wt 95,000. 4. A variety of chemicals was tested as possible activators or inhibitors. 5. The enzyme is strongly inhibited by aldono 1-5 lactones and DMDP. 6. The kinetic evidences suggest a substrate activation model and the existence of two active sites (a "gluco-fuco" site and a "galacto" site). 7. The activation energies were calculated from beta-glucosidase and beta-galactosidase activities.     

Energy loss in an aestivating population of the tropical snail pila globosa

The dried peripheral area of pond Idumban (62 ha) increased from 3.2 ha in January to 3 1.9 ha in April. Pila globosa, which were abundant in the littoral area, did not commence aestivation during this period, perhaps due to low temperature and/or high dissolved oxygen content. The number of aestivating snails averaged 0.5/m² in May, 1973 (3.6% of the total population) and it increased to 1.1/M² in September (26.2%). Biomass of the snail increased from 3.5 to 19.9 g dry weight (including shell)/M². Number of aestivating snails increased from 0.4/m² (5.2% of the total population) in May 1974 to 0.8/m² (11.1%) in July and the biomass from 4.1 g/m² to 10.7 g/m². Availability of dried area for aestivation increased from 5.3 to 23.7 ha in 1973 and from 13.5 to 30.2 ha in 1974.Monthly observations made on the marked snails forced to aestivate at 7.5, 15.0, 22.5 and 30.0 cm depth in the pond during May, revealed that temperature above 35°C and moisture below 5% were critical. Mortality and weight loss decreased in the snails forced to aestivate at increasing depth. Random observations indicated that 83% of the aestivating snails buried themselves at 15 cm depth in the pond. On the whole, 98,480 snails (592 Kg) and 115,270 (758 Kg) died during aestivation in 1973 and 1974 respectively. Of the total weight loss, the energy lost via metabolism contributed only a small fraction of 2.2% (12 Kg) and 2.1% (15 Kg) during these years. Considering the total aestivation area, the snails which succumbed averaged only 0.4/m²/year (2.5 g/m²/year). On an average, dry substance equivalent to about 2.6 mg dry weight/ g dry weight of snail/ day (3.7 gcal/ g live snail/ day) was lost on metabolism by the aestivating snails, i.e. the metabolic level of the aestivating snail was about 1 / 18th of that of the actively feeding snail.     

Heterogeneity in human acid beta-glucosidase revealed by cellulose-acetate electrophoresis

Cellulose-acetate gel electrophoresis, a technique commonly used for the separation of human acid hydrolases, was applied to study heterogeneity in acid beta-glucosidase (EC 3.2.1.45). With this technique, three forms of beta-glucosidase were distinguishable in extracts of several tissues. The most anodic beta-glucosidase activity (band 3) represents the broad-specificity beta-glucosidase that is not deficient in Gaucher disease and is not inhibited by conduritol B-epoxide (CBE). The beta-glucosidase activity was deficient in Gaucher disease. A third beta-glucosidase activity with an intermediate mobility (band 2) was also inhibited by CBE and deficient in Gaucher disease. Band 1 and band 2 beta-glucosidase thus represent different forms of glucocerebrosidase. By adding phosphatidylserine and sphingolipid activator protein (SAP-2), monomeric glucocerebrosidase could be completely converted into a form that comigrated with band 2 beta-glucosidase of tissue extracts. The addition of phosphatidylserine only also resulted in a changed mobility of the monomeric enzyme, but the migration in this case differed from that of band 2 beta-glucosidase of tissue extracts. The electrophoretic profile of beta-glucosidase activity of tissue extracts changed upon ethanol/chloroform extraction: the two glucocerebrosidase forms were converted into a band with a mobility identical to that of band 1 beta-glucosidase. Our findings indicate that the interaction of glucocerebrosidase with phospholipid and SAP-2 has major effects on the mobility of the enzyme in the cellulose-acetate gel electrophoresis system. The findings with the cellulose-acetate gel electrophoretic system are discussed in relation to the heterogeneity in glucocerebrosidase observed with sucrose density gradient analysis, immunochemical methods and isoelectric focussing studies.     

Purification and characterization of the extracellular beta-glucosidase produced by Candida wickerhamii

Candida wickerhamii NRRL Y-2563 produced a cell-bound beta-glucosidase when grown in complex media containing 50 g of cellobiose per liter. The majority of the enzyme was located on the cell surface and was released into the supernatant upon treatment of intact cells with Zymolyase 60,000. Only about 10% of the total activity was associated with the cytoplasm. The enzyme was purified to homogeneity, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had an apparent native molecular mass of about 198,000 Da and appeared to be composed of two subunits with approximate molecular masses of 94,000 Da. The beta-glucosidase contained approximately 30.5% (w/w) carbohydrate. Mannose was the only detected neutral carbohydrate associated with the purified enzyme. The enzyme demonstrated optimal activity at a pH of 4.0 to 5.0. The Km of the purified beta-glucosidase was 6.74 X 10(-2) M for cellobiose and 4.17 X 10(-3) M for p-nitrophenyl-beta-D-glucopyranoside. Glucose did not appear to inhibit the enzyme.     

The effects of aestivation on the catalytic and regulatory properties of pyruvate kinase from Helix aspersa.

1. Pyruvate kinase was partially purified from the foot, mantle, and digestive gland of active and aestivating snails. 2. At pH 7.0 the apparent Km values for phosphoenolpyruvate (PEP) were 0.064 mmol/l for the enzyme from foot and 0.071 mmol/l for the enzyme from mantle; those for ADP were 0.35 mmol/l for the foot enzyme and 0.33 mmol/l for the mantle enzyme. 3. Both enzymes were inhibited by alanine, and this could be reversed by fructose 1,6-bisphosphate (FBP), although FBP alone was a weak activator. 4. Decreasing the pH to 6.5 markedly increased the inhibition by alanine and reduced the response to FBP. 5. The enzymes from these tissues of aestivating snails showed a small decrease in their affinity for PEP and a small increase in the effectiveness of alanine as an inhibitor. 6. These changes are indicative of a down-regulation of this enzyme which is consistent with the observations in other species during metabolic depression. 7. In contrast the enzyme from the digestive gland of active animals showed sigmoidal saturation kinetics for PEP with a S0.5 of 1.2 mmol/l, but had a markedly higher affinity for PEP, S0.5 = 0.20 mmol/l during aestivation. This may be indicative of other metabolic changes occurring in the digestive gland.     

Protein phosphorylation patterns during aestivation in the land snailOtala lactea

Protein phosphorylation patterns were investigated in whole tissues and subcellular fractions of active and aestivatingOtala lactea (Müller) (Pulmonata, Helicidae). Measurement of overall protein phosphorylation showed that incorporation of³²P increased until the second day after injection and remained constant for the remaining 4 days of the time course. Comparison of tissues from aestivating and active snails on day 3 showed a decreased protein phosphorylation in aestivating snails (44% of active). No differences in total and protein-associated radioactivity for foot, mantle or haemolymph were observed. Subcellular fractionation of the hepatopancreas localized the changes to plasma membrane, microsomal, and cytosolic fractions: values for aestivating animals were reduced to 71, 37 and 58% of the corresponding active values. Separation of the individual subcellular fractions on isoelectric focusing columns revealed differences in the phosphate incorporation patterns. Plasma membrane from aestivating animal hepatopancreas had a lower overall level of incorporation and fewer radioactive peaks in the pH 7–10 region than did the plasma membrane fraction from active animals. SDS-PAGE analysis of plasma membrane fractions from active and aestivating snails showed a relative decrease in phosphorylation between 60–80 kDa and 30–40 kDa. IEF analysis of cytosolic proteins from aestivating snail hepatopancreas also showed peaks of radioactivity that were apparently shifted by 0.3 pH units toward higher pI values. Increased phosphate incorporation was observed at a peak that corresponded to the pI value for pyruvate kinase in aestivating snails but definite assignment of peaks was not possible. SDS-PAGE analysis of cytosolic proteins showed an aestivation-related decrease in relative protein phosphorylation between 30–35 kDa and 40–45 kDa. A relative increase in phosphorylation during aestivation was observed for proteins between 16–22 kDa. Overall, the data indicate that snails dramatically alter their protein phosphorylation pattern in hepatopancreas during aestivation. (Mol Cell Biochem143: 7–13, 1995)Abbreviations CY cytosol - dpm radioactive disintegrations per minute - IEF isoelectrofocusing - GP glycogen phosphorylase - MC microsomes - MT mitochondria - PAGE polyacrylamide gel electrophoresis - PKF phosphofructokinase - PK pyruvate kinase - PM plasma membrane - SDS sodium dodecyl sulphate     

水牛瘤胃宏基因组的一个新的b-葡萄糖苷酶基因umcel3G的克隆、表达及其表达产物的酶学特性

以木质纤维素为原料、应用同步糖化共发酵工艺发酵生产酒精时需要酸性中低温高活力纤维素酶包括b-葡萄糖苷酶。本工作分6次构建了水牛瘤胃未培养微生物宏基因组文库, 获得1.26×105个克隆, 文库含外源DNA的总长度 约为4.8×106 kb。从文库中筛选到118个表达b-葡萄糖苷酶活性的独立克隆。发现其中8个克隆表达的b-葡萄糖苷酶在pH5.0、37oC条件下活性较强。对其中一个克隆进行了亚克隆, 序列分析发现一个2223 bp的潜在的编码b-葡萄糖苷酶基因(umcel3G)的开放阅读框(ORF), 其编码产物的氨基酸序列与来自于 Bacillus sp.的一个b-葡萄糖苷酶同源性最高, 具有60%的一致性和73%的相似性。该ORF在E.coli中的表达产物Umcel3G的分子量与预测大小相似, 酶谱分析表明该表达产物具有b-葡萄糖苷酶活性, 证实该基因为一个b-葡萄糖苷酶基因。测定了用Ni-NTA纯化的Umcel3G的酶学特性, 其最适pH和最适温度分别为6.0~6.5和45oC。一些金属离子如Ca2+、Zn2+能显著提高该酶的酶活, 而另外一些金属离子如Fe3+、Cu2+能抑制Umcel3G的活性。在pH4.5、35oC和5 mmol/L的 Ca2+存在的条件下, 用Ni-NTA纯化的重组酶的比活为22.8 IU/mg, 说明该酶在用SSCF工艺发酵生产酒精中有潜在的应用价值。     

Glycoprotein enzymes secreted by Aspergillus fumigatus: purification and properties of a second beta-glucosidase.

A second extracellular beta-glucosidase (betalarge) of Aspergillus fumigatus was purified to homogeneity and shown to be a glycoprotein, as determined by polyacrylamide gel electrophoresis followed by staining for protein and for carbohydrate. Its molecular weight was approximately 340,000 by gel filtration, while sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave an apparent molecular weight of 170,000, suggesting that the enzyme has two subunits. The glucosidase contained covalently bound sugars consisting of about 2 mol of glucosamine and 16 mol of mannose per mol of protein. The carbohydrate was found to be attached to the peptide via glucosaminyl leads to peptide linkage, possibly to asparagine residues. At pH 4.5 this enzyme readily hydrolyzed p-nitrophenyl-beta-D-glucopyranoside (Km = 0.88 mM) and cleaved two glucose disaccharides: gentiobiose (beta,1 leads to 6; Km = 0.75 mM) and cellobiose (beta,1 leads to 4; Km = 0.84 mM). Although its activity is similar to that of a previously purified beta-glucosidase (betasmall), the two enzymes differ with respect to their pH activity profiles, substrate specificities, and molecular weights. Also double diffusion tests with anti-betasmall antiserum and both purified beta-glucosidases revealed a nonidentical cross-reaction. Microcomplement fixation of native and periodate-oxidized betasmall suggested that the oligosaccharide chain(s) was not a major antigenic site.     

Production, purification, and characterization of a highly glucose-tolerant novel beta-glucosidase from Candida peltata.

Candida peltata (NRRL Y-6888) produced beta-glucosidase when grown in liquid culture on various substrates (glucose, xylose, L-arabinose, cellobiose, sucrose, and maltose). An extracellular beta-glucosidase was purified 1,800-fold to homogeneity from the culture supernatant of the yeast grown on glucose by salting out with ammonium sulfate, ion-exchange chromatography with DEAE Bio-Gel A agarose, Bio-Gel A-0.5m gel filtration, and cellobiose-Sepharose affinity chromatography. The enzyme was a monomeric protein with an apparent molecular weight of 43,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel filtration. It was optimally active at pH 5.0 and 50 degrees C and had a specific activity of 108 mumol.min-1.mg of protein-1 against p-nitrophenyl-beta-D-glucoside (pNP beta G). The purified beta-glucosidase readily hydrolyzed pNP beta G, cellobiose, cellotriose, cellotetraose, cellopentaose, and cellohexaose, with Km values of 2.3, 66, 39, 35, 21, and 18 mM, respectively. The enzyme was highly tolerant to glucose inhibition, with a Ki of 1.4 M (252 mg/ml). Substrate inhibition was not observed with 40 mM pNP beta G or 15% cellobiose. The enzyme did not require divalent cations for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol at an optimal concentration (0.75%, vol/vol) stimulated the initial enzyme activity by only 11%. Cellobiose (10%, wt/vol) was almost completely hydrolyzed to glucose by the purified beta-glucosidase (1.5 U/ml) in both the absence and presence of glucose (6%). Glucose production was enhanced by 8.3% when microcrystalline cellulose (2%, wt/vol) was treated for 24 h with a commercial cellulase preparation (cellulase, 5 U/ml; beta-glucosidase, 0.45 U/ml) that was supplemented with purified beta-glucosidase (0.4 U/ml).     

Comparative study of glucosidases from the thermophilic fungus Thermoascus aurantiacus Miehe. Purification and characterization of intracellular beta-glucosidase

Intracellular beta-glucosidase was extracted from the mycelium of Th. aurantiacus, concentrated by DEAE-cellulose treatment, separated from alpha-glucosidase by hydroxylapatite chromatography and purified to electrophoretic homogeneity. Optimally active at 75 degrees C and pH 4.2, beta-glucosidase displayed complex kinetics with p-nitrophenyl-beta-glucoside which inhibited the enzyme at concentrations greater than 0.5 mM. With cellobiose the kinetics were practically hyperbolic at 70 degrees C (Hill coefficient nH = 1.09 and Km = 0.83 mM), but faint inhibition was observed at 50 degrees C. beta-glucosidase shares with alpha-glucosidase a high number of physicochemical properties: with similar aminoacid composition, very close isoelectric point (4.5 and 4.2), high molecular weight in the native state (175,000 and 140,000), the two enzymes showed the same behaviour on DEAE-cellulose, were equally stable at high temperature and were dissociated by 6 M urea to still active proteins. Furthermore, the carbohydrate contents of beta-glucosidase (17.6%) is not far from that previously determined for some forms of alpha-glucosidase (14-16%).     

Functional significance of AMP deaminase and adenosine deaminase in the aestivating freshwater snail, Pila globosa (swainson): possible neuroendocrine involvement

The functional significance of AMP deaminase and adenosine deaminase has been studied in hepatopancreas of active, aestivated and ganglionic extracts-administered snails. The activity levels of both enzymes decreased in aestivating snails. Active snails injected with ganglionic extracts of aestivated snails also showed decreased activity. Contrastingly, the hepatopancreas of aestivated snails when treated with ganglionic extracts of active snails showed increased specific activities of both enzymes. The decrease or increase in the specific activities varied with different ganglionic extracts and the significance of the same is discussed.     

Production, Purification, and Properties of a Thermostable beta-Glucosidase from a Color Variant Strain of Aureobasidium pullulans

A color variant strain of Aureobasidium pullulans (NRRL Y-12974) produced beta-glucosidase activity when grown in liquid culture on a variety of carbon sources, such as cellobiose, xylose, arabinose, lactose, sucrose, maltose, glucose, xylitol, xylan, cellulose, starch, and pullulan. An extracellular beta-glucosidase was purified 129-fold to homogeneity from the cell-free culture broth of the organism grown on corn bran. The purification protocol included ammonium sulfate treatment, CM Bio-Gel A agarose column chromatography, and gel filtrations on Bio-Gel A-0.5m and Sephacryl S-200. The beta-glucosidase was a glycoprotein with native molecular weight of 340,000 and was composed of two subunits with molecular weights of about 165,000. The enzyme displayed optimal activity at 75 degrees C and pH 4.5 and had a specific activity of 315 mumol . min . mg of protein under these conditions. The purified beta-glucosidase was active against p-nitrophenyl-beta-d-glucoside, cellobiose, cellotriose, cellotetraose, cellopentaose, cellohexaose, and celloheptaose, with K(m) values of 1.17, 1.00, 0.34, 0.36, 0.64, 0.68, and 1.65 mM, respectively. The enzyme activity was competitively inhibited by glucose (K(i) = 5.65 mM), while fructose, arabinose, galactose, mannose, and xylose (each at 56 mM) and sucrose and lactose (each at 29 mM) were not inhibitory. The enzyme did not require a metal ion for activity, and its activity was not affected by p-chloromercuribenzoate (0.2 mM), EDTA (10 mM), or dithiothreitol (10 mM). Ethanol (7.5%, vol/vol) stimulated the initial enzyme activity by 15%. Glucose production was enhanced by 7.9% when microcrystalline cellulose (2%, wt/vol) was treated for 48 h with a commercial cellulase preparation (5 U/ml) that was supplemented with the purified beta-glucosidase (0.21 U/ml) from A. pullulans.     

Human lysosomal beta-glucosidase: purification by affinity chromatography

Two Sepharose-bound substrate analogs, 6'-aminohexanoyl-(2-N-sphingosyl-O-beta-D-glucoside) and 6'-aminohexyl-dodecanedioyl-1-(2-N-sphingosyl-1-O-beta-D-glu coside), were synthesized and used sequentially for the affinity purification of lysosomal beta-glucosidase (N-acyl-sphingosyl-1-O-beta-D-glucoside:glucohydrolase, EC 3.2.1.45). The capacities of these nondegradable affinity supports were 0.1 and 0.15 mg enzyme/ml settled gel, respectively. The purified enzyme had a specific activity of 75 mumol min-1 mg-1. The preparation had a single protein band with a molecular weight of 67,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, evidencing its apparent homogeneity. Isoelectric focusing on granular gels revealed four molecular forms of the enzyme with pI values of 4.0, 4.5, 4.7, and 5.8 to 6.2. The purified enzyme hydrolyzed glucosyl ceramide and 4-methylumbelliferyl-beta-D-glucoside with Km and Vmax values of 0.6 and 2.5 mM, and 101 and 26.1 mumol min-1 mg-1, respectively. The enzyme also hydrolyzed octyl beta-glucoside, a linear mixed-type inhibitor of the enzyme. Binding constants (Ki) were determined for the inhibitors, sphingosyl-1-O-beta-D-glucoside (Ki = 20 microM) and its N-hexyl derivative (Ki = 0.3 microM). The enzyme had a half-life of 65 and 30 min at 50 degrees C and pH 5.0 or 6.0, respectively. In addition, two other classes of ligands were used for the purification of lysosomal beta-glucosidase, and their capacities and specificities were compared to those of the substrate analog affinity supports. These included (i) the alkyl amine inhibitors octylamine, decylamine, and tetradecylamine; and (ii) the inhibitors, 6-aminohexanoyl-beta-glucosylamine and aminododecanoyl-1-(2-N-sphingosyl-1-O-beta-D-glucoside). Compared to these other ligand columns, the substrate analog affinity supports had about 100- to 1000-fold greater capacities or afforded 8- to 40-fold greater purification of human lysosomal beta-glucosidase.     

Isolation and properties of an extracellular beta-glucosidase from the polycentric rumen fungus Orpinomyces sp. strain PC-2.

An extracellular beta-glucosidase (EC 3.2.1.21) was purified from culture filtrate of the anaerobic rumen fungus Orpinomyces sp. strain PC-2 grown on 0.3% (wt vol-1) Avicel by using Q Sepharose anion-exchange chromatography, ammonium sulfate precipitation, chromatofocusing ion-exchange chromatography, and Superose 12 gel filtration. The enzyme is monomeric with a M(r) of 85,400, as estimated by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, has a pI of 3.95, and contains about 8.5% (wt vol-1) carbohydrate. The N terminus appears to be blocked. The enzyme catalyzes the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (PNPG). The Km and Vmax values with cellobiose as the substrate at pH 6.0 and 40 degrees C are 0.25 mM and 27.1 mumol.min-1 x mg-1, respectively; with PNPG as the substrate, the corresponding values are of 0.35 mM and 27.7 mumol.min-1 x mg-1. Glucose (Ki = 8.75 mM, with PNPG as the substrate) and gluconolactone (Ki = 1.68 x 10(-2) and 2.57 mM, with PNPG and cellobiose as the substrates, respectively) are competitive inhibitors. Optimal activity with PNPG and cellobiose as the substrates is at pH 6.2 and 50 degrees C. The enzyme has high activity against sophorose (beta-1,2-glucobiose) and laminaribiose (beta-1,3-glucobiose) but has no activity against gentiobiose (beta-1,6-glucobiose). The activity of the beta-glucosidase is stimulated by Mg2+, Mn2+, Co2+, and Ni2+ and inhibited by Ag+, Fe2+, Cu2+, Hg2+, SDS, and p-chloromercuribenzoate.     

The haemocyanin of the whelk, Busycon contrarium (Conrad): aggregation states and subunit structure

1. The haemocyanin of the left-handed whelk Busycon contrarium (Conrad) exists largely as six or more multi-decameric aggregates characterized by sedimentation coefficients of approximately 105S, 132S, 155S, 170S, 185S and about 200-220S. 2. These aggregates represent di- to hepta- or octa-decameric assemblies of the basic haemocyanin decamer having a mol. wt of 4.3 x 10(6)-4.5 x 10(6). 3. The fully dissociated subunits in 8.0 M urea (pH 8.5) and at pH 11.1, 0.01 M EDTA have mol. wts of 4.78 x 10(5) and 4.62 x 10(5), close to one-tenth of the mol. wt of the basic decameric unit of most gastropod haemocyanins. 4. The pH dependence of the mol. wts (Mw), studied by light-scattering at the constant protein concentration of 0.010%, exhibit bell-shaped pH transition profiles with mol. wt values of about 16 x 10(6) in the presence of 0.01 M Mg2+, in the pH region from about pH 4.5-8.0; in the absence of stabilizing divalent ions the observed mol. wt is about 10 x 10(6) at pH 4.5-7.0. Below pH 4.5 and above 7.0-8.0 there is a sharp drop in mol. wt to about 4 x 10(5)-4.5 x 10(5). 5. The transition profiles observed with both the urea and salt series of probes investigated at concentration = 0.010% are found to produce aggregation at low reagent concentrations with mol. wt changes from about 9 x 10(6)-12 x 10(6)-14 x 10(6), followed by a decrease in mol. wt below 4.3 x 10(6)-4.5 x 10(6) of the haemocyanin decamers.(ABSTRACT TRUNCATED AT 250 WORDS)