Isolation and Some Properties of a beta-d-Xylosidase from Clostridium acetobutylicum ATCC 824

A beta-d-xylosidase from C. acetobutylicum ATCC 824 was purified by column chromatography on CM-Sepharose, hydroxylapatite, Phenyl Sepharose, and Sephadex G-200. The enzyme had an apparent molecular weight of 224,000 as estimated by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the enzyme consisted of two subunits of 85,000 and one subunit of 63,000 daltons. It exhibited optimal activity at pH 6.0 to 6.5 and 45 degrees C. the enzyme had an isoelectric point of 5.85. It hydrolyzed p-nitrophenylxyloside readily with a K(m) of 3.7 mM. The enzyme hydrolyzed xylo-oligosaccharides with chain lengths of 2 to 6 units by cleaving a single xylose from the chain end. It showed little or no activity against xylan, carboxymethyl cellulose, and other p-nitrophenylglycosides.     

Purification and characterization of recombinant human interleukin-1 beta produced in Escherichia coli

Recombinant human interleukin-1 beta (rIL-1 beta) produced in Escherichia coli was purified to homogeneity by a combination of mass ion exchange column chromatography, ion exchange and gel filtration high performance liquid chromatography. The purified rIL-1 beta had a molecular weight of 18 kD on SDS-polyacrylamide gel electrophoresis and an isoelectric point of 6.9 on analytical isoelectric focusing. These values were almost same as those of natural interleukin-1 beta. The amino acid composition and amino acid sequence of the amino terminal region were consistent with those deduced from the cDNA sequence. In addition, the primary structure was confirmed by peptide mapping with lysyl-endopeptidase on reverse phase HPLC. Besides rIL-1 beta with amino terminal Ala, two molecular species, [Met0] rIL-1 beta and [desAla1] rIL-1 beta, were also obtained. Biological and physicochemical properties of the three species of rIL-1 beta were compared.     

Isolation of mRNA from bovine pituitary. The cell-free synthesis of the alpha and beta subunits of luteinizing hormone

RNA derived from bovine steer pituitary was translated in wheat germ cell-free extracts containing [35S]methionine. Antisera generated against purified denatured alpha and beta subunits of lutropin were used to demonstrate the synthesis of both proteins in vitro. The immunoprecipitated products of the cell-free system were resolved on sodium dodecyl sulfate/polyacrylamide gels and it was observed that the molecular weight of the immunoprecipitated alpha subunit protein was approximately 14,000, while that of the beta protein was estimated to be 16,000. Since the molecular weights of authentic alpha and beta subunits are 10,600 and 14,000 respectively, the cell-free products presumably represented their pre-protein forms. The ratio of the immunoprecipitated subunit pre-proteins was dependent on the magnesium concentration in the translation mixtures; at 2.1 mM, translation of lutropin alpha and beta mRNAs was comparable. RNA isolated from cow pituitary tissue directed the synthesis of fivefold less of the alpha and beta immunoprecipitated proteins than did steer RNA. Since the blood levels of gonadal steroids are higher in the cow, the results supported the hypothesis that lutropin alpha and beta mRNA biosynthesis is repressed by these steroids. The data also suggest that synthesis of lutropin alpha and beta subunits is coordinately expressed in certain physiological situations.     

UDP-Xylose-stimulated glucuronyltransferase activity in wheat microsomal membranes: characterization and role in glucurono(arabino)xylan biosynthesis

Microsomal membranes from etiolated wheat (Triticum aestivum) seedlings cooperatively incorporated xylose (Xyl), arabinose, and glucuronic acid residues from their corresponding uridine 5'-diphosphosugars into an ethanol-insoluble glucurono(arabino)xylan (GAX)-like product. A glucuronyltransferase activity that is enhanced by the presence of UDP-Xyl was also identified in these microsomes. Wheat glucuronyltransferase activity was optimal at pH 7 and required manganese ions, and several lines of evidence suggest its involvement in GAX-like biosynthesis. The GAX characteristics of the 14C-product were confirmed by digestion with a purified endo-xylanase from Aspergillus awamori (endo-xylanase III) and by total acid hydrolysis, resulting in a Xyl:arabinose:glucuronic acid molar ratio of approximately 105:34:1. Endo-xylanase III released only three types of oligosaccharides in addition to free Xyl. No radiolabel was released as xylobiose, xylotriose, or xylotetraose, indicating the absence of long stretches of unbranched Xyl residues in the nascent GAX-like product. High-pH anion exchange chromatography analysis of the resulting oligosaccharides along with known arabinoxylan oligosaccharide standards suggests that a portion of the nascent GAX-like product has a relatively regular structure. The other portion of the [14C]GAX-like polymer was resistant to proteinase K, endo-polygalacturonase, and endo-xylanase III (GH11 family) but was degraded by Driselase, supporting the hypothesis that the xylan backbone in this portion of the product is most likely highly substituted. Size exclusion chromatography indicated that the nascent GAX-like polymer had an apparent molecular mass of approximately 10 to 15 kD; however, mature GAXs from wheat cell walls had larger apparent molecular masses (>66 kD).     

Partial purification and properties of an endo-xylanase from cucumber seeds

An endo-xylanase. 1,4-β-D-xylan xylanohydrolase (EC 3.2.1.8) from immature cucumber L. cv. Heinz 3534) seeds was partially purified using ammonium sulfate fractionation and chromatography on SP-Sephadex and Sephadex G-100 in order to determine its role in xylan metabolism during development. Attempts to further purify the enzyme using chromatography on DEAE-Sephadex, Bio-Gel HTP hydroxylapatite, Sephadex G-200 and con A-Sepharose 4B and native polyacrylamide gel electrophoresis resulted in a significant decrease or complete loss of enzyme activity. Endo-xylanase had a native molecular weight of 96 kDa as determined by gel filtration, exhibited optimal activity at pH 5.0 and 48°C, and was most stable from pH 4.0 to 5.0. Using beechwood 4-o-methyl-d -glucurono-d -xylan dyed with Remazol Brilliant Blue R as substrate, the K_m was estimated to be 0.70 mg ml?¹. HgCl₂ at 1 mM inhibited endo-xylanase completely. Other metal ions inhibited the enzyme in the order Cu²⁺ > Fe³⁺ > Zn²⁺ > Ca²⁺ > Mn²⁺. The ethanol-soluble products of endo-xylanase action on beechwood xylan were isolated and characterized by consecutive chromatography on Bio-Gel P-10 and P-2. The major reaction products were xylo-oligosaccharides [degree of polymerization (dp) > 10] but traces of xylobiose and free xylose were also isolated. The formation of xylo-oligosaccharides indicated that the reaction was catalyzed primarily by an endoxylanase. The partially pure enzyme had no activity towards other cell wall polysaccharides such as cellulose, carboxymethyl cellulose, sodium carboxyl cellulose, potato starch, orange pectin, polygalacturonic acid, arabinogalactan and β-giucan. However, it was able to hydrolyze larchwood and oat spelts xylan and a polysaccharide component from purified cucumber cell walls. The ability to utilize a substrate from cucumber cell walls supports the hypothesis that endo-xylanase is involved in the development of cucumber seeds.     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Changes in the Accumulation of [alpha]- and [beta]-Tubulin Isotypes during Cotton Fiber Development

The expression of [alpha]- and [beta]-tubulin proteins in developing fibers and several other tissues of cotton (Gossypium hirsutum, cv Texas Marker 1) have been analyzed by immunoblots of one- and two-dimensional gels utilizing anti-tubulin antibodies as probes. As a percentage of total protein, fibers had greater amounts of tubulin than did hypocotyls, roots, leaves, or cotyledons. Both [alpha]- and [beta]-tubulin, having apparent molecular masses of approximately 50 kD and isoelectric points between pH 5 and pH 6, were resolved on a single two-dimensional gel. Under the conditions used, [alpha]-tubulin was less acidic in the isoelectric focusing dimension and migrated slightly faster in the sodium dodecyl sulfate dimension than did [beta]-tubulin. Nine [alpha]-tubulin isotypes that formed two distinct groups were identified on immunoblots of two-dimensional gels. The three most abundant [alpha]-tubulin isotypes were common to all tissues examined. Seven distinct [beta]-tubulin isotypes were also identified. Although their level of accumulation differed, four of the [beta]-tubulin isotypes were common to all tissues. Preferential accumulation of isotypes was more apparent in fibers than in the other tissues examined. Two [alpha]-tubulin isotypes and two [beta]-tubulin isotypes showed preferential accumulation in 10- and 20-d postanthesis fibers, respectively.     

ADP-ribosylation of microtubule proteins as catalyzed by cholera toxin

Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8-3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5'-AMP, respectively labeled with the corresponding isotope. Two-dimensional separation by isoelectric focusing and SDS-PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP-ribosyl residues into the tubulin subunits. SDS-PAGE of peptides derived from [32P]ADP-ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule-associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP-ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.     

Purification and characterization of a carboxylesterase from rabbit liver lysosomes

Carboxylesterase [EC 3.1.1.1] was purified from rabbit liver lysosomes by means of detergent solubilization, and by hydroxyapatite, phenyl-Sepharose and chromatofocusing column chromatographies. The purified enzyme appeared to be homogeneous on SDS-polyacrylamide gel electrophoresis and its molecular weight was estimated to be 58,000. This enzyme was eluted at an isoelectric point of approximately 5.8 by chromatofocusing, and exhibited a broad pH optimum of between 6.0 and 9.0. The enzyme hydrolyzed 4-methylumbelliferyl esters of saturated fatty acids (C2-C12), and it also hydrolyzed p-nitrophenylacetate, methyl butyrate, and tributyrin, but not acetanilide. Its activity was completely inhibited by diisopropyl-fluorophosphate (DFP) and phenylmethylsulfonyl fluoride (PMSF) at 10(-4) M, but was not affected by eserine, or by alpha- or beta-naphthyl acetate at 10(-3) M. Various metal ions (Mg2+, Mn2+, Ca2+, Co2+, Cu2+, Zn2+, Ni2+) at 10(-3) M also had no effect on the enzyme activity.     

Purification and partial characterization of a plasma inhibitor of tonin

A plasma inhibitor of tonin activity in the rat, was purified by ammonium sulfate precipitation, ion-exchange of chromatography, and gel filtration. Its purity was investigated by analytical electrophoresis on polyacrylamide gel and by ultracentrifugation sedimentation velocity. The molecular weight (360 000) of the purified inhibitor was determined by sodium dodecyl sulfate electrophoresis and its isoelectric point (4.5) by gel isoelectrofocusing. The Stokes radius (640 nm) was evaluated by gel filtration studies and a frictional ratio (f/fo) of 1.95 was calculated from the molecular weight and Stokes radius. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by the purified inhibitor was noncompetitive and does not exceed 70%. Electrophoresis showed the same mobility for [125I]tonin bound to plasma proteins and for [125I]tonin bound to the purified inhibitor. The inhibitor may be a protein resembling half of the dimeric protease inhibitor rat alpha 1-macroglobulin or human alpha 2-macroglobulin.     

Purification and characterization of an extracellular β-D-xylosidase from Aspergillus carbonarius

The production of an extracellular -D-xylosidase (-D-xyloside xylohydrolase, EC 3.2.1.37) by four Aspergillus strains (A. carbonarius, A. nidulans, A. niger and A. oryzae) grown on wheat bran medium was compared. The highest amount of the enzyme was found in the culture of A. carbonarius. The -D-xylosidase from A. carbonarius was purified to homogeneity by a rapid procedure, using hydrophobic interaction chromatography, chromatofocusing and affinity chromatography. The purified enzyme possessed not only -D-xylosidase activity, but also -L-arabinosidase activity. Mixed substrate experiments revealed that a single active centre was responsible for the splitting of the corresponding synthetic substrates. The molecular weight of the purified enzyme proved to be 100,000 Da, as estimated by SDS–PAGE. The isoelectric point was at pH 4.4. The pH and temperature optima were 4.0 and 60 °C, respectively. The enzyme remained stable over a pH range of 3.5–6.5 and up to 50 °C for 30 min. The Michaelis constant for p-nitrophenyl -D-xyloside was 0.198 mM. Kinetic studies demonstrated that the lack of the C-5 hydroxylmethyl group and the configuration of the C-4 hydroxyl group on the pyranoside ring play an important role in both substrate binding and splitting.     

Hemorrhagic toxin from the venom of Agkistrodon bilineatus (common cantil)

1. Hemorrhagic toxin was isolated from Agkistrodon bilineatus (Common cantil) venom using a three-step purification procedure to obtain 32.8 mg of purified hemorrhagic toxin from 700 mg of crude venom. 2. The purified toxin was homogeneous by disc polyacrylamide gel electrophoresis at pH 8.3, and by isoelectric focusing. 3. Hemorrhagic toxin possessed lethal, hemorrhagic and proteolytic activities. These activities of this toxin were inhibited by ethylenediaminetetraacetic acid (EDTA) and ethyleneglycol-bis-(beta-aminoethylether)N,N'-tetraacetic acid (EGTA), but not by cysteine or soybean trypsin inhibitor (SBTI). 4. Its molecular weight was approximately 48 kDa and the isoelectric point was 4.2. 5. Purified preparation hydrolyzed the Asn(3)--Gln(4), His(10)--Leu(11), Ala(14)--Leu(15), Tyr(16)--Leu(17), Arg(22)--Gly(23) and Phe(24)--Phe(25) bonds of oxidized insulin B. chain. 6. The A alpha chain of fibrinogen was first split and B beta chain was cleaved later by this toxin. 7. Hemorrhagic toxin contains 1 mol of zinc and 2 mol of calcium per mol of protein.     

Purification and characterization of predominant G-protein from bovine lung membranes. Biochemical and immunochemical comparison with Gi1 and Go purified from brain.

The predominant guanine nucleotide-binding protein (G-protein) of bovine lung membranes, termed GL, has been purified and compared biochemically, immunochemically and functionally with Gi and Go purified from rabbit brain. The purified GL appeared to have a similar subunit structure to Gi and Go, being composed of alpha, beta and possibly gamma subunits. On Coomassie Blue-stained SDS/polyacrylamide gels and immunoblots, the alpha subunit of GL (GL alpha) displayed an intermediate mobility (40 kDa) between those of Gi and Go (Gi alpha and Go alpha). GL alpha was [32P]ADP-ribosylated in the presence of pertussis toxin and [32P]NAD+. Analysis of [32P]ADP-ribosylated alpha subunits by SDS/polyacrylamide-gel electrophoresis and isoelectric focusing showed that GL alpha was distinct from Gi alpha and Go alpha, but very similar to the predominant G-protein in neutrophil membranes. Immunochemical characterization also revealed that GL was distinct from Gi and Go, but was indistinguishable from the G-protein of neutrophils, which has been tentatively identified as Gi2 [Goldsmith, Gierschik, Milligan, Unson, Vinitsky, Maleck & Spiegel (1987) J. Biol. Chem. 262, 14683-14688]. In functional studies, higher Mg2+ concentrations were required for guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[35S]) binding to GL than were required for nucleotide binding to Go, whereas Gi showed a Mg2+-dependence similar to that of GL. The kinetics of GTP[35S] binding to GL was quite different from those of Gi and Go; t1/2 values of maximal binding were 30, 15 and 5 min respectively. In contrast, the rate of hydrolysis of [gamma-32P]GTP by GL (t1/2 approximately 1 min) was approx. 4 times faster than that by Gi or Go. These results indicated that the predominant G-protein purified from lung is structurally and functionally distinct from Gi and Go of brain, but structurally indistinguishable from Gi2 of neutrophils.     

An α-L-arabinofuranosidase of Trichoderma reesei

An α-L-arabinofuranosidase (EC 3.2.1.55) of Trichoderma reesei was purified to homogeneity by cation- and anion-exchange chromatography. The enzyme had a molecular weight of 53 kDa as estimated by SDS electrophoresis. The isoelectric point of the enzyme was 7.5 and its pH optimum was 4.0. The enzyme hydrolyzed beet arabinan and released arabinose from wheat straw arabinoxylan.     

Thermostable, salt tolerant, wide pH range novel chitobiase from Vibrio parahemolyticus: isolation, characterization, molecular cloning, and expression.

A chitobiase gene from Vibrio parahemolyticus was cloned into plasmid pUC18 in Escherichia coli strain DH5 alpha. The plasmid construct, pC120, contained a 6.4 kb Vibrio DNA insert. The recombinant gene expressed chitobiase [EC 3.2.1.30] activity similar to that found in the native Vibrio. The enzyme was purified by ion exchange, hydroxylapatite and gel permeation chromatographies, and exhibited an apparent molecular weight of 80 kDa on SDS-polyacrylamide gel electrophoresis. Chitobiose and 6 more substrates, including beta-N-acetyl galactosamine glycosides, were hydrolyzed by the recombinant chitobiase, indicating its putative classification as an hexosaminidase [EC 3.2.1.52]. The enzyme was resistant to denaturation by 2 M NaCl, thermostable at 45 degrees C and active over a very unusual (for glycosyl hydrolases) pH range, from 4 to 10. The purified cloned chitobiase gave 4 closely focussed bands on an isoelectric focusing gel, at pH 4 to 6.5. The N-terminal 43 amino acid sequence shows no homology with other proteins in commercial databanks or in the literature, and from its N-terminal sequence, appears to be a novel protein, unrelated in sequence to chitobiases from other Vibrios reported and unrelated to hexosaminidases from other organisms.     

Peptide elongation factor 1 from yeasts: purification and biochemical characterization of peptide elongation factors 1 alpha and 1 beta (gamma) from Saccharomyces carlsbergensis and Schizosaccharomyces pombe

Cytoplasmic elongation factor 1 alpha (EF-1 alpha) [corrected] was purified to homogeneity in high yield from the two different yeasts Saccharomyces carlsbergensis (S. carls.) and Schizosaccharomyces pombe (S. pombe). The purification was easily achieved by CM-Sephadex column chromatography of the breakthrough fractions from DEAE-Sephadex chromatography of cell-free extracts. The basic proteins have a molecular weight of 47,000 for the S. carls. factor and of 49,000 for the S. pombe factor. While the purified yeast EF-1 alpha s function analogously to other eukaryotic factors and the E. coli EF-Tu in Phe-tRNA binding and polyphenylalanine synthesis, the yeast factor unusually hydrolyzed GTP on yeast ribosomes upon addition of Phe-tRNA in the absence of poly(U) as mRNA. This novelty is probably owing to the yeast ribosomes, which are assumed to lack elongation factor 3-equivalent component(s). Trypsin and chymotrypsin selectively cleaved the two yeast factors to generate resistant fragments with the same molecular weight of 43,000 (by trypsin) and of 44,000 (by chymotrypsin), respectively. Those cleavage sites were characteristically protected by the presence of several ligands bound to EF-1 alpha such as GDP, GTP, and aminoacyl-tRNA. Based on the sequence analysis of the fragments generated by the two proteases, the partial amino acid sequence of the S. carls. EF-1 alpha was deduced to be in accordance with the N-terminal region covering positions (1) to 94 and two Lys residues at the C-terminal end of the predicted total sequence of the Saccharomyces cerevisiae (S. cerev.) factor derived from DNA analysis, except for a few N-terminal residues, confirming the predicted S. cerev. sequence at the protein level. EF-1 beta and EF-1 beta gamma were isolated and highly purified as biologically active entities from the two yeasts. EF-1 beta s from the two yeasts have the same molecular weight of 27,000, whereas component gamma of the S. carls. EF-1 beta gamma showed a higher molecular weight (47,000) than that of the S. pombe factor (40,000). It was also shown that a stoichiometric complex was formed between EF-1 alpha and EF-1 beta gamma from S. pombe. Furthermore, a considerable amount of Phe-tRNA binding activity was distributed in the EF-1H (probably EF-1 alpha beta gamma) fraction from freshly prepared cell-free extracts of yeast.     

Pteroylpolyglutamate hydrolase from human jejunal brush borders. Purification and characterization

Pteroylpolyglutamate hydrolase was solubilized with Triton X-100 from human jejunal mucosal brush borders and purified approximately 5,000-fold using organomercurial affinity chromatography, DEAE-cellulose chromatography, and gel filtration. The apparent molecular weight of the purified enzyme in the Triton micelle was estimated as 700,000 using Bio-Gel A-1.5m gel filtration. Sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis followed by Coomassie stain demonstrated two polypeptide bands at 145,000 and 115,000 daltons. The purified enzyme had an isoelectric point of 7.2, was maximally active at pH 5.5, and was stable above pH 6.5 and at temperatures up to 65 degrees C for at least 90 min. Human jejunal brush-border pteroylpolyglutamate hydrolase is an exopeptidase which liberated [14C]Glu as the sole labeled product of PteGlu2[14C]Glue (where PteGlun represents pteroylpolyglutamate), failed to liberate a radioactive product from PteGlu2[14C]GluLeu2, and released all possible labeled PteGlun products during incubation with Pte[14C]GluGlu6 with the accumulation of Pte[14C]Glu. PteGlu2, PteGlu3, and PteGlu7 were substrates, each with Km = 0.6 microM, whereas PteGlu was a weak inhibitor of the hydrolysis of PteGlu3 with Ki = 20 microM. Components of the pteroyl moiety, Glu, and short chain Glun in alpha or gamma linkages were not inhibitory. The enzyme was activated by Zn2+ or Co2+. The properties of brush-border pteroylpolyglutamate hydrolase are different from those described for the soluble intracellular pteroylpolyglutamate hydrolase in other species and in human mucosa, yet are consistent with previous data on the process of hydrolysis of PteGlun in the intact human intestine.     

Expression and characterization of functional recombinant bovine follicle-stimulating hormone (boFSHalpha/beta) produced in the milk of transgenic rabbits

Bovine follicle-stimulating hormone (boFSH) is a heterodimeric glycoprotein that belongs to the pituitary gonadotropins. Bioactive FSH is composed of alpha and beta subunits which require extensive N-glycosylation and sialylation. The mammary gland of transgenic livestock is an attractive source for the synthesis of post-translationally modified proteins. Two mammary gland-specific gene constructs with the cDNA for the boFSH alpha (boFSHalpha) and beta (boFSHbeta) subunits controlled by bovine alpha-s1 casein regulatory sequences were co-microinjected into fertilized rabbit oocytes. Two FSHalpha/FSHbeta double transgenic rabbit lines were established. The transgene expression was strictly lactation and mammary gland specific. Protein analysis revealed the presence of the boFSH heterodimer in the milk of transgenic rabbits showing a molecular weight similar to that of purified pituitary gland derived boFSH (boFSH-P). Subunit specific antibodies detected both polypeptides with the expected molecular sizes. Biochemical characterization demonstrated the expected isoelectric points of the recombinant boFSH. The presence of the post-translationally added terminal sialic acid residues was indicated by wheat germ agglutinin (WGA) lectin Western blotting. The biological activity of the recombinant mammary gland produced boFSH was determined using a FSH-dependent reporter cell line. The bioactivity of the recombinant boFSH was comparable to that of purified boFSH-P.     

Characterization and purification of thermostable beta-glucosidase from Clostridium thermocellum.

A β-glucosidase was isolated from $\text{Clostridium thermocellum}$; the enzyme was localized in the periplasmic space.It was purified in a five-step procedure including ion-exchange chromatography on DEAE-Cellulose, chromatography on HA-Ultrogel and DEAE-Sephadex, gel filtration on AcA 34 Ultrogel and isoelectric focusing.The final preparation was purified 944-fold with a recovery of about 5% of the initial enzyme activity.Polyacrylamide disc electrophoresis of the purified enzyme gave a single band at pH 8.3. The enzyme is active towards cellobiose and p-nitrophenyl-β-D-glucoside(PNPG) and developed maximum activities at pH 6.0 and 65°C. A molecular weight of 50,000 daltons was estimated by gel filtration and the enzyme was isoelectric at pH 4.68.     

A novel lactococcal bacteriocin whose activity depends on the complementary action of two peptides.

A lactococcal bacteriocin, termed lactococcin G, was purified to homogeneity by a simple four-step purification procedure that includes ammonium sulfate precipitation, binding to a cation exchanger and octyl-Sepharose CL-4B, and reverse-phase chromatography. The final yield was about 20%, and nearly a 7,000-fold increase in the specific activity was obtained. The bacteriocin activity was associated with three peptides, termed alpha 1, alpha 2, and beta, which were separated by reverse-phase chromatography. Judging from their amino acid sequences, alpha 1 and alpha 2 were the same gene product. Differences in their configurations presumably resulted in alpha 2 having a slightly lower affinity for the reverse-phase column than alpha 1 and a reduced bacteriocin activity when combined with beta. Bacteriocin activity required the complementary action of both the alpha and the beta peptides. When neither alpha 1 nor beta was in excess, about 0.3 nM alpha 1 and 0.04 nM beta induced 50% growth inhibition, suggesting that they might interact in a 7:1 or 8:1 ratio. As judged by the amino acid sequence, alpha 1 has an isoelectric point of 10.9, an extinction coefficient of 1.3 x 10(4) M-1 cm-1, and a molecular weight of 4,346 (39 amino acid residues long). Similarly, beta has an isoelectric point of 10.4, an extinction coefficient of 2.4 x 10(4) M-1 cm-1, and a molecular weight of 4110 (35 amino acid residues long). Molecular weights of 4,376 and 4,109 for alpha 1 and beta, respectively, were obtained by mass spectrometry. The N-terminal halves of both the alpha and beta peptides may form amphiphilic alpha-helices, suggesting that the peptides are pore-forming toxins that create cell membrane channels through a "barrel-stave" mechanism. The C-terminal halves of both peptides consist largely of polar amino acids.