Isolation, characterization, and amino-terminal amino acid sequence analysis of human neutrophil cathepsin G from normal donors

Human neutrophil cathepsin G from normal donors has been purified 82-fold using an isolation procedure which included sequential sodium chloride extraction, Aprotonin-Sepharose affinity chromatography, CM-cellulose ion-exchange chromatography, and AcA44 gel filtration chromatography. The inclusion of this last purification step was crucial for separating inactive lower molecular weight species from the active forms of neutrophil cathepsin G and resulted in a higher specific activity of the final preparation. SDS polyacrylamide gradient gel electrophoresis of the purified reduced protein demonstrated three discrete polypeptides of Mr 31,000, 30,000, and 29,500. Peptide analysis of tryptic digests indicated that these three polypeptides are structurally related to each other and represent microheterogeneity of the purified protein. The cathepsin G peptide maps were distinctly different from the peptide maps of neutrophil elastase. The apparent isoelectric points of these forms as determined by two-dimensional electrophoresis was approximately 8.0. Utilizing microsequencing techniques, the first 25 residues of normal neutrophil cathepsin G have been determined and shown to be identical (except for residue 11) with the sequence of 21 residues of cathepsin G isolated from leukemic myeloid cells. A high degree of homology was found when the amino-terminal regions of neutrophil cathepsin G, rat mast cell protease II (65%) and two human serine proteinases, factor D (52%) and neutrophil elastase (48%), were compared. A precipitating monospecific antiserum to cathepsin G was produced by repeated immunizations of guinea pigs. This antiserum has been used in immunoblotting experiments to demonstrate that the intracellular form(s) of this enzyme is the same approximate Mr as the purified enzyme, and to develop a solid-phase radioimmunoassay for measuring neutrophil cathepsin G in the range 5-50 ng/ml.     

Purification and characterization of the phosphate/hydroxyl ion antiport protein from rat liver mitochondria.

The phosphate transport protein was purified from rat liver mitochondria by extraction in an 8% (v/v) Triton X-100 buffer followed by adsorption chromatography on hydroxyapatite and Celite. SDS/polyacrylamide-gel electrophoresis (10%, w/v) demonstrated that the purified polypeptide was apparently homogeneous when stained with Coomassie Blue and had a subunit Mr of 34,000. However, lectin overlay analysis of this gel with 125I-labelled concanavalin A demonstrated the presence of several low- and high-Mr glycoprotein contaminants. To overcome this problem, mitochondria were pre-extracted with a 0.5% (v/v) Triton X-100 buffer as an additional step in the purification of phosphate transport protein. SDS/polyacrylamide gradient gel electrophoresis (14-20%, w/v) of the hydroxyapatite and Celite eluates revealed one major band of Mr 34,000 when stained with Coomassie Blue. The known thiol group sensitivity of the phosphate transporter was employed to characterize the isolated polypeptide further. Labelling studies with N-[2-3H]ethylmaleimide showed that only the 34,000-Mr band was labelled in both the hydroxyapatite and Celite fractions, when purified from rat liver mitochondria. Further confirmation of its identity has been provided with an antiserum directed against the 34,000-Mr protein. Specific partial inhibition of phosphate uptake, as measured by iso-osmotic swelling in the presence of (NH4)2HPO4, was achieved when mitoplasts (mitochondria minus outer membrane) were incubated with this antiserum. Finally, amino acid analysis of the rat liver mitochondrial phosphate/hydroxyl ion antiport protein indicates that it is similar in composition to the equivalent protein isolated from ox heart.     

Purification of branched chain aminotransferase from rat heart mitochondria

This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.     

Quantitative assay of deoxyribonuclease activity after isoelectric focusing in polyacrylamide gels: pH control and effects of enzyme diffusion

A method for the quantitative assay of nuclease activity in crude cell lysates after isoelectric focusing (IEF) in polyacrylamide slab gels is described. After IEF, an agarose overlay gel containing DNA is placed on the IEF gel and the nuclease activity quantified by the loss of ethidium bromide fluorescence of the DNA. With this method a linear response was obtained for 1 to 10 ng of DNase I. Various methods of pH equilibration after IEF were also evaluated. The use of a high buffer concentration in the overlay gel is recommended to control the pH during the enzyme reaction. An analytical solution for the diffusion of enzymes from the IEF gel to the overlay gel is also presented and an equation that may be used to choose optimum times for transfer of the enzyme from the IEF gel to the overlay gel is given.     

Biospecific adsorption of hepatic DT-diaphorase on immobilized dicoumarol. II. Purification of mitochondrial and microsomal DT-diaphorase from 3-methylcholanthrene-treated rats

Liver mitochondrial and microsomal DT-diaphorase have been purified from 3-methylcholanthrene-treated rats. A 1150-fold and 3500-fold purification of mitochondrial and microsomal DT-diaphorase, respectively, is achieved after solubilization of the membranes with deoxycholate followed by affinity chromatography on azodicoumarol Sepharose 6B and subsequent gel filtration on Sephadex G-100. From this purification procedure, 65–70% of mitochondrial DT-diaphorase is recovered and the purified enzyme has a specific activity comparable to that of cytosolic DT-diaphorase; i.e., 50.4 kat/kg protein. Microsomal DT-diaphorase is obtained with a yield of 45% and a specific activity of 15.5 kat/kg protein.Purified mitochondrial DT-diaphorase exhibits an absorption spectrum characteristic of a flavoprotein and very similar to that of the cytosolic enzyme. Purification of both mitochondrial and microsomal DT-diaphorase results in fractions enriched in a polypeptide with a molecular weight of 28,000 which comigrates with purified cytosolic DT-diaphorase on SDS-polyacrylamide gel electrophoresis. Employing antiserum raised against cytosolic DT-diaphorase, immunological identity between DT-diaphorase isolated from the three cell fractions is observed with both the Ouchterlony immunodiffusion technique and fused rocket immunoelectrophoresis. The latter method also reveals that DT-diaphorase isolated from mitochondria and microsomes contains several antigenic forms identical to those observed in purified cytosolic DT-diaphorase. Furthermore, this antiserum inhibits DT-diaphorase to about the same extent whether the enzyme is isolated from mitochondria, microsomes, or cytosol. In addition, this antiserum efficiently inhibits membrane-bound microsomal DT-diaphorase.     

Purification and characterization of a phosphodiesterase from Bothrops alternatus snake venom

A phosphodiesterase was purified from the venom of the snake Bothrops alternatus by a combination of gel filtration and ion exchange chromatographies. In SDS-PAGE, the enzyme gave a single band with a molecular mass of 105 kDa, which was unaltered in the presence of -mercaptoethanol, indicating that the protein contained no subunits. A single protein band was also observed in native PAGE. There were no contaminating 59-nucleotidase, alkaline phosphatase and protease activities. The enzyme was recognized by commercial bothropic antiserum and gave a single band in immunoblotting. The enzyme had a pH optimum in the range of 7.5–9.5 and the optimum temperature was 60°C, with activity being rapidly lost within 1 min at 70°C. The K_m of the enzyme was 2.69 mM. PDE activity was potentiated by cobalt and, to a lesser extent, by calcium, whereas copper, manganese, zinc, EDTA, and -mercaptoethanol were inhibitory. These properties show that this enzyme is very similar to that isolated from other snake venoms.     

Purification and characterization of an endo-polygalacturonase from Verticillium albo-atrum

A polygalacturonase was isolated from the culture filtrate of the fungal plant pathogen Verticillium albo-atrum and purified 22-fold to homogeneity as judged by SDS-electrophoresis. The enzyme was a basic protein with a molecular weight of 37 kDa, an isoelectric point ≥8·6 and containing 1·7% carbohydrate. The enzyme was an endo-polygalacturonase and hydrolysed a wide range of pectic substrates including polygalacturonic acid, 93% methylated pectin and pectins in tomato cell walls. The best substrate was 31% methylated pectin. Relative reaction rates on pectins with different degrees of methylation could be explained by considering both the number of susceptible bonds and non-specific enzyme-substrate interactions. The principal products of long-term hydrolysis were di- and mono-galacturonate. Maximum activity was observed at pH 4·6–5·0 and 46 °C. However, the enzyme lost activity above 30 °C in the absence of substrate. Enzyme activity was very sensitive to changes in ionic strength at low salt levels. It was stable in the pH range 3–11 at 30 °C.     

Purification and some properties of ornithine decarboxylase from rat liver

Ornithine decarboxylase (EC 4.1.1.17) was purified to near homogeniety from livers of thioacetamide- and dl-α-hydrazino-δ-aminovaleric acid-treated rats by using three types of affinity chromatography with pyridoxamine phosphate-Sepharose, pyridoxamine phosphate-dipropylenetriamine-Sepharose and heparin-Sepharose. This procedure gave a purification of about 3.5·10⁵-fold with an 8% yield; the specific activity of the final enzyme preparation was 1,1·10⁶ nmol CO₂/h per mg protein. The purified enzyme gave a single band of protein which coincided with activity peak on polyacrylamide gel electrophoresis and also gave a single major band on SDS-polyacrylamide gel electrophoresis. A single precipitin line was formed between the purified enzyme and an antiserum raised against a partially purified enzyme, on Ouchterlony immunodiffusion. The molecular weight of the enzyme was estimated to be 105 000 by polyacrylamide gel electrophoresis at several different gel concentrations; the dissociated subunits had molecular weights of 50 000 on SDS-polyacrylmide gels. The isoelectric point of the enzyme was pH 4.1.     

Purification of aspartate transcarbamylase from Drosophila melanogaster.

A purification procedure is described by which aspartate transcarbamylase was obtained from cultured cells of Drosophila melanogaster as part of a high-molecular-weight enzyme complex. The complex is shown to contain several polypeptides. An antiserum directed against the complex enzyme inhibited in vitro the activity of aspartate transcarbamylase, carbamylphosphate synthetase and dihydro-orotase which were shown to copurify on a sucrose gradient and by gel electrophoresis. A fast preparation procedure using this antiserum yielded a 220 000-molecular-weight protein in addition to the polypeptides present in the complex. A purification procedure is also described to obtain aspartate transcarbamylase from second instar larvae of Drosophila. At this stage, the enzyme is not complexed with carbamylphosphate synthetase and dihydro-orotase but exhibits the same molecular weight as the aspartate transcarbamylase moiety found in the high-molecular-weight complex of cultured cells.     

Human prolyl hydroxylase. Purification, partial characterization and preparation of antiserum to the enzyme.

Prolyl hydroxylase was purified from human foetal skin and from a mixture of human foetal tissues by the affinity chromatography procedure using poly(L-proline). The enzyme from both sources was pure, when examined by polyacrylamide gel electrophoresis, as a native protein or in the presence of sodium dodecylsulphate, and enzyme activity recovery varied from 38% to 70% with seven enzyme preparations. The enzyme synthesized from 61.0 mumol to 82.7 mumol hydroxyproline mg protein-1 h-1 degrees C with a saturating concentration of (Pro-Pro-Gly)5 as substrate. The molecular weight of the enzyme was identical with that of the chick prolyl hydroxylase when studied by gel filtration, and the molecular weights of the subunits of the enzyme were about 61000 and 64000 as determined by sodium dodecylsulphate-polyacrylamide gel electrophoresis. The amino acid composition of the human enzyme was very similar to that of the chick prolyl hydroxylase. Antisera to human and chick prolyl hydroxylases were prepared in rabbits. A single precipitin line was seen between the antiserum to human prolyl hydroxylase and the human enzyme in double immunodiffusion, and no cross-reactivity was detected between the human chick enzymes by this technique. However, a distinct cross-reactivity was observed between the human and chick enzymes in inhibition experiments.     

Partial purification, characterization and regulation of cellulolytic enzymes from Trichoderma longibrachiatum

A net purification of 9·46-, 18·6- and 16·7-fold for filter paper (FP) hydrolytic activity, carboxymethyl (CM) cellulase and β-glucosidase, respectively was achieved through ion exchange and gel chromatographies. The purified enzyme preparation showed an optimal pH of 5·0 for CM cellulase and 5·5 for the other two components. The enzyme activities increased up to 60°–65°C for the three enzyme components and they were stable at 30° or 40°C and pH 4·5 to 5·0 after 20–30 min treatment. The four enzyme components, that is, two FP activities (unadsorbed and adsorbed), a CM cellulase and a β-glucosidase, had K_m values of 47·6 mg, 33·3 mg, 4·0 mg and 0·18 mmol/l with V _max of 4, 1·28, 66·5 and 1·28 units per mg protein. The molecular weights as determined with SDS-PAGE were found to be 44000, 38000, 55000 and 63000 for the above four enzyme components in the same sequence. A distinct type of synergistic action was observed between these components by their action on dewaxed cotton. Glycerol at 1% strongly repressed the formation of all the cellulolytic enzymes. The role of proteolytic enzymes in in vitro inactivation of cellulases was not apparent.     

Sandwich configuration of type I collagen suppresses progesterone production in primary cultured porcine granulosa cells by reducing gene expression of cytochrome P450 cholesterol side-chain cleavage enzyme

When porcine granulosa cells were cultured on type I collagen (TIC)-coated dishes, progesterone was continuously secreted in the culture medium. However, when cells were overlaid with a TIC gel, progesterone production was decreased to 34% (day 3) and 16% (day 4) of the value measured for cells without the overlay. The effect of TIC gel overlay on cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which catalyzes the conversion of cholesterol to pregnenolone and is thought to be the rate-limiting enzyme in the conversion of cholesterol to progesterone, was examined. P450scc gene expression in cells overlaid with a TIC gel was decreased to 62% (day 3) and 36% (day 4) of the value measured for cells without the overlay. Amounts of P450scc were also reduced in the cells overlaid with a TIC gel. When pregnenolone, the direct precursor of progesterone, was added to the culture medium, the increase in progesterone production by cells overlaid with a TIC gel was much greater than that for cells without a TIC gel and a statistical difference in progesterone production was no longer observed between the two groups of cells. Treatment of the cells with human FSH (hFSH) enhanced progesterone production in a dose-dependent manner, irrespective of the presence of a TIC gel overlay. Moreover, hFSH induced P450scc gene expression in cells with and without a TIC gel overlay. These results indicate that a TIC gel overlay reduces progesterone production in granulosa cells via the suppression of P450scc gene expression. This supports the possibility that the existence of a TIC gel on the apical side of granulosa cells prevents the spontaneous luteinization of granulosa cells cultured on TIC-coated dishes. The fact that hFSH overcomes the suppressive effect of the TIC gel overlay on progesterone production may explain the mechanism for the subtle rise in serum progesterone concentration in the late follicle phase of the "in vitro fertilization" program.     

Specific Antiserum and Monoclonal Antibodies Against the Taurine Biosynthesis Enzyme Cysteine Sulfinate Decarboxylase: Identity of Brain and Liver Enzyme

Cysteine sulfinate decarboxylase (CSD), the putative biosynthetic enzyme for taurine, was purified 1,800-fold with a 1% yield from rat liver, where it was found to be 20-fold enriched compared with brain. The final fraction was homogeneous, as ascertained through sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reverse-phase HPLC. An antiserum was raised in the rabbit that (a) quantitatively immunoprecipitated CSD activity and (b) immunolabeled only one band (MW = 51,000) on an immunoblot from liver homogenate. Monoclonal antibodies were also raised that recognized the CSD protein and immunolabeled the same 51-kilodalton protein on an immunoblot from liver homogenate. In a brain extract, two CSD activities had been previously found and named CSDI and CSDII, according to their chromatographic elution patterns. We have compared the properties of CSDI from brain--the most likely enzyme involved in the biosynthesis of taurine in the brain, according to previous investigations-and CSD from liver: Both activities (a) were similarly eluted on ion-exchange and hydroxyapatite chromatographies, (b) showed the same elution pattern on gel filtration with an apparent native molecular weight of approximately 63,000, and (c) were immunoprecipitated in a strictly identical manner by the antiserum against liver CSD. Moreover, this antiserum as well as the monoclonal antibodies immunolabeled a single band (51 kilodaltons) on an immunoblot from brain CSD-enriched fraction or liver fraction. All these data show that CSDI from brain and liver CSD are the same monomeric enzyme. They also indicate that a specific antiserum against rat liver CSD has been raised that can be used for immunocytochemical visualization of CSD-containing cells in the brain.     

Cloning of Thermomonospora fusca genes coding for beta 1-4 endoglucanases E1, E2 and E5

Thermomonospora fusca chromosomal DNA was partially digested with EcoRI and fragments in the size range from 4 to 15 kb were isolated, ligated into lambda gtWES.lambda B arms, packaged, and the recombinant phages plated on Escherichia coli. The plaques were screened for carboxymethyl cellulase (CMCase) activity by a gel overlay procedure, and 25 plaques were positive among the 15,000 plaques that were screened. Positive phages were amplified and used to prepare infected E. coli extracts which were assayed for CMCase activity before and after treatment with antisera prepared against five purified T. fusca beta 1-4 endoglucanases (E1-E5). One phage produced an enzyme that was inhibited by E1 antiserum, nine of the phages produced enzymes that were inhibited by E2 antiserum, 14 produced enzymes that were inhibited by E5 antiserum and the enzyme produced by the other phages was not inhibited by any of the five antisera. The DNA insert present in the phage coding for E1 was cut into a number of different fragments which were subcloned into E. coli first using lambda gtWES.lambda B and then plasmid pBR322. The smallest active subclone, pTE12, contained a 3.1-kb insert. The insert present in one of the phages coding for E2 was also subcloned and the smallest active subclone pTE23 contained a 2-kb insert. E. coli HB101 containing plasmid pTE12 or pTE23 produced enzymes that were identical to E1 and E2, respectively, in all the properties tested.     

Affinity Purification of Synenkephalin-Containing Peptides Including a Novel 23.3-Kilodalton Species

Abstract: Affinity chromatography has been used for rapid and high-yield purification of synenkephalin (proenkephalin 1 -70) containing peptides present in bovine adrenal medulla (BAM) chromaffin granular lysate. A column of CN-Br-activated Sepharose 4B coupled to synenkephalin antiserum bound synenkephalin immunoreactivity which was eluted by a stepwise gradient of 50 mM ammonium acetate containing 20% (vol/vol) acetonitrile over the pH range 7–3. Synenkephalin immunoreactivity emerged as two peaks, eluting at pH 5.5 and 4.5. Characterization of the two peaks by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting indicated that the pH 5.5 peak contained principally low-molecular-weight proenkephalin species (8.6 and 12.6 kilodaltons), whereas the pH 4.5 peak contained, in addition, high-molecular-weight proenkephalin species (18.2 and 23.3 kilodaltons). The 8.6- and 12.6- kilodalton species were isolated from the pH 5.5 peak by TSK gel filtration HPLC, whereas the pH 4.5 peak was further purified by passage over successive affinity columns coupled to antiserum against BAM 22P (proenkephalin 182–203) and [Met⁵]-enkephalin-Arg⁶-Gly⁷-Leu⁸. The former column retains the 23.3-kilodalton species, whereas the latter column retains the 18.2-kilodalton species. The 23.3- kilodalton peptide represents a novel putative proenkephalin intermediate (proenkephalin-1–206), containing [Leu⁵]- enkephalin at the C-terminus.     

Physiological mechanisms of buoyancy in eggs from brackish water cod

Newly fertilized eggs of brackish water (Gotland, Baltic Sea) and marine (Lofoten, Norway) cod were investigated with regard to specific gravity, wet and dry weight, water content, chorion weight, and content of protein, free amino acids (FAA), and ions. The eggs had neutral buoyancies equivalent to a salinity of 14^.3% (range 11^.5–16^.2%) in brackish water, and 33^.0% (range 31^.8–34^.5%) in the marine environment. A buoyancy model was developed and showed that this difference was mainly caused by differences in egg water content which was 96.6 ± 0^.47% and 92.7 ± 0.45% in the brackish and marine eggs, respectively. The higher water content of the brackish eggs resulted from increased water uptake during final oocyte maturation due to higher intracellular contents of FAA, Cl ^– and NH₄⁺. SDS polyacrylamide gel electrophoresis of eggs and oocytes, and measurements of egg protein content suggested that the FAA pool of both egg types originated from hydrolysis of specific yolk proteins. The main contributor seemed to be a protein with a molecular weight of 100 kDa.     

Plant plasma membrane proteins : immunological characterization of a major 75 kilodalton protein group

A major 75 kD protein group from the tomato plasma membrane was semipurified on polyacrylamide gels and used to raise a rabbit antiserum. The resulting antiserum recognized a single 75 kilodalton band from phase partitioned tomato plasma membrane (from both suspension cells and mature, green fruit) after resolution on one-dimensional polyacrylamide gels. Two-dimensional polyacrylamide gel analysis of proteins from tomato plasma membrane showed that the 75 kilodalton antiserum recognized a group of proteins ranging from 63.1 to 88.2 kilodaltons (mean = 75.6 kilodaltons) and with isoelectric point values ranging from 5.7 to 6.3. No other spots were visible on the two-dimensional blots. This antiserum was shown to bind protoplast surface epitopes by indirect immunofluorescence. The presence of this protein group in both monocotyledonous and dicotyledonous plants was established by immunoblotting the tomato 75 kilodalton antiserum against proteins obtained from plasma membrane-enriched fractions from corn roots and soybean roots. The data suggest that this 75 kilodalton protein group is a major proteinaceous component of the plant plasma membrane.     

Purification and molecular characterization of bovine pregastric lipase

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44-48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing approximately 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.     

Use of continuous-elution gel electrophoresis as a preparative tool for blot overlay analysis

Blot overlay techniques have long been used to directly visualize protein-protein interactions within membrane complexes. However, this approach is often hampered by the limited quantities of purified membrane proteins available for conjugation with marker molecules. Here we applied continuous-elution gel electrophoresis as a preparative alternative to isolate sufficient amounts of a homogeneous protein sample to be used as a peroxidase-labeled probe in blot overlays. Microsomal muscle proteins ranging from approximately 20 to 600 kDa were electrophoretically separated and various marker proteins present in eluted fractions were identified by immunoblotting. Since the supramolecular structure of calsequestrin has recently been determined, this terminal cisternae protein was isolated as a model protein for studying protein-protein interactions. In blot overlay assays, peroxidase-conjugated calsequestrin specifically bound to the ryanodine receptor, triadin, calsequestrin itself, and junctin, illustrating that the biological binding affinities are retained in electrophoretically prepared muscle proteins. Potential applications for differential blot overlay approaches and for analyzing pathophysiological preparations from dystrophic muscle were evaluated. Since continuous-elution gel electrophoresis can separate a wide range of differently sized proteins from subcellular fractions, our report indicates that this technique can be utilized for the rapid identification of protein-protein interactions in future high-throughput analyses of subproteomes.     

Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8

Keratinolytic activity was detected in the culture broth of feather-degrading thermophilic Streptomyces thermoviolaceus SD8. The crude enzyme was concentrated by precipitation with 80% saturation of ammonium sulphate and desalted by SephadexG-10–120 gel chromatography followed by lyophilization. The specific activity of the enzyme was enhanced 50-fold. PAGE analysis indicated a monomeric form with a molecular weight of 40 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 °C, respectively. The enzyme was stable at a pH range of 6·5–8·5 and up to 65 °C. The enzyme could hydrolyse fibrin, muscle, collagen, nail and hair and could produce leucine, threonine and tyrosine from feather. It could be a useful enzyme for waste treatment by promoting hydrolysis of the above substances in the sewage, or it could be used for animal feed preparation.