The primary structure of glycoprotein III from bovine adrenal medullary chromaffin granules. Sequence similarity with human serum protein-40,40 and rat Sertoli cell glycoprotein.

Glycoprotein III (GpIII) was purified from the soluble fraction of bovine chromaffin granules, the secretory vesicles of the adrenal medulla, by chromatography using wheat germ agglutinin-Sepharose followed by reverse-phase high performance liquid chromatography (HPLC). Characterization of this glycoprotein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reverse-phase HPLC, amino acid analysis and partial NH2-terminal sequence analysis indicated that GPIII was a disulfide-linked heterodimer with 37-kDa subunits. Analysis of in vitro translation products of adrenal medullary poly(A)+ RNA by immunoprecipitation using an anti-GpIII serum and sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that both subunits are synthesized from a single precursor. Partial NH2-terminal sequence analysis allowed construction of oligonucleotides which were used as primers for a polymerase chain reaction to generate a GpIII-specific DNA probe. This probe was used to isolate a cDNA clone encoding the GpIII precursor from a bovine adrenal medullary cDNA library. The predicted amino acid sequence of GpIII has greater than 80% similarity to human serum protein-40,40, a protein implicated in the complement system, and to a major secretory product of Sertoli cells, glycoprotein 2, which is thought to play a role in spermatogenesis. Northern blot analysis confirmed that RNA encoding GpIII is also abundant in liver, testis, and brain.     

Major proteins released by a protein-producing bacterium, Bacillus brevis 47, are derived from cell wall protein

B. brevis 47 secretes a vast amount of protein consisting mainly of two kinds with approximate molecular weights of 130,000 and 150,000. The two major extracellular proteins were indistinguishable from those of cell wall protein by SDS-polyacrylamide gel electrophoresis. Based on the results of analysis of amino acid composition, limited proteolysis followed by electrophoresis, and the cross-reactivity of antisera, we conclude that the 130K and 150K extracellular proteins are derived from the respective cell wall proteins. Furthermore, the NH2-terminal amino acid analysis suggests that the two major extracellular proteins are released from the cell wall without any modification of the NH2-terminal portion.     

Purification and characterization of 81K, heat stable calmodulin-binding protein from bovine brain

Heat stable calmodulin-binding protein has been purified from Triton X-100 soluble particulate fraction of bovine brain. Considerable purification was achieved with calmodulin coupled Sepharose 4B affinity chromatography. SDS-PAGE of the purified protein revealed the apparent homogeneity being 92% at Mr 81,000. Isoelectric focusing of purified 81K protein gave isoelectric point of 4.3. The amino acid composition was notable for high contents of acidic amino acids (15.0 mol% of glutamic acid and 8.1 mol% of aspartic acid) and 17.4 mol% of alanine. On alkaline 1 M urea gel electrophoresis, mobility of the purified 81K protein in the presence of Ca2+ and calmodulin became lower than 81K protein alone toward the anode; however, Ca2+ solely did not affect the mobility of this protein. Similarly, S-100 protein and troponin C showed the interaction with 81K protein and a decrease of mobility in the presence of Ca2+ in alkaline urea PAGE. Binding assay of 125I-labeled calmodulin revealed that 81K protein could bind to an equimolar of 125I-calmodulin as apparent dissociation constant (Kd) of 0.65 x 10(-6) M.     

Purification and characterization of the nuclear cytidine 5'-monophosphate N-acetylneuraminic acid synthetase from rat liver.

N-Acetylneuraminic acid cytidylyltransferase (EC 2.7.7.43) (CAMP-NeuAc synthetase) from rat liver catalyzes the formation of cytidine monophosphate N-acetylneuraminic acid from CTP and NeuAc. We have purified this enzyme to apparent homogeneity (241-fold) using gel filtration on Sephacryl S-200 and two types of affinity chromatographies (Reactive Brown-10 Agarose and Blue Sepharose CL-6B columns). The pure enzyme, whose amino acid composition and NH2-terminal amino acid sequence are also established, migrates as a single protein band on non-denaturing polyacrylamide gel electrophoresis. The molecular mass of the native enzyme, estimated by gel filtration, was 116 +/- 2 kDa whereas its Mr in sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 58 +/- 1 kDa. CMP-NeuAc synthetase requires Mg2+ for catalysis although this ion can be replaced by Mn2+, Ca2+, or Co2+. The optimal pH was 8.0 in the presence of 10 mM Mg2+ and 5 mM dithiothreitol. The apparent Km for CTP and NeuAc are 1.5 and 1.3 mM, respectively. The enzyme also converts N-glycolylneuraminic acid to its corresponding CMP-sialic acid (Km, 2.6 mM), whereas CMP-NeuAc, high CTP concentrations, and other nucleotides (CDP, CMP, ATP, UTP, GTP, and TTP) inhibited the enzyme to different extents.     

A modified procedure for the purification of clostridial collagenase.

A method is described for the purification of clostridial collagenase from a crude enzyme preparation employing cation exchange chromatography on SP Sephadex, anion exchange chromatography on DEAE cellulose and gel filtration on Sephacryl S-200. Emphasis was placed on purity using continuous shallow gradients for the ion exchange separations to increase resolution and monitoring eluates both with respect to ultraviolet light absorption at 230 nm and analytical disc gel acrylamide electrophoresis. In addition, protein fractions were assayed for collagenolytic and non-specific proteolytic activity. The purity of the final preparation was assessed by acrylamide electrophoresis, gel filtration and amino acid analysis. The isolated enzyme hydrolyzed between 30 and 40% of rat tail tendon collagen in 1 h at 37 degrees C and lacked measurable trypsin or elastase-like activity.     

WATER-SOLUBLE AND PENTANOL-EXTRACTABLE PROTEINS IN HUMAN BRAIN NORMAL TISSUE AND HUMAN BRAIN TUMOURS, WITH SPECIAL REFERENCE TO S-100 PROTEIN

Disc electrophoretic separation of water-soluble and pentanol-extractable protein from normal human brain and human brain tumours (glioblastoma, neurinoma and medulloblastoma) on 10 per cent polyacrylamide gels showed minor differences between tissues. After disc electrophoresis ependymomal tumour cells contained high concentrations of a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein. After electrophoresis of normal brain grey matter in a continuous buffer system, a rapidly migrating anodic protein fraction which was immunologically distinct from S-100 protein was found, and this protein fraction had a similar relative mobility to that of ependymomal tumour cells. This protein fraction was present to a low extent in human normal white matter, but absent from neurinoma and glioblastoma. In a continuous buffer system at least two separable protein fractions, immunologically equivalent to S-100 protein, were observed in normal human brain. The more anodic of these two fractions was shown to be present in relatively high amounts in neurinomas, and may be of Schwann cell origin. Additional S-100 protein could be extracted from residual material remaining after removal of water-soluble proteins; 2.8-10 per cent of the water-soluble S-100 in normal material, and 0.1-0.6 per cent of that present in tumour material, was extractable from the water-insoluble residue by pentanol.     

Isolation and physico-chemical properties of lactoferrin from swine neutrophils

Lactoferrin, a non-heme iron-binding protein was isolated from pig neutrophils. The purification procedure included initial extraction of the protein in the presence of cetyltrimethylammonium bromide followed by chromatography on carboxymethyl-cellulose and Sephadex G-100. The thus obtained protein was found to be homogeneous on polyacrylamide gel (PAAG) electrophoresis at acidic values of pH. PAAG electrophoresis in the presence of sodium dodecyl sulfate revealed a single component with a molecular weight of 75 000-80 000. The resulting protein is capable of binding two atoms of iron molecule. The absorbance spectra for the pig neutrophil lactoferrin are identical to those for cow milk lactoferrin in the visible region and have a maximum at 465 nm. The amino acid composition of pig lactoferrin was determined. Isoelectric focusing of the protein obtained in a PAAG stabilized pH gradient revealed a component with pI of about 6.8. A single precipitin line was observed with rabbit antipig lactoferrin when examined by immunodiffusion. No immunological cross-reactions were observed between pig lactoferrin and bovine lactoferrin.     

The heterogeneity of rat high density lipoproteins.

Ubiquitin was first found in nuclei in protein A24 where its carboxyl terminal is covalently bound to histone 2A by an isopeptide linkage (Goldknopf, I. L. and Busch, H. (1977) Proc. Natl. Acad. Sci. USA $\text{74}$, 864–868). Two-dimensional polyacrylamide gel electrophoresis of the 0.4 N H₂SO₄ soluble proteins from fractionated rat liver chromatin showed that protein A24 and histones H1, H2A, H2B, H3 and H4 were present in fractions P1 and P2 and markedly diminished in relative amounts in fraction S2. Conversely, a spot designated Ub was found in fraction S2 along with an increased amount and number of non-histone proteins. The Ub spot was not found in chromatin fractions P1 and P2. Ub was identified as ubiquitin by migration on two-dimensional gels and after purification by preparative polyacrylamide gel electrophoresis by its methionine NH₂-terminal amino acid and its amino acid composition.     

A rapid and simple method for the isolation of S100a0 (αα) protein by high-performance liquid chromatography

A rapid and simple method to isolate S100a₀ protein from the mixture of bovine S100 protein (S100a₀, S100a and S100b) is described. The S100 mixture purified from bovine brain was applied to an anion-exchange column, equilibrated with 50 mM Tris HC1 buffer (pH 8.0) in a high-performance liquid chromatography (HPLC) system. S100a₀, S100a and S100b proteins could be eluted separately from the column, which were identified by the immunoassay method, by the Tris-HC1 buffer containing a linear concentration gradient (0.25–0.4) M of NaCl. Immunoreactive S100a₀ protein was found in two peak fractions, and each S100a₀ fraction could be isolated (S100a₀-1 and S100a₀-2). Both fractions of S100a₀ protein showed a single band at the same position on acrylamide gel electrophoresis, and eluted in a single peak in the same fractions upon gel-filtration column chromatography. There was no significant difference in the amino acid composition between the two S100a₀ fractions. Since the S100a₀-1 fraction aged for several months at 4°C in the presence of 0.1% NaN₃ was found to contain four protein peaks including the fraction corresponding to the S100a₀-2 fraction, the difference between the two S100a₀ fractions is probably due to some modification of amino acid residues in the molecule, which may occur both in vivo and in vitro.     

Purification and characterization of dopamine beta-hydroxylase from bovine adrenal medulla.

A new purification procedure that permits large-scale purification of dopamine beta-hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1% Triton X-100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE-cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis. The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2-terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2-terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2-terminal amino acids do not support the suggestion that dopamine beta-hydroxylase and chromogranin A contain identical peptide chains.     

Subunit structure and amino acid composition of xylose isomerase from Streptomyces albus.

The subunit structure and amino acid composition of xylose isomerase from Streptomyces albus have been examined. A native molecular weight of 165,000 determined by sedimentation equilibrium was reduced to 43,000 when the protein was treated with 6 M guanidine hydrochloride. No further reduction in molecular weight was observed when potential disulfide bridges of xylose isomerase were reduced and alkylated, indicating that the protein was devoid of interchain disulfide bonds. NH2-terminal analysis using [3H]dansyl chloride showed 0.86 residues of methionine per Mr equals 41,500 unit. Analysis of the native protein with an automated protein sequenator revealed the presence of only one degradable polypeptide chain. Fractionation of the soluble tryptic peptides of S-[14C]carboxymethyl xylose isomerase by ion exchange chromatography and one-dimensional paper electrophoresis yielded 37 to 43 peptides. When the acid-insoluble tryptic peptides were dissolved and analyzed using gel filtration techniques, and additional four peptides were found. A unique radioactive tryptic peptide containing S-carboxymethylcysteine was found among the soluble peptides, confirming cysteine as the limiting amino acid residue in the amino acid composition of xylose isomerase. On the basis of its lysine and arginine content, the number of tryptic peptides is consistent with the hypothesis that the native xylose isomerase is a tetramer of four very similar or identical subunits of Mr equals 41,500, associated by noncovalent bonds.     

Isolation and characterization of major intrinsic microsomal membrane proteins.

Treatment of the membrane matrix derived from hepatic microsomes with buffered 1 M urea resulted in the selective extraction of a group of proteins together with a portion of the membrane lipid. Thorough chemical characterization of this fraction has been performed, and the proteins have been fractionated by two different procedures. The first of these, preparative polyacrylamide gel electrophoresis, has produced five highly homogeneous membrane proteins which have been characterized with regard to molecular weight, electrophoretic behavior in five different polyacrylamide systems, NH2 terminus, relative carbohydrate content, isoelectric point, and amino acid composition. The five proteins of this group fell in the molecular weight range of 54,000 to 96,000 and had isoelectric points ranging from pH 4.9 to pH 6.7. Further fractionation of the urea-soluble proteins by gel filtration in a sodium dodecyl sulfate-containing medium resulted in the isolation of four homogeneous molecular weight classes of proteins which have been characterized with respect to various physicochemical parameters. The major membrane glycoprotein (apparent molecular weight, 171,000) was isolated by this procedure and found to contain approximately equal amounts of NH2-terminal glycine and serine. suggesting the presence of at least two polypeptide chains in this molecular weight region. From the urea-insoluble fraction of the membrane comprising approximately 80% of the total protein, five intrinsic polypeptides designated S-5 through S-9 were isolated. S-5 (54,000) and S-6 (49,000) represent the most prominent components in the microsomal membrane, accounting for close to 30% of the total protein. Also isolated and characterized is the smallest membrane protein (S-9), a hydrophobic polypeptide of molecular weight 16,000. All of the urea-insoluble proteins are glycoproteins, and S-7 (35,000) gives the second most intense stain for carbohydrate of all proteins in the microsomal membrane.     

Purification and some physicochemical properties of staphylococcal enterotoxin D.

A method was developed for the isolation of staphylococcal enterotoxin D in highly purified form from cultures of Staphylococcus aureus strain 1151m. The method involves removal of the toxin from the culture supernatant fluid with the ion-exchange resin CG-50 followed by chromatography on carboxymethylcellulose (twice) and by gel filtration on Sephadex G-75 (twice). The purified toxin is homogeneous by polyacrylamide gel and sodium dodecyl sulfate-polyacrylamide gel electrophoresis and double gel diffusion tests. It is a simple, colorless, antigenic protein with an isoelectric point of 7.4 as determined by isoelectric focusing. Its molecular weight was determined to be 27 300 +/- 700 by molecular sieve chromatography on Sephadex G-100 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its serological activity is stable over a wide range of pH values (1.2--10.7). The enterotoxin consists of 236 amino acid residues and contains no free sulfhydryl groups. End-group analysis showed serine to be the NH2-terminal amino acid and lysine to be the COOH-terminal amino acid.     

PURIFICATION AND COMPARISON OF 2'',3''-CYCLIC NUCLEOTIDE 3''-PHOSPHOHYDROLASES FROM BOVINE BRAIN AND SPINAL CORD

The membrane-bound enzyme 2′,3′-cyclic nucleotide 3′-phosphohydrolase has been purified from acetone powders of bovine white matter and spinal cord. Affinity chromatography on AMP-Sepharose has been used as the final step in the chromatographic purifications. The yield was about 3 mg of purified enzyme per 100 g of tissue in each instance. The enzymes from the two sources were indistinguishable by chromatography, gel electrophoresis, and amino acid analysis; the enzyme from spinal cord, however, has shown a specific activity of 225 units/mg compared to 342 units/mg for the enzyme from white matter. Both proteins had a molecular weight of 100,000 by gel filtration and 50,000 by sodium dodecyl sulfate-gel electrophoresis under reducing conditions. The properties of the enzyme, including amino acid composition determined on the purified soluble protein and on the protein purified by sodium dodecyl sulfate-gel electrophoresis, were those of a basic hydrophobic protein.     

ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D

Bovine brain cathepsin D was purified 1774-fold with a 19% recovery by affinity chromatography on immobilized pepstatin. Approximately 2 mg of enzyme protein were isolated from 150 g (wet weight) of bovine brain. The enzyme eluted from gel filtration as a single peak with a molecular weight of 40,000–42,000. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the predominant band migrated with a molecular weight of 48,000: however, less distinct bands were also present in the molecular weight ranges of 31,000 and 13,000. The isolated enzyme had isoelectric points over a range of pH 5–7 with 3 major peaks occurring at pH 5.6, 6.1, and 6.6. The amino acid composition of brain cathepsin D showed substantial differences from that reported for cathepsin D isolated from bovine spleen. Amino-terminal sequence analysis revealed an Asp-Val-lle sequence by Edman degradation. With hemoglobin as the substrate the enzyme had an apparent K, of 60mM.     

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.     

脊髓和周围神经可溶性酸性蛋白质的研究

 利用微型双向电泳、SDS电泳、免疫印迹法、DEAE-Sephadex色谱、高效液相色谱及氨基酸分析等方法,对牛脊髓(中枢神经)和马尾神经(周围神经)的可溶性酸性蛋白质进行了研究。结果表明在牛脊髓和马尾神经中有钙调素(CaM)、S-100蛋白和神经元特异烯醇化酶(NSE)等可溶性酸性蛋白质存在;脊髓中这些酸性蛋白质的含量远较马尾神经为高。     

Purification and molecular characterization of human transcobalamin II

Transcobalamin II (TCII) has been purified from Cohn fraction III of human plasma by batchwise binding to and then elution from carboxymethyl-Sephadex, affinity chromatography using photo-labile aminopropyl cobalamin coupled to activated Sephacryl S-200, and finally chromatography through carboxymethyl cellulose. The yield was approximately 80%. The addition of protease inhibitors in all steps of the purification procedure and extensive washing of the carboxymethyl-Sephadex prior to eluting the TCII minimized degradation of the protein and the final preparation of holo-TCII contained 1 mol of cobalamin/mol of protein. A single polypeptide of 43,000 daltons was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The NH2-terminal 19 amino acids have been determined for human TCII. 12 of the amino acids are homologous with rabbit TCII and six are homologous with human R-binder, but there is no homology with human intrinsic factor.     

Purification and characterization of a new member of the S-100 protein family from human placenta.

A novel Ca(2+)-binding protein which is termed S-100P was purified from human placenta with a hydrophobic column followed by an anion exchange column and reverse phase high performance liquid chromatography (HPLC). Molecular mass of the protein was 10 kDa according to sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Using immunoblotting technique, anti-human calcyclin antibodies did not bind to the S-100P. Isoelectric point of S-100P was pI = 4.6. S-100P did not formed disulfide-linked dimer. Calcium binding ability was proved by UV difference spectrometry, urea/alkaline gel electrophoresis, and 45Ca overlay technique. A ninety amino acid sequence of S-100P was determined. It is 49% identical with human S-100 beta, 38% with human calcyclin, and 37% with human cystic fibrosis antigen.     

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.