Boar acrosin, II: Amino acid composition, amino terminal residue and molecular weight estimations by ultracentrifugation.

The molecular weight of boar acrosin in neutral solution was estimated to be 41000 +/- 1000 by high-speed sedimentation equilibrium analysis. This result is in good agreement with the value found earlier[1] by sodium dodecylsulfate polyacrylamide gel electrophoresis. The sedimentation coefficeint of acrosin obtained by active enzyme centrifugation of partly purified preparations is in accordance with the sedimentation coefficient of the pure preparation estimated by conventional sedimentation velocity analysis. The sedimentation coefficient of acrosin is considerably decreased in slightly acidic solution (pH 4), indicating that changes in the tertiary structure occur upon acidification. The amino acid composition of the acrosin preparation homogeneous by electrophoretic and chromatographic criteria and in sedimentation studies was determined. Valine was found as the unique N-terminal amino acid. However, in microheterogeneous forms of acrosin, alanine and methionine were also detected in end group analysis.     

Molecular cloning and expression of the gene encoding family 19 chitinase from Streptomyces sp. J-13-3

The gene encoding chitinase from Streptomyces sp. (strain J-13-3) was cloned and its nucleotide structure was analyzed. The chitinase consisted of 298 amino acids containing a signal peptides (29 amino acids) and a mature protein (269 amino acids), and had calculated molecular mass of 31,081 Da. The calculated molecular mass (28,229 Da) of the mature protein was almost same as that of the native chitinase determined by matrix-assisted laser desorption ionization time-of-flight mass spectrometer. Comparison of the encoded amino acid sequences with those of other chitinases showed that J-13-3 chitinase was a member of the glycosyl-hydrolase family 19 chitinases and the mature protein had a chitin binding domain (65 amino acids) containing AKWWTQ motif and a catalytic domain (204 amino acids). The J-13-3 strain had a single chitinase gene. The chitinase (298 amino acids) with C-terminal His tag was overexpressed in Escherichia coli BL21(DE3) cells. The recombinant chitinase purified from the cell extract had identical N-terminal amino acid sequence of the mature protein in spite of confirmation of the nucleotide sequence, suggesting that the signal peptide sequence is successfully cut off at the predicted site by signal peptidase from E. coli and will be a useful genetic tool in protein engineering for production of soluble recombinant protein. The optimum temperature and pH ranges of the purified chitinase were at 35-40 degrees C and 5.5-6.0, respectively. The purified chitinase hydrolyzed colloidal chitin and trimer to hexamer of N-acetylglucosamine and also inhibited the hyphal extension of Tricoderma reesei.     

Isolation and partial characterization of a 700 kilodalton human colon carcinoma associated antigen

An antigen of high molecular weight (CA-3) was isolated from the cytosol fraction of GW-39 human colon tumor cells by antibody affinity chromatography. CA-3 was characterized by an acidic pI value of 4.5-4.9, a molecular weight of 700 kilodaltons and a sedimentation coefficient of 13S. It contained all of the commonly occurring amino acids and had an acidic to basic amino acid ratio of 1.4. CA-3 was resistant to dissociation by reducing agents as well as by sodium dodecylsulfate. Quantitation of CA-3 by a radioimmunoassay employing rabbit anti-CA-3 antiserum revealed a marked elevation of CA-3 in the cytosol extracts of human primary colon carcinoma in comparison to normal colon. The molecular properties of CA-3 are compared to those of carcinoembryonic antigen, high molecular weight colon specific antigen CSAp and two other high molecular weight proteins, fibronectin and conglutinin. Colon antigen CA-3 appears to be different from these other molecules in terms of its molecular weight, sedimentation value, isoelectric point and amino acid composition.     

Purification and properties of Bacillus subtilis aspartate transcarbamylase.

Aspartate transcarbamylase from Bacillus subtilis has been purified to apparent homogeneity. A subunit molecular weight of 33,500 +/- 1,000 was obtained from electrophoresis in polyarcylamide gels containing sodium dodecyl sulfate and from sedimentation equilibrium analysis of the protein dissolved in 6 M guanidine hydrochloride. The molecular weight of the native enzyme was determined to be 102,000 +/- 2,000 by sedimentation velocity and sedimentation equilibrium analysis. Aspartate transcarbamylase thus appears to be a trimeric protein; cross-linking with dimethyl suberimidate and electrophoretic analysis confirmed this structure. B. subtilis aspartate transcarbamylase has an amino acid composition quite similar to that of the catalytic subunit from Escherichia coli aspartate transcarbamylase; only the content of four amino acids is substantially different. The denaturated enzyme has one free sulfhydryl group. Aspartate transcarbamylase exhibited Michaelis-Menten kinetics and was neither inhibited nor activated by nucleotides. Several anions stimulated activity 2- to 5-fold. Immunochemical studies indicated very little similarity between B. subtilis and E. coli aspartate transcarbamylase or E. coli aspartate transcarbamylase catalytic subunit.     

Physical parameters and chemical composition of porcine pancreatic elastase II.

Some molecular properties of the elastase II preparation, homogenous in ultracentrifugation, have been determined. The molecular weight is 25 000, the sedimentation coefficient and the diffusion coefficient are 3.69-10(-13) s(-1) and 12.09-10(-7) cm2/s, respectively. The partial specific volume was 0.716 g/cm3, and the axial ratio is 1.95. Elastase II exhibited a considerably lower content of arginine, tyrosine, and valine, and a higher content of proline, serine and conjugated carbohydrates than elastase I. The N-terminal amino acid of the enzyme is leucine, and its isoelectric point was 10.7.     

Purification and characterization of a trypsin inhibitor from Solanum tuberosum.

A trypsin inhibitor isolated from a potato acetone powder has been purified by affinity chromatography. This protein inhibits trypsin mole per mole. To a lesser extent it combines also with chymotrypsin and elastase. For trypsin, K1 = 8 X 10(-7) M. The inhibitor has a single polypeptide chain of 207 amino acid residues. It contains no sugar or free sulfhydryl groups. Its extinction coefficient E2801% = 10.3 and its isoelectric point is 6.9. Its molecular weight is of the order of 21 000-22000, as determined by sedimentation equilbrium, by inhibition experiment or from its amino acid composition. These same techniques, taken together with the single band observed at different pH on polyacrylamide gel electrophoresis, indicate that the protein purified is monodisperse. However, the finding of two N-terminal amino acid residues, leucine and aspartic acid, and the different stoichometry observed during the interaction of the inhibitor, either with trypsin or with chymotrypsin and elastase, raises the possibility that our preparation is contaminated by a polyvalent inhibitor not detectable by physiochemical methods.     

Monoclonal antibody Y13-259 recognizes an epitope of the p21 ras molecule not directly involved in the GTP-binding activity of the protein.

The p21 products of ras proto-oncogenes are GTP-binding proteins with associated GTPase activity. Recent studies have indicated that ras p21 may be required for the initiation of normal cell DNA synthesis, since microinjection of a monoclonal antibody, Y13-259, blocks serum stimulation of DNA synthesis in quiescent cell cultures (L. S. Mulcahy, M.R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We localized the structural domain within the p21 molecule recognized by the Y13-259 monoclonal antibody. By analysis of a series of bacterially expressed p21 deletion mutants, the monoclonal antibody was found to interact with a region between positions 70 and 89 in the p21 amino acid sequence. By comparison of the coding sequences of different p21 proteins recognized by this monoclonal antibody, a highly conserved amino acid region between positions 70 and 81 was found to be the most likely site for the epitope detected by the Y13-259 antibody. This monoclonal antibody was further shown not to interfere directly with in vitro biochemical functions of the molecule, including GTP binding, GTPase, and autokinase activities.     

Purification and physicochemical properties of fatty acid synthetase and acetyl-CoA carboxylase from lactating rabbit mammary gland.

Fatty acid synthetase was purified 13-fold from lactating rabbit mammary glands by a procedure which involved chromatography on DEAE-cellulose, ammonium sulphate precipitation and gel filtration on Sepharose 4B. The preparation was completed within two days and over 100 mg of enzyme was isolated from 100--150 g of mammary tissue, which represented a yield of over 40%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.3 S, the absorption coefficient, A280nm1%, measured refractometrically was 10.0 +/- 0.1, and the amino acid composition was determined. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 252,000 +/- 6,000, and the molecular weight of the native enzyme measured by sedimentation equilibrium was 515,000. These experiments indicate that at the concentrations which exist in mammary tissue (2--4 mg/ml) fatty acid synthetase is a dimer. The purified enzyme did however show a tendency to dissociate to a monomeric 9-9S species on storage for several days or following exposure to a low ionic strength buffer at pH 8.3. There was only a small quantity of alkali labile phosphate (0.2 molecules per subunit) bound covalently to the purified enzyme. Acetyl-CoA carboxylase was purified 300-fold in a 50% yield within 24 h by ammonium sulphate and polyethylene glycol precipitations [Hardie, D.G. and Cohen, P. (1978) FEBS Lett. 91, 1--7]. The preparation was in a state approaching homogeneity as judged by polyacrylamide gel electrophoresis, gel filtration on Sepharose 4B and ultracentrifugal analysis. The sedimentation constant, S20,w, was 50.5 S, the absorption index, A280nm1%, was 14.5 +/- 0.7, and the amino acid composition was determined. The subunit molecular weight of acetyl-CoA carboxylase determined by gel electrophoresis in the presence of sodium dodecyl sulphate was identical to that of fatty acid synthetase (252,000) as shown by electrophoresis of a mixture of the two proteins. The preparations also contained two minor components of molecular weight 235,000 and 225,000, which appear to be derived from the major species of mol. wt 252,000. A large emount of phosphate (3.2 molecules per subunit) was found to be bound covalently to the purified enzyme. The properties of fatty acid synthetase and acetyl-CoA carboxylase are compared to those obtained by other workers.     

Saponins from Steganotaenia araliacea.

Six saponins have been isolated and identified from the leaves of Steganotaenia araliacea. They were identified as 3-O-[beta-D-galactopyranosyl(1----2)-(beta-D-galactopyranosyl (1----3))-beta-D-glucuronopyranosyl]-21-O-tigloyl and -21-O-angeloyl-R1-barrigenol, 3-O-[beta-D-glucopyranosyl(1----2)-(beta-D-xylopyranosyl (1----3))-beta-D-glucuronopyranosyl]-21-O-tigloyl and -21-O-angeloyl-R1-barrigenol, 3-O-[beta-D-glucopyranosyl(1----2)-(beta-D-glucopyranosyl-(1----3))-(alp ha-L- rhamnopyranosyl(1----4))-beta-D-glucopyranosyl] steganogenin and 3-O-[(beta-D-galactopyranosyl(1----2)-beta-D-glucuronopyranosyl]-2 8-O- beta-D-glucopyranosyl olean-12-ene-28-oic acid. Steganogenin is a new 17,22-seco-oleanolic acid derivative. The structures of the saponins were established by analysis of their 1H and 13C NMR spectra with the help of 2D-experiments and by Californium Plasma Desorption Mass Spectrometry.     

Studies on an antineoplastic fraction from human urine. Characterization of the major protein in this fraction.

A fraction has been isolated from human urine which exhibits antiproliferative activity against human tumour cell lines without affecting the growth of several normal diploid cell lines or tumour cells of mouse or hamster origin. The major protein present in this fraction has been characterized and tentatively designated antineoplastic urinary protein (ANUP). An S020,W value of 3.69 S was obtained by sedimentation velocity analysis, and a subunit molecular mass of 16 300 Da was obtained by sedimentation equilibrium and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Centrifugation data also indicated that the protein self-associates. The amino acid analysis of ANUP was consistent with its low pI (4.2) as determined by chromatofocusing analysis. Furthermore, the amino acid composition exhibited some features similar to collagen, as shown by high levels of proline and glycine, the absence of cysteine, and the presence of low levels of hydroxyproline.     

Interaction of RNA with transformed glucocorticoid receptor. II. Identification of the RNA as transfer RNA

An endogenous RNA (designated as PIVB RNA), which is capable of associating with the 4 S glucocorticoid receptor (GR) to generate the 6 S form, has been purified from AtT-20 cells (Ali, M., and Vedeckis, W. V. (1987) J. Biol. Chem., 262, 6771-6777). We describe here the physiochemical properties, GR-RNA interaction characteristics, and the chemical identification of PIVB RNA. 32P-Labeled PIVB RNA was similar to transfer RNA (tRNA) in its sedimentation coefficient (4 S) on sucrose gradients, electrophoretic mobility on formaldehyde-agarose gels, and receptor binding characteristics. The amino acid acceptor activity of PIVB RNA displayed a typical tRNA-dependent saturation curve and was 2-3-fold higher than that of homologous rabbit liver tRNA when tested using rabbit liver aminoacyl-tRNA synthetase. The purified [3H] aminoacyl-PIVB complex was also capable of binding to the 4 S GR to generate the 6 S form. The analysis of PIVB RNA on an acrylamide-urea sequencing gel revealed that it contained a major tRNA of 76 nucleotides and other minor tRNA species of 74 and 78 nucleotides. The identity of the tRNA present in the PIVB RNA was indirectly deduced by analyzing the 3H-amino acids, liberated from the [3H]aminoacyl-PIVB RNA (tRNA) complex, and subsequent analysis on an amino acid analyzer. PIVB RNA mainly contained tRNAArg (51.8%), tRNALys (17.1%), and tRNAHis (9.2%) which together accounted for 78% of the total PIVB tRNA. The remaining 22% of tRNA was contributed by threonine, valine, aspartic acid, alanine, and phenylalanine tRNAs. The GR displayed no species specificity, and tRNA samples from mouse, cow, rabbit, yeast, and Escherichia coli can bind to the mouse 4 S GR to generate the 6 S form. However, PIVB RNA did not affect the sedimentation profiles of albumin, chymotrypsinogen, and histone, indicating that PIVB RNA does not bind to all proteins. Thus, there may exist some specificity both at the level of protein (GR) and the selection of RNA (tRNA). The GR binding to PIVB RNA occurred at low (nM) receptor concentration, and PIVB RNA showed limited capacity to shift 4 S GR to the 6 S form. 22.4 X 10(-11) mol of PIVB RNA can completely shift 4.8 X 10(-13) mol of 4 S GR to 6 S. That is, PIVB RNA has to be in a 500-600-fold excess over the amounts of GR to observe a stable 6 S GR X RNA complex on sucrose gradients. These results conclusively demonstrate that the transformed GR specifically binds to endogenous tRNA.     

Purification and physico-chemical properties of glutamine synthetase from pea chloroplasts]

A highly purified, practically homogeneous glutamine synthetase was isolated from pea leaf chloroplasts. The enzyme purity was assayed by polyacrylamide gel electrophoresis and analytical ultracentrifugation. The sedimentation coefficient is 16,3S. The sedimentation equilibrium analysis showed that the molecular weight of the enzyme is equal to 480 000. The minimal molecular weights of the enzyme as calculated from the data of polyacrylamide gel electrophoresis in the presence of SDS and the amino acid analysis were found to be 62 000 and 60 000, respectively. The enzyme contains a large amount of dicarboxylic and sulfur-containing amino-acids. The N-terminal amino acid is glycine. The isoelectric point for the enzyme lies within the pH range of 4,2-4-4.     

A clottable protein (coagulogen) from amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus). Its isolation and biochemical properties.

A clottable protein, named coagulogen, was highly purified from the amoebocyte lysate of Japanese horseshoe crab (Tachypleus tridentatus) by a method similar to that used for the lysate of Limulus polyphemus amoebocytes. The isolated material gave a single protein band on analytical gel electrophoresis at pH 3.2, gel electrofocusing, and sodium dodecyl sulfate (SDS) gel electrophoresis with or without 2-mercaptoethanol. It was 90 percent coagulable, and the total yield from 10 ml of the amoebocyte lysate was about 40 mg. The sedimentation coefficient of purified coagulogen was 2.6 S and its molecular weight was estimated to be about 15,300 by sedimentation equilibrium analysis. The molecular weight estimated by SDS-gel electrophoretic analysis was 19,500 +/- 1,000. This discrepancy was apparently due to abnormal mobility arising from the basic nature of this protein on electrophoresis. The protein had a high isoelectric point of pH 10.0 +/- 0.2, as measured by the isoelectric focusing technique. It consisted of a total of 132 to 135 amino acid residues and contained high levels of basic amino acids, which accounted for more than 16 per cent of the total amino acid residues. No methionine was detected. High contents of valine, half-cystine, glutamic acid (glutamine), and phenylalanine were found. The N-terminal sequence of the first three residues of the coagulogen was Ala-Asx-Thr, and its C-terminal residues was identified as phenylalanine, indicating that it consists of a single polypeptide chain. It is of interest that the first three N-terminal residues are homologous with those of the Aalpha-chain of non-human primate fibrinogen.     

The isolation and partial characterization of chymotrypsinogen from the pancreas of the ostrich (Struthio camelus)

1. Cationic chymotrypsinogen from the pancreas of the ostrich was purified to homogeneity by sulfuric acid extraction of pancrei, (NH4)2SO4 fractionation and SP-Sephadex C-50 and Sephadex G-100 chromatography. 2. The final preparation was homogeneous when subjected to SDS-PAGE, isoelectric focusing and sedimentation equilibrium centrifugation. The Mmin value obtained from amino acid analysis was 25,572 Da. A mean sedimentation coefficient of 2.575 S was obtained by sedimentation velocity centrifugation. 3. N-terminal analysis by dansylation showed an Ala residue which is the N-terminal of a neochymotrypsinogen. 4. The effects of pH, temperature and inhibitors (LBTI, PMSF, TPCK and DFP) on the chymotryptic activity were examined. A Km-value for ATEE as substrate was found to be 0.57 mM.     

Purification, characterization, and quantitation of the antigen employed in the immunodiffusion test for diagnosis of equine infectious anemia.

Equine infectious anemia (EIA) antigen extracted from the spleen of horses infected with EIA virus was purified by pH treatment, (NH4)2SO4 fractionation and affinity chromatography. The homogeneity of the antigen was indicated by sedimentation rate and sedimentation equilibrium experiments. A S20,w of 0.51 was determined and a molecular weight of 7600 was calculated from sedimentation equilibrium analysis. The amino acid composition of the pure antigen indicated the antigen is an acidic protein. Employing radical immunodiffusion (RID) and pure antigen a method for quantitating antigen content of antigen containing preparations was developed.     

Characterization of T-Even Bacteriophage Substructures: I. Tail Fibers and Tail Tubes

T-even bacteriophages were grown and purified in bulk quantities. The protein coats were disrupted into their component substructures by treatment with 67% dimethyl sulfoxide (DMSO). Tail fibers and tubes were purified on glycerol-CsCl-D(2)O gradients and examined with respect to sedimentation properties, subunit molecular weights, amino acid composition, isoelectric points, and morphology. It was found that intact tail fibers had a sedimentation coefficient of 12 to 13S and that dissociated fibers consisted of three classes of proteins having molecular weights of 150 K +/- 10, 42 K +/- 4, and 28 K +/- 3 daltons. A model was constructed in which the 150-K subunit folded back on itself twice to give a three-stranded rope. Each 150-K subunit then represented a half-fiber and it was proposed that the role of the 42- and 28-K subunits was to hold each half-fiber together as well as serve as a possible link with other substructures. Isoelectric point studies also indicated that there were three different proteins with pI values of 3.5, 5.7, and 8.0. Amino acid analyses indicated that fibers had a composition distinct from other phage substructures. In addition, a striking difference was noted in the content of tryptophan among the phages examined. T4B had three to five times more tryptophan than did T2L, T2H, T4D, and T6. Intact tail tubes had an S(20,w) of 31 to 38S and dissociated tubes consisted of three proteins of molecular weights 57 K +/- 5, 38 K +/- 4, and 25 K +/- 3 daltons. Based on degradation studies with DMSO, it was proposed that these three proteins were arranged in a helical array yielding the tube structure. Isoelectric point studies indicated that there were three major proteins in the tube whose pI values were 5.1, 5.7, and 8.5. No significant differences were observed in the amino acid content of tubes obtained from all the T-even bacteriophages.     

Critical amino acids of p21 protein are located within beta-turns: further evaluation

It has been shown that malignant activation of ras proto-oncogenes was mediated by point mutations which resulted in the single amino acid conversions at positions 12, 13 or 61 of the ras gene products (p21 proteins). By analyzing randomly mutated ras genes, it has been demonstrated that amino acid substitutions at residues 12, 13, 59 and 63 activated p21. Furthermore, it has been shown that residues 16, 116 and 119 in p21 played critical roles in the guanine nucleotide binding and, consequently, the ability of the protein to induce changes characteristic of cellular transformation. By using the protein conformational prediction method of Chou and Fasman, the present work predicts that these critical amino acids, except glutamic acid at position 63, are located within beta-turns. The major "hot spots" for ras activation are codons 12 and 61. The author has predicted in an earlier paper that the single amino acid conversions at positions 12 and 61 would occur at beta-turn conformation consisting of residues 10-13 and 58-61, respectively. In the present study, probabilities of beta-turn occurrence at residues 10-13 or 58-61 of the p21 proteins encoded by various ras genes are compared. The probability for the normal p21 containing glycine as residue 12 is greatest, and the cancer-associated variants show less probabilities. The single amino acid substitutions at position 61 do not cause so decreased probabilities of beta-turn potential at residues 58-61, except the replacement by histidine. Histidine at position 61 is not predicted as occurring within a beta-turn.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of a novel sequence change in the major form of alpha-MSH isolated from the intermediate pituitary of the reptile, Anolis carolinensis.

Intermediate pituitaries of the reptile, Anolis carolinensis, were separately pulse labeled with [3H]Trp and [3H]Tyr. The major form of alpha-MSH was purified by immunoprecipitation and isolated by reverse phase HPLC. Tryptic peptide analysis indicated that the [3H]Trp-labeled C-terminal fragment of Anolis alpha-MSH had the same retention time as mammalian ACTH(9-13) amide; however, the [3H]Tyr-labeled N-terminal fragment did not coelute with either mammalian ACTH(1-8) or N-acetyl-ACTH(1-8). Purification of alpha-MSH from 76 Anolis intermediate pituitaries confirmed that a sequence change had occurred in the N-terminal region of Anolis alpha-MSH. The tissues were acid extracted and purified by Sephadex G-25 chromatography and reverse phase HPLC to yield 4.5 micrograms of purified Anolis alpha-MSH for amino acid composition analysis and automated Edman degradation sequence analysis. The major form of Anolis alpha-MSH is nonacetylated and has the following novel primary sequence: Ser-Tyr-Ala-Met-Glu-His-Phe-Arg-Trp-Gly-Lys-Pro(Val-amide). The presence of Val-amide was verified by immunological analysis, tryptic peptide analysis and amino acid composition analysis.