Changes in the blood vessel wall elastin of spontaneously hypertensive (SHR) rats

It has been demonstrated that both elastin and tropoelastin preparations obtained from aortae of spontaneously hypertensive rats at the stage of established hypertension differ in their amino acid composition from age-matching controls. The differences refer to an increased proportion of polar amino acids, particularly aspartic and glutamic acid (about a two-fold increase compared to the controls) and arginine and tyrosine (1.5 times the control value). On the other hand, this increase is compensated for by a decrease in the valine concentration. Furthermore, direct estimation of the number of val-pro sequence in different elastin preparations indicated a drop from 49.3 to 29.2 per 1,000 residues in normotensive controls and preparations obtained from spontaneously hypertensive rats respectively.     

Physical and chemical characterization of a horse serum carboxylesterase

The serine carboxylesterase from horse serum was characterized by amino acid composition, peptide mapping, molecular and subunit weights, and sequencing of the amino acids around the essential serine residue at the active site. A protocol was developed for using reversed-phase high-performance liquid chromatography as the final step to obtain homogeneous preparations of horse serum carboxylesterase. Amounts sufficient for determining the amino acid composition and for peptide maps were obtained from a partially purified starting material which contained approximately 55% carboxylesterase. The amino acid composition, like the subunit weight (70,800 +/- 1400), was similar to the corresponding values reported for other serine carboxylesterases. However, the amino acid sequence of the tryptic digest fragment containing the essential nucleophilic seryl residue differed significantly from the corresponding sequences of other mammalian serine carboxylesterases.     

Evidence for the identity of hirudin isolated after kidney passage with the starting material

Natural, sulfated and recombinant, non-sulfated hirudin preparations re-isolated from urine after kidney passage were compared with the corresponding administered preparations by reversed-phase high-performance liquid chromatography, and by amino acid sequence and composition analysis. It could be demonstrated that the hirudins were excreted in unmodified form. Natural hirudin could be fractionated by the chromatographic system used into a large number of isoinhibitor forms, some of which were shown to differ from each other in their amino acid sequence.     

Purification and molecular characterization of alcohol dehydrogenase from Drosophila hydei: Conservation in the biochemical features of the enzyme in several species of Drosophila

Alcohol dehydrogenase (ADH) has been purified from Drosophila hydei. Biochemical investigations show that the native enzyme is a dimer consisting of two identical subunits with molecular weight 27,000. The pH optimum values of pure enzyme preparations are 7.9 and 9.4. The pI values are 8.83 and 8.41. Substrate specificities have been characterized. Km(app) values are lowest for propan-2-ol and butan-2-ol and Vmax(app) values are highest for these two substrates. The amino acid composition has been determined. Peptide mapping experiments performed after trypsin digestion of the enzyme allow the identification of 24 peptides. Peptides comprising 64% of the amino acid residues have also been purified by high-performance liquid chromatography (HPLC), and their N-terminal residues and amino acid composition determined. Results are compared with the amino acid sequence of ADH from D. melanogaster Adhs [Thatcher, D. R. (1980). Biochem. J. 187:875]. When data on the biochemical and structural characterization of ADH from D. hydei are compared with data from other species of Drosophila, clear homologies are observed.     

Beta-cyanoalanine synthase: Its purification and basic physico-chemical properties]

A method has been developed for the purification of beta-cyano-L-alanine synthase from etiolated 10-day-old seedlings of blue lupine. High purity preparations of the enzyme were obtained with specific activity exceeding 4000-fold that of the seedling homogenate. Preparations were homogeneous on electrophoresis in polyacrylamide gel. The yield of total activity after purification was approximately 20%. Glutamic acid is the enzyme's only N-terminal amino acid; the molecular weight of the enzyme (both native and treated with 6 M urea) is 52000. The synthase containes one mole of pyridoxal-P per mole of protein; its isoelectric point is situated at pH 4,8. The enzyme's absorption spectrum has a maximum at 410 nm i.e., in the characteristic range of many pyridoxal-U-containing enzymes. Data on the amino acid composition of the enzyme are presented.     

Comparative characterization of proteolytic enzymatic preparations by the extent of hydrolysis of microbial protein

Proteolytic enzymatic preparations obtained from fungi and bacteria were compared by their ability to hydrolyze yeast protein. Fungal preparations were more effective. There was a more than twofold increase in the level of amine nitrogen in cell biomass hydrolysates in comparison to that induced by bacterial preparations. The amino acid composition of these hydrolysates was studied. Amyloprotooryzin, a preparation fromAspergillus oryzae 387, displayed the highest potency in profound protein hydrolysis: the concentration of free amino acids released was 34.7% in comparison to 20.6% induced by amyloryzin and 10.5% by protosubtilin.     

Comparative characteristics of proteolytic enzyme preparations by degree of hydrolysis of a microbial protein

Proteolytic enzymatic preparations obtained from fungi and bacteria were compared by their ability to hydrolyze yeast protein. Fungal preparations were more effective. There was a more than twofold increase in the level of amine nitrogen in cell biomass hydrolysates in of cell biomass comparison to that induced by bacterial preparations. The amino acid composition of these hydrolysates was studied. Amyloprotooryzin, a preparation from Aspergillus oryzae 387, displayed the highest potency in profound protein hydrolysis: the concentration of free amino acids released was 34.7% in comparison to 20.6% induced by amyloryzin and 10.5% by protosubtilin.     

Amino acid composition and zinc content of milk-clotting protease from Bacillus mesentericus strain 76

The milk-clotting protease from Bacillus mesentericus strain 76 is free of carbohydrate and lacks cysteine and cystine. The amino acid composition indicates a single peptide chain with 304 residues. Five amino acids--aspartic acid, threonine, serine, glycine and alanine--represent one-half of the total residues. The enzyme contains 35 aromatic amino acids and 103 ionic amino acids. The observed constant value of the ratio Menzyme:Azinc is 1:1 for active and acid denatured enzyme preparations, indicating that 0.1 M acetic acid causes denaturation of the enzyme but it can not eliminate the zinc ions.     

The major nucleoside triphosphatase of nuclear scaffold is distinct from actin

The major nucleoside triphosphatase of rat liver nuclear scaffold, a 46 kD protein thought to participate in nucleocytoplasmic RNA translocation, is distinct from immunologically-identified scaffold actin on Western blots, has a substantially different amino acid composition, and its enzymatic activity is not affected by anti-actin antibodies. Thus, although the contractile protein actin is found in nuclear scaffold and appears to interact with RNA, it is not associated with the nucleoside triphosphatase activity in such preparations.     

Human plasma carboxypeptidase N. Isolation and characterization.

Human plasma carboxypeptidase N has been purified 2,600-fold from pooled, outdated plasma in a 30% yield. Isolation was accomplished by chromatography on DEAE-cellulose and on a p-aminobenzoyl-L-arginine-Sepharose 6B affinity column. Carbohydrate accounts for 17% of the weight calculated from its amino acid and carbohydrate composition. The enzyme appears to consist of three subunits of Mr = 83,000, 55,000, and 49,000 and contains a significant amount of bound zinc. Purified enzyme preparations are very sensitive to proteolytic degradation but are stable for at least 3 months at 4 degrees.     

Human liver alcohol dehydrogenase: purification, composition, and catalytic features.

Alcohol dehydrogenase has been purified from human liver by affinity chromatography. Ultracentrifugation, Sephadex G-200 chromatography, and amino acid analyses of multiple preparations demonstrate homogeneity of molecular weight. Sodium dodecyl sulfate disc gel electrophoresis reveals a single species of molecular weight 42 000. Based on a molecular weight of 85 000 for the dimer obtained from the amino acid composition and a molar absorptivity of A280nm0.1% = 0.58, the enzyme contains 3.6-4.2 g-atoms of zinc, as determined by emission spectrography, microwave-induced emission, and atomic absorption spectrometry. Inhibition by o-phenanthroline, (ethylenedinitrilo)tetraacetic acid, and alpha,alpha'-bipyridine demonstrates that zinc is essential to enzymatic function. Detailed kinetic analyses using primary alcohols of the homologous series CH3(CH2)nOH, n = 0-5, and the corresponding aldehydes as substrates show that KM values become smaller as n increases. This suggest that hydrophobic interactions play a role in substrate binding. The availability of well-defined preparations of human liver alcohol dehydrogenase now allows definitive genetic and functional studies of this enzyme to elucidate human ethanol metabolism.     

Chemical composition of gas vesicles isolated from Anabaena flos-aquae

Summary Chemical analysis of purified preparations of gas vesicles isolated from the filamentous blue-green alga Anabaena flos-aquae has shown that they are similar in composition to those isolated from the unicellular alga Microcystis aeruginosa by Jones and Jost (1970), being proteinaceous structures, free of lipid and carbohydrate. The gas vesicle protein from Anabaena contains the same 14 amino acids, in broadly the same proportions; in addition there is a small proportion of proline. No sulphur-containing amino acids are present. The empirical formula, suggested by the amino acid ratios, indicates a molecule of 15000 MW.     

Carbohydrate content of insoluble elastins prepared from adult bovine and calf ligamentum nuchae and tropoelastin isolated from copper-deficient procine aorta

1. Insoluble elastin has been prepared by several different methods from adult bovine and calf ligamentum nuchae. Highly purified tropoelastin has been prepared from copper-deficient porcine aorta. 2. Amino acid analyses indicated that all preparations, except that obtained from calf ligamentum nuchae by using an EDTA extraction followed by collagenase digestion (preparation E6), were typical of pure elastin having high concentrations of hydrophobic and low concentrations of hydrophilic amino acids. Preparation E6 was found to contain approx. 40% collagen. 3. The determination and composition of the carbohydrates associated with these preparations is reported. With the exception of preparation E6, the insoluble elastins contained only trace amounts of neutral sugars (0.13-0.35%, w/w) and amino sugars (0.01-0.06%, w/w). The porcine tropoelastin contained virtually no carbohydrate. 4. The results suggest that carbohydrate analyses can yield valuable information about the purity of elastin preparations.     

Isolation of plasma membrane glycoprotein from bovine thymocytes

Glycoprotein was isolated from a purified thymocyte membrane preparation by two methods: lithium diiodosalicylate-phenol extraction and hot 75% ethanol extraction. A higher yield of membrane sialic acid was obtained by the latter method. The preparations had similar apparent molecular weights on sodium dodecyl sulfate gel electrophoresis. Both had similar receptor activities against a panel of hemagglutinins, although the 75% ethanol extract was more active on a weight basis. However, there were significant differences in carbohydrate and amino acid compositions of the two thymocyte extracts. The lithium diiodosalicylate-extracted material had much more glucose, ribose, and glycine than the ethanol extract. The glycoprotein preparations from thymocytes were quite distinct from the glycoprotein of bovine erythrocytes in both composition and receptor properties.     

CELL WALL AND PEPTIDOGLYCAN FROM Lactobacillus fermenti

Cell walls from Lactobacillus fermenti were prepared by differential centrifugation of disrupted cells, with and without trypsin treatment. Approximately 16% of the dry weight of walls was found in a crude trichloroacetic acid extract of the walls; half of this amount remained upon further purification. The purufied extract lacked alanine, but contained substantial amounts of glucosamine. The walls constituted 23 to 33% of the dry weight of the cell. The chemical composition of the various types of wall preparations and of the peptidoglycan from them was studied. The peptidoglycan contained equimolar proportions of glucosamine, muramic acid, l-alanine, d-glutamic acid, and lysine, with somewhat lower proportions of d-aspartic acid and d-alanine. The chemical composition of the peptidoglycan is similar to that reported for three other lactobacilli. In addition to the major constituents of walls and peptidoglycan, there were several minor amino acids. The protein and the amounts of the minor amino acids decreased, and among these threonine and arginine were completely absent from preparations obtained with trypsin. Such preparations contained higher proportions of the d-isomers of alanine, glutamic acid, and aspartic acid as compared to walls and peptidoglycan prepared without trypsin. In addition, walls isolated with the use of trypsin were susceptible to lysozyme, whereas those prepared without trypsin were not. However, the trypsin treatment did not result in any change of the ultrastructure as revealed by electron microscope studies.     

Comparative studies on the soluble proteins of adenovirus type 1

The soluble proteins of adenovirus type 1 have been separated and purified. Their antigenic characteristics were compared in different precipitation experiments performed in electric field. Both two-dimension immune electrophoresis and rocket electrophoresis can successfully be applied for quick diagnostic purposes. Quantitative determination of virus proteins is also feasible by rocket electrophoresis. The isoelectric point values of hexon, penton and fibre were pI 4.55, pI 4.69 and pI 7.07, respectively. The amino acid composition of type 1 adenovirus and its capsid components was determined from separated and purified protein preparations. The former differed in amino acid composition from the tissues used for virus propagation.     

Studies on the structure of the Lp(a) lipoprotein: Isolation and partial characterization of the Lp(a)-specific antigen

Lp(a), a polymorphic lipoprotein of human serum, has been isolated from strongly positive normal sera. The described procedure utilizes a serum pool of several liters. The purified lipoprotein has been investigated in its intact and totally delipidized form. Protein constituents could be characterized as apolipoprotein-B, apolipoprotein-AIII, and the Lp(a)-specific, dissociable polypeptide. Albumin could only be found in some of the preparations. The Lp(a)-specific polypeptide could be separated and characterized by its amino acid composition.     

Qualitative Characterization of Some Peptides Inducing Morphogenesis in the Nematode- Trapping Fungus Arthrobotrys oligospora

Some peptides inducing capture organ formation in Arthrobotrys oligospora were characterized with regard to their amino acid contents. The N-terminal, C-terminal and total amino acids were determined as their dinitrophenyl-derivatives by thin layer chromatography on silica gel in two different solvents. The amino acid composition was further confirmed by gas-liquid chromatography. The peptides investigated had a high proportion of non-polar and aromatic residues. Thus, leucine, isoleucine, valine, proline, and tyrosine were present in all the peptides. In addition, phenyl-alanine, glycine, and alanine occurred in some preparations. Tyrosine, valine, and phenylalanine were found in N-terminal position, and leucine or isoleucine were C-terminals.     

Mammalian brain plasma membranes : Isolation, enzymatic, and chemical characterization

Two variants of a simplified procedure for the isolation of plasma membrane fractions from monkey and rat brains, are described. The preparations show marked enrichments in the marker enzymes, (Na⁺-K⁺) adenosine triphosphatase, acetylcholinesterase, 5′-nucleotidase and adenylate cyclase. Lipid analysis and a protein electrophoretic pattern are presented. An enzymatic check has been made to assess for contamination by other cellular organelles. The amino acid composition of brain membrane proteins show a resemblance to the reported composition of erythrocyte ghost proteins but differ from myelin proteins.     

Characterization of cuticular proteins in the red flour beetle, Tribolium castaneum

We are characterizing the cuticular proteins of Tribolium castaneum (Herbst) (Coleoptera:Tenebrionidae) to determine their role in the function of the exoskeleton. Based on qualitative analyses of cuticles, we focused on the sodium dodecyl sulfate (SDS)-extractable proteins. A small-scale cuticle "mini-prep" procedure was devised that yields preparations virtually free of contaminating cellular material compared to hand-dissected preparations, as assessed by fluorescent microscopy using DAPI to stain nuclei. Proteins extracted in 1% SDS from various developmental stages (last larval instar, pupal, adult) were analyzed by one-dimensional denaturing polyacrylamide gel electrophoresis and by two-dimensional gel electrophoresis. The cuticular protein profiles show both similarities and differences among the stages examined. The amino acid composition, glycosylation, and partial amino acid sequence of several abundant cuticular proteins indicate similarity to cuticular proteins of other insects.