Amino Acid Composition Features Research Articles

Collagen – The Predominant Protein of the Tectorial Membrane

Evidence is presented that, in contrast to the traditional view, a large proportion of the proteins of the tectorial membrane (TM) consists of collagen, primarily type II: characteristic amino acid composition of TM, including high levels of glycine, hydroxyproline and hydroxylysine; comigration of the main TM protein with appropriate collagen standards in two-dimensional gel electrophoresis; digestion of the main TM protein band following treatment with bacterial collagenase; one- and two-dimensional peptide mapping of cyanogen bromide digests of the TM exhibits patterns characteristic for collagen type II.

Production and characterization of antibodies against murine dentine phosphoprotein.

Experiments were designed to produce and characterize a polyclonal antibody directed against mouse dentine phosphoprotein, the major non-collagenous protein of the dentine extracellular matrix. Dental extracellular matrix proteins from 2-day-postnatal Swiss-Webster-mouse tooth organs were extracted with 0.5 M-acetic acid, followed by 4 M-guanidinium chloride/0.5 M-EDTA. Mouse dentine phosphoprotein yields were further increased by precipitation with 1 M-CaCl2. Final purification was achieved by excising and eluting dentine phosphoprotein polypeptide bands from preparative sodium dodecyl sulphate/urea/polyacrylamide gels. Mouse dentine phosphoprotein is a single component of approx. 72 kDa and has a characteristic amino acid composition of 33% aspartic acid and 55% serine/phosphoserine. A polyclonal antibody was raised in rabbits against purified mouse dentine phosphoprotein and was shown to be monospecific by enzyme-linked immunoabsorbent, dot-immunobinding and 'Western transfer' assays. This antibody was used to detect the expression and localization of dentine phosphoprotein in 1-day-postnatal mouse tooth organs. This antigen was localized intracellularly within the monolayer of odontoblasts, which line the perimeter of the dental papilla mesenchyme, and within the odontoblastic cell processes, which traverse the predentine matrix. Newly forming mineralized dentine matrix was also cross-reactive with the dentine phosphoprotein specific antibody. The non-mineralized predentine matrix did not contain any detectable cross-reactive antigens.

CL glycoprotein is the tissue form of type VI collagen.

CL glycoprotein (CLGP), the 140,000-dalton collagenous glycoprotein, has been isolated from fetal bovine aorta and nuchal ligament, in milligram amounts in its reduced and alkylated form, using a multistage procedure. This material exhibited a characteristic amino acid composition with a consistent ratio of hydroxylysine to hydroxyproline (approximately 1:1). Digestion of CLGP with bacterial collagenase yielded three discrete noncollagenous fragments. Monospecific anti-CLGP antiserum exhibited strong cross-reactivity with the pepsin-resistant polypeptides of type VI collagen. CLGP was also prepared in the unreduced disulfide-bonded form and in a partially reduced form, using brief treatment with cysteine. On treatment with pepsin these preparations yielded resistant peptides corresponding in size to the longer and shorter forms, respectively, of type VI collagen. A slightly larger, soluble form of CLGP (Mr = 150,000) was detected in the media from cultures of aortic smooth muscle cells and nuchal ligament fibroblasts. The evidence indicates that CLGP is the native form in which type VI collagen is present in the tissues and that it consists of three structurally distinct polypeptide chains, each about 140 kDa in mass, which are disulfide bonded into a triple-helical molecule. The CLGP molecules appear to be present in the tissues as dimers and larger aggregates, stabilized by intermolecular disulfide bonding. The distribution of type VI collagen will thus be as described in our earlier immunofluorescence studies with anti-CLGP antiserum (Gibson, M.A., and Cleary, E.G. (1983) Collagen Relat. Res. 3, 469-488).

Lysis protein encoded by plasmid ColA-CA31. Gene sequence and export.

A gene, cal, coding for a polypeptide needed for the release of colicin A from Escherichia coli cells has been identified by transposon insertion. The cal gene was located on the ColA plasmid map adjacent to cai, the gene coding for colicin A immunity protein, and therefore 592 bases downstream from caa, the structural gene for colicin A. Transcription of cal is in the same direction as caa, that is in the opposite direction to cai. Its sequence has been determined and the predicted amino acid composition features a basic N-terminal end followed by a serie of hydrophobic residues similar to the signal sequence in precursors of exported proteins. The C-terminal part also contains a core of hydrophobic residues. The overall amino acid sequence of the cal protein is homologous to that of lytic proteins encoded by the related plasmids pColE1, and pCloDF13. The cal protein has been identified on urea-SDS-polyacrylamide gels by selective labelling with various radioactive amino acids and its synthesis is co-induced with that of colicin A. The cal protein undergoes slow processing with loss of the N-terminal "signal" region and the mature form is released into the medium together with colicin A.

Amino-acid sequences of typical peptides from wheat prolamines and glutelins

Peptides with characteristic amino acid compositions, obtained in high yields from chymotryptic hydrolyzates of prolamines and glutelins from wheat, were sequenced. Examples of typical partial sequences are PQQP, QPQQ, QPFP for the prolamines and SQQQQ, GQ, GQQ, YPTS for the glutelins. Sequences rich in G seem to be of special importance for glutelin.

Presence of connectin-like protein in white blood cells and platelets.

Connectin is known to be an anchoring protein of actomyosin filaments in skeletal muscle. Attempts were made to extract this protein from white blood cells and platelets in order to investigate its properties. The purified connectin-like protein obtained had a rubbery appearance and was insoluble in water and immobile on SDS gel-electrophoresis. Amino acid composition and electron microscopic features of the protein were similar to those of skeletal muscles. FITC-labeled antibody to the protein showed a positive reaction to the membrane fractions of red cells, white cells and platelets. In moving cells fluorescence was observed not only in pseudopod, but also in whole cytoplasm. No changes in the fluorescence patterns were observed with respect to moving stages or cell types. The presence of the protein beneath the cell membrane was also confirmed electron micrographically.

Structure-function Relationships in the Evolution of Elastin

The evolution of the structure of the rubber-like protein elastin, found in connective tissues which are subjected to periodic physiological stress, was studied with respect to its phylogenetic distribution, fiber morphology and arrangement, response to deformation, and amino acid composition. Aortae and other tissues from several vertebrates and invertebrates were examined for the presence of elastin, which was defined on the basis of a characteristic amino acid composition, the presence of the unique crosslinks desmosine and isodesmosine, and by histologic criteria. The protein was present in all vertebrates except the primitive jawless fishes and was absent from all invertebrates which were examined. In addition, the morphology of aortic elastin fibers differed markedly among the vertebrate families. Biochemical analysis revealed increases in both the degree of crosslinking and hydrophobicity in elastins from higher vertebrates (mammals, birds) as compared to those from bony fish. Mammalian elastin displayed an increased tendency toward coacervation (polymerization into aggregated structures) at 37 degrees C and behaved differently from a conventional elastomer when stretched in a microcalorimeter. Selection for an increasingly hydrophobic elastin appears to have paralleled the development of a highly-pressurized, closed circulatory system in homeothermic animals. The data do not support a common genetic origin for elastin and other connective tissue proteins. Significant variations in amino acid composition among aortic elastins from different species, however, indicate that genetically distinct elastin types could have arisen by divergence from a common ancestral gene.

Isolation, purification, and properties of respiratory mucus glycoproteins

The major glycoprotein from human tracheobronchial secretions and from primary explant cultures of human tracheal epithelium has been purified to apparent homogeneity. Mucin was solubilized in buffer and fractionated on Sepharose CL-4B, followed by CsBr density gradient centrifugation of the void volume fraction. High- and low-density fractions were obtained in ratios ranging from 2:1 to 5:1. The high-density (1.46) fraction appeared homogeneous by exclusion chromatography and recentrifugation in CsBr and had an amino acid composition characteristic of a mucin-type structure (threonine, serine, proline, glycine, and alanine comprise two-thirds of the amino acid residues). The carbohydrate, which is nearly 80% by weight, was O-glycosidically linked via GalNAc, sulfated (5.4% by weight), and contained fucose, galactose, glucosamine, galactosamine, and sialic acid. The low-density fraction had an amino acid composition distinct from that of the high-density fraction (threonine, serine, proline, glycine, and alanine comprise 51% of the amino acid residues) and a lower sulfate content. The size distribution of the saccharides in the low-density fraction was similar to that of the high-density fraction; the same sugars were present although the ratios were different. The low-density fraction contained 3 times more noncovalently associated lipid than did the high-density fraction. Several distinct classes of lipids were identified. Neutral lipids (mono-, di-, and triglycerides, cholesterol, and cholesteryl esters) comprised 56% of weight of the total lipid. Glycolipids and phospholipids were also identified. Palmitate (16:0), stearate (18:0), and oleate (18:1) were the major fatty acids in all classes of lipids.

Reduced Soluble Proteins Associated with Maize Endosperm Protein Bodies

Endosperm protein bodies from developing maize were purified by discontinuous sucrose gradient centrifugation and the protein content analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAE). Major proteins detected were zein polypeptides plus a component with Mr 28 000 and a doublet around Mr 58 000. These proteins were present only in the protein body fraction of the sucrose gradient. Treatment of protein bodies with the reducing agent dithiothreitol (DTT) in aqueous buffer dissolved the components with Mr 28 000 and 58 000, plus minor ones, but not zein. The reduced soluble proteins were separated by DEAE-Sephacel chromatography into three fractions: two of these contained the component with Mr 28 000, and the third the components around Mr 58 000 plus minor ones. Proteins from the three fractions had characteristic amino acid compositions, markedly different from those of zein polypeptides. Chymotryptic digestion experiments performed on protein bodies under various conditions, and two-dimensional electrophoresis of proteins from protein bodies suggested that the major zein polypeptides, the protein with Mr 28 000 and the other reduced soluble proteins have different native organizations.

Secondary structure predictions for silkmoth chorion proteins

The complete primary structure of many silkmoth chorion proteins is now known. These proteins are products of two major multigene famlies, A and B. Here we predict the secondary structure of representative A and B chorion proteins, using six different predictive methods. In both families of proteins β-sheet structure predominates. The proteins can be divided into a number of distinct regions or domains, according to the degree of evolutionary constancy in sequence, the amino acid composition and secondary structure features. Both families are characterized by a central ‘core’ region which is highly structured and enriched in valine and alanine. ‘Arms’ are more variable, presumably reflecting protein-specific functions. Cysteines are found predominantly near the extremes of the molecules. β-turns are predicted frequently, and may often connect short anti-parallel β-sheet strands.

Purification and pharmacological characterization of a cardiotoxin-like protein from Formosan banded krait ( Bungarus multicinctus) venom

By ion exchange chromatography followed by gel filtration, a polypeptide toxin was purified from Formosan banded krait ( Bungarus multicinctus) venom. The toxin had a molecular weight of 7000 ± 300 and showed an amino acid composition characteristic of cardiotoxin from cobra venom. The i.p. ld 50 value of the toxin was 2.5 (1.9–3.2) mg per kg mouse. Pharmacological studies showed that the toxin (10 μg/ml) induced contracture in chick and mouse skeletal muscles, depolarized the cell membrane of the mouse diaphragm, arrested the contraction of spontaneously beating atria and the electrically driven ventricle strip of the rat, and caused direct hemolysis of guinea-pig erythrocytes. From these chemical and pharmacological characterizations it was concluded that this toxin has characteristics similar to those of cobra venom cardiotoxins.

Purification, composition, and31P NMR spectroscopic properties of a noncollagenous phosphoprotein isolated from chicken bone matrix

Fractionation of the EDTA-soluble, noncollagenous proteins of the organic matrix of chicken bone by Sephadex G-100 molecular sieving has revealed that the majority of the organic phosphorus is present in two fractions, from one of which a homogeneous phosphoprotein has been isolated. The purified phosphoprotein has an apparent molecular weight of 12,000 and contains both O-phosphoserine and O-phosphothreonine. 31P-NMR spectroscopy demonstrates that all of the organic phosphorus exists in the form of phosphomonoesters which have an average pK2 of 6.8. The phosphoprotein is highly acidic due to its high content of dicarboxylic acids in addition to the presence of organic phosphorus. The characteristic amino acid composition of the phosphoprotein establishes its noncollagenous nature and highlights the differences among bone, dentin, and enamel phosphoproteins. The absence of gamma-carboxyglutamic acid distinguishes it from osteocalcin, the noncollagenous gamma-carboxyglutamic acid-containing peptide of bone matrix.

Isolation and characterization of the amino and carboxyl proximal fragments of the adenosine cyclic 3' ,5'-phosphate receptor protein of Escherichia coli.

The cyclic AMP receptor protein (CRP) is a positive and negative regulatory protein for gene expression in Escherichia coli. The protein has been cleaved proteolytically to determine the relation between CRP structure and function. In the presence of sodium dodecyl sulfate (NaDodSO4), chymotrypsin dissects CRP into two stable fragments of molecular weight 9500 (9.5K) and 13 000 (13K). After removal of NaDodSO4, the two fragments are resolved by Bio-Rex 70 chromatography in 6 M urea. Analyses of the terminal amino acids released from each fragment and cyanogen bromide cleavage products indicate that the 9.5K fragment is amino proximal in CRP while the 13K fragment is carboxyl proximal. Notable features of amino acid composition are the relatively high amount of arginine and methionine in the 13K fragment and the retention in the 9.5K fragment of the two tryptophans present in the CRP subunit. Following isoelectric focusing in 8 M urea, the 9.5K fragment, 22.5K CRP, and 13K fragment migrate to pH 5.5, 8.3, and 10.3, respectively. While CRP is a cAMP-stimulated DNA binding protein, the 13K fragment binds to DNA in the presence and absence of cAMP. The 9.5K fragment associates to form dimers and decamers. These data are consonant with a model in which the DNA binding domain is present in the carboxyl proximal region of CRP while the amino proximal region contains the subunit-subunit interaction sites and much of the cAMP binding domain.

Calmodulin is a major component of extruded trichocysts from Paramecium tetraurelia.

Extruded trichocysts are composed of a family of proteins with molecular weights between 15,000 and 20,000. We have used heat treatment and affinity chromatography on fluphenazine-Sepharose to purify calmodulinlike proteins from whole cells and from extruded trichocysts. The purified protein from trichocysts is indistinguishable from that of whole cells; it is heat-stable, activates brain phosphodiesterase in a Ca++-dependent fashion, changes mobility on SDS polyacrylamide gels in the presence of Ca++, contains 1 mol of trimethyllysine/17 kdaltons, and has the amino acid composition characteristic of calmodulins. Calmodulin is a major component of purified, extruded trichocysts, of which it represents between 1 and 10% by mass. The other proteins of the trichocyst also resemble calmodulin in several properties. Possible roles for calmodulin in the Ca++-activated extrusion of trichocysts is discussed.

Location of an intermolecular crosslink in bovine bone collagen.

A peptide containing 59 amino acid residues with a stoichiometric amount of dihydroxylysinonorleucine (0.7 mole) and hydroxylysinonorleucine (0.2 mole) was isolated from reduced 3H labelled bovine bone collagen sequentially cleaved with CNBr and trypsin. Further cleavage of the isolated crosslinked peptide with periodate yielded a radioactive peptide of 45 residues and a non-radioactive peptide of 16 residues. From the characteristic amino acid composition of these peptides it was deduced that the peptide was derived from an intermolecularly crosslinked region between lysyl or hydroxylysl residues in the carboxy-terminal extension of alpha 1-CB6 (17C residue) and alpha 1-CB5 (87th residue). This finding supports the observation that the alpha 1-CB6 peak was prominent on carboxymethyl cellulose chromatography of the CNBr digest of bone collagen only after limited pepsin digestion, and is consistent with the results obtained from a smaller crosslinked peptide previously isolated from calf bone collagen.

Radioimmunoassay studies of human apolipoprotein E.

This report describes the development and first applications of a sensitive and specific double antibody radioimmunoassay for human apoplipoprotein E (apoE). ApoE was purified from the very low density lipoproteins of hypertriglyceridemic patients by heparin-agarose affinity chromatography, DEAE-cellulose chromatography, and preparative polyacrylamide gel electrophoresis. The purified apoprotein had an amino acid composition characteristic of apoE and resulted in the production of monospecific antisera when injected into rabbits. The radioimmunoassay, which was carried out in the presence of 5 mM sodium decyl sulfate, had a working range of 0.8-12 ng. The withinassay coefficient of variation was 9% and the coefficient of variation for systematic between-assay variability was 3%. Prior delipidation of samples with organic solvents did not alter their immunoreactivity. In 26 normal volunteers, the mean plasma apoE concentration was 36 +/- 13 microgram/ml. Hyperlipidemic patients (n = 68) had higher mean apoE levels. A single patient with type III hyperlipoproteinemia had a plasma apoE level of 664 microgram/ml. The plasma apoE level was independently related to plasma cholesterol and triglyceride levels in a population of 108 normal and nonchylomicronemic hyperlipidemic patients. The multiple correlation coefficient for this relationship was 0.73. Thus, variation in plasma cholesterol and triglyceride concentrations described 53% of the variation in apoE concentrations in this population. The lipoprotein distribution of apoE was investigated by agarose column chromatography and ultracentrifugation of plasma. Agarose column chromatography demonstrated that all or nearly all plasma apoE is associated with lipoproteins. In plasma from normal volunteers and hypercholesterolemic patients, apoE was found in two discrete lipoprotein classes: very low density lipoproteins and a set of lipoprotein particles with size and density characteristics similar to HDL2. In hypertriglyceridemic patients, nearly all apoE was associated with the triglyceride-rich lipoproteins.

Isolation and function of a low molecular weight protein of mung bean embryonic axes

A low molecular weight protein from dry mung bean (Vigna radiata) embryonic axes has been purified to near homogeneity by chromatography on DEAE-cellulose and hydroxylapatite. It shows a molecular weight of about 12,000 in sodium dodecyl sulfate-polyacrylamide gels and a sedimentation coefficient of about 2 S in sucrose gradients. This protein occurs in greater amounts in dry axes than in dry cotyledons, and it dramatically disappears during early germination of the seed. Affinity chromatography tests do not indicate it as a trypsin inhibitor or as a glycoprotein. It is a water-soluble cytoplasmic protein exhibiting an amino acid composition characteristic of storage proteins with a high content of glutamic acid/glutamine. We suggest that it is a low molecular weight storage albumin.

ズワイガニの有機成分 ＩＩ エキス中の遊離アミノ酸

The taste of snow crabs depends on the sex of the crab, the organs eaten, the cooking methods used, etc. We analysed the free amino acids in aqueous methanol extracts from the raw and boiled crabs, and from individual organs of the male and the female. A common feature of the free amino acid composition in the extracts from the muscle was the high con-tents of taurine, glycine, and alanine; their sum accounted for nearly 50% of the total free amino acids. In the extracts from the muscle of raw crabs, a large quantity of arginine was detected, but in those from boiled ones, only a very little was found. On the contrary, only a little citrulline and ornithine were detected in the extracts from the muscles of raw crabs, but a great deal was found in those from boiled ones. In the extracts from the raw and boiled hepatopancreas of both oya-gani and mizu-gani, a large quantity of sarcosine was involved, and in the extracts from the raw hepatopancreas of both, a considerable amount of glutamic acid was detected.

Studies on flavor components in boiled crabs. I. Amino acids and related compounds in the extracts.

In order to elucidate the flavor components characteristic of boiled crabs, the suitable method of extraction was examined and extracts were prepared from different edible parts of five species of crab. The amino acid composition of the extracts was analyzed as the first step of a series of of investigations on the flavor components. A common feature of the free amino acid composition of the leg meat extracts was a very high content of glycine and arginine, with somewhat lower amounts of proline and taurine; the former two comprised about 50% of the total of free amino acids. In the extracts of the hepatopancreas and ovary, the abundant amino acids were taurine, glycine, and arginine. After acid hydrolysis of the extracts, the total of free amino acids showed a 10-20% increase in the leg meat and the ovary and a 50-70% increase in the hepatopancreas, The main amino acids to increase were glutamic and aspartic acids.

Isolation and characterization of type III collagen from bovine cardiac muscle

1. 1. Bovine cardiac muscle collagen was solubilized by limited digestion with pepsin and subsequently fractionated by differential salt precipitation. 2. 2. Chromatographie studies on CM-cellulose and agarose of pepsin-soluble collagen and its salt-precipitated fractions showed it to be composed of type I collagen and a species with a mol. wt of 285,000 daltons, having an amino acid composition characteristic of type III collagen. 3. 3. The molecular structure of the 285,000-dalton component was confirmed as [α1(III)] 3 after reduction with dithiothreitol to a chains having the same amino acid composition. 4. 4. Cyanogen bromide peptides of pepsin-soluble collagen which co-eluted on CM-cellulose and were identified as α1(I)-CB8 and α1(III)-CB8 showed a molar ratio of 2:1 indicating that 25% of the collagen molecules solubilized are [α1(III)] 3.