The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.     

Purification and characterization of a spinach-leaf protein capable of transferring phospholipids from liposomes to mitochondria or chloroplasts

A phospholipid transfer protein has been purified 195-fold from an extract of spinach leaves. This protein is capable of transferring phosphatidylcholine, phosphatidylinositol, phosphatidylglycerol and phosphatidylethanolamine from liposomes to mitochondria. In addition to this protein, a minor part of the total activity was associated with a less purified fraction. The pure protein has an isoelectric point of 9.0 +/- 0.2 determined by a chromatofocusing technique. Electrophoresis on sodium dodecyl sulfate/polyacrylamide gel showed that the protein is homogeneous and has an apparent molecular weight of 9000 +/- 1000, in agreement with the value (8832) calculated from the amino acid composition. This composition is characterized by a high amount of alanine and glycine and by the absence of phenylalanine, whereas arginine, glutamine, histidine and methionine are minor components. The spinach protein is also able to transfer phosphatidylcholine and phosphatidylglycerol from liposomes to intact chloroplasts. This observation reinforces the hypothesis that plastid phospholipids are partly imported from outside the organelle by a transfer process.     

Separation of Component-Polypeptides of Glutenin by Isoelectric Focusing

1. By isoelectric focusing S-cyanoethyl glutenin was observed to be composed of various component-polypeptides having a pI spectrum in a pH range from 6 to 9.

2. During isoelectric focusing a precipitation zone was built up in the column in spite of the presence of 6 m urea. The amount of the precipitate formed was less with S-cyanoethyl glutenin than with S-sulfo glutenin.

3. S-Cyanoethyl glutenin was divided into eight fractions by isoelectric focusing. By starch-gel electrophoresis it was suggested that Fractions I, III and P were mainly composed of a single component.

4. Major N-terminal amino acids of Fractions I, III and P were phenylalanine, glycine and alanine, respectively. In the amino acid composition, distinct differences were observed in the respective fractions, especially in Fraction P. Fraction P showed a much higher content of basic amino acids and a lower content of glutamic acid in comparison with the other two.     

A novel matrix protein family participating in the prismatic layer framework formation of pearl oyster, Pinctada fucata

Understanding the molecular composition and the formation mechanism of shell matrix framework is of great interest for biomineralization in mollusk shell. The cDNAs encoding a novel matrix protein family (KRMP) were cloned from the mantle of pearl oyster, Pinctada fucata. Analysis of the deduced amino acid sequences revealed that KRMP have a high proportion of lysine, glycine, and tyrosine, and their predict isoelectric points are higher than any other identified shell matrix protein to our knowledge. The deduced amino acid sequences of KRMP can be divided into three regions, including an N-terminal signal peptide, a lysine-rich basic region interacting with acidic proteins or CO(3)(2-), and a Gly/Tyr-rich region involved in the protein cross-link via quinone-tanning process. RT-PCR and in situ hybridization demonstrated that KRMP mRNA was specifically expressed in the mantle edge, involved in the prismatic layer formation. Taken together, it seems that KRMP is a matrix protein family participating in the framework formation of prismatic layer.     

Synthesis of Aspartyl Tripeptide Esters in Relation to Structural Features of Sweet Peptides

The distribution of multiple forms of β-amylase in some varieties or species of soybean seeds was examined by the gel isoelectric focusing method. Seven components (1′, 1, 2, 3, 4, 5 and 6) were found. Their respective isoelectric points were 5.07, 5.15, 5.25, 5.40, 5.55, 5.70 and 5.93±0.04. The varieties or species of soybean seeds were separated into two types by their zymograph: the low pI type and high pI type. Component 6 was purified from commercial defatted soybean meal containing all seven components by ion-exchange column chromatography and by gel filtration, and compared with previously purified components 2 and 4. Components 2, 4 and 6 had the same molecular weight and immunological properties but some differences were found in their amino acid compositions and enzymatic properties. The C-terminal amino acid of components 2 and 6 was glycine but that of component 4 was alanine. It was concluded from these results that differences between components 2, 4 and 6 were caused by charged amino acid substitution.     

Isolation and partial characterization of heparan sulphate proteoglycan from the human glomerular basement membrane.

Heparan sulphate proteoglycan was solubilized from human glomerular basement membranes by guanidine extraction and purified by ion-exchange chromatography and gel filtration. The yield of proteoglycan was approx. 2 mg/g of basement membrane. The glycoconjugate had an apparent molecular mass of 200-400 kDa and consisted of about 75% protein and 25% heparan sulphate. The amino acid composition was characterized by a high content of glycine, proline, alanine and glutamic acid. Hydrolysis with trifluoromethanesulphonic acid yielded core proteins of 160 and 110 kDa (and minor bands of 90 and 60 kDa). Alkaline NaBH4 treatment of the proteoglycan released heparan sulphate chains with an average molecular mass of 18 kDa. HNO2 oxidation of these chains yielded oligosaccharides of about 5 kDa, whereas heparitinase digestion resulted in a more complete degradation. The data suggest a clustering of N-sulphate groups in the peripheral regions of the glycosaminoglycan chains. A polyclonal antiserum raised against the intact proteoglycan showed reactivity against the core protein. It stained all basement membranes in an intense linear fashion in immunohistochemical studies on frozen kidney sections from man and various mammalian species.     

Purification and properties of a binding protein for branched-chain amino acids in Pseudomonas aeruginosa.

A binding protein for branched-chain amino acids was purified to a homogeneous state from shock fluid of Pseudomonas aeruginosa PML14. It was a monomeric protein with an apparent molecular weight of 4.3 x 10(4) or 4.0 x 10(4) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or gel filtration, respectively. The isoelectric point was determined to be pH 4.1 by electrofocusing. Amino acid analysis of the protein showed that aspartic acid, glutamic acid, glycine, and alanine were major components and that the protein contained only one residue each of tryptophan and cysteine per molecule. The binding protein contained no sugar. The binding activity of the protein was specific for the branched-chain amino acids. The protein also bound alanine and threonine with lower affinity. The dissociation constants of this protein for leucine, isoleucine, and valine were found to be 0.4, 0.3, and 0.5 microM, respectively. Mutants defective in the production of the binding protein were identified among the mutants deficient in a transport system for branched-chain amino acids (LIV-I). The revertants from these mutants to LIV-I-positive phenotype simultaneously recovered normal levels of the binding protein. These findings suggest strongly the association of the binding protein with the LIV-I transport system.     

Biochemical characterization of five glycoprotein molecules of the porcine zona pellucida

Five glycoproteinmolecules with the molecular masses of 17 000; 38 000; 42 000; 50 000 and 67 000 were purified by high performance liquid chromatography following solubilization of isolated porcine zonae pellucidae by treatment with lithium-3,5-diiodosalicylate. The N-terminal amino acid residues were identified as arginine for 67 000, alanine for 50 000, arginine for 42 000, alanine for 38 000 and histidine for 17 000. The glycopeptides 42 000 and 17 000 were found to be rich on carbohydrates and 67 000 contained 7, 16% sialic acids. The latter moieties were tentatively identified as 5-N-acetylneuraminic acid, 5-N-glycolylneuraminic acid and 5-N-acetyl-7,8,9 tri-O-acetylneuraminic acid. The five components of the zona were resolved by thin layer chromatography in a solvent system of propanol/butanol/HCl (2:1:1) and showed Rf-values of 0.17, 0.42, 0.46, 0.50 and 0.55 respectively. The glycoprotein with the molecular mass of 38 000 possesses spermatozoal receptor properties. This receptor molecule showed a pI of 5.9 upon isoelectric focusing.     

Analysis of rat serum apolipoproteins by isoelectric focusing. II. Studies on the low molecular weight subunits.

The low molecular weight proteins of rat apo HDL and apo VLDL have been isolated and analyzed by the technique of isoelectric focusing. Sephadex fractions from apo HDL (HS-3) and apo VLDL (VS-3) that contain these proteins reveal three major bands with apparent isoelectric points of pH 4.50, 4.67, and 4.74, as well as three minor bands at pH 4.43, 4.57, and 4.61. In addition, apo HDL has a major band at pI of 4.83. DEAE-Cellulose chromatography was used to prepare purified fractions of these components that were characterized by N-terminal analyses and molecular weight determinantions by SDS gel electrophoresis. The major low molecular weight components of apo HDL were focused on a slab gel and the bands were identified as A-II (pI 4.83), C-II (pI 4.74), C-III-0 (pI 4.67), and C-III-3 (pI 4.50). Neuraminidase treatment of apo HDL, followed by isoelectric focusing, suggested that the other bands, which have not previously been reported, may be additional forms of the C-III protein, differing only in their content of sialic acid.     

Diverse roles of glycine residues conserved in photoactive yellow proteins

The role of glycine residues was studied by alanine-scanning mutagenesis using photoactive yellow protein, a structural prototype of PER ARNT SIM domain proteins, as a template. Mutation of glycine located close to the end of beta-strands with dihedral angles disallowed for alanine (Gly-37, Gly-59, Gly-86, and Gly-115) induces destabilization of the protein structure. On the other hand, substitution for Gly-77 and Gly-82, incorporated into the fifth alpha-helix, slows the photocycle by 15-20 times, suggesting that these residues regulate the light-induced structural switch between dark-state structure and signaling-state structure. Most importantly, a significant amount of G29A is in the bleached state and showed a 1000-fold slower photocycle. As O(epsilon2) of the carboxylic acid of Glu-46 is close enough for contact with C(alpha) of Gly-29, alanine mutation perturbs this packing. Fourier transform infrared spectroscopy demonstrated that the C=O(epsilon2) stretching mode of Glu-46 is 6 cm(-1) upshifted in G29A, suggesting that C(alpha) of Gly-29 acts as a proton donor for the C(alpha)-H...O(epsilon2) hydrogen bond with Glu-46, which stabilizes the dark-state structure. During the photocycle, Glu-46 becomes negatively charged by donating a proton to the chromophore, resulting in breakage of this hydrophobic packing and consequent conformational change of the protein.     

Radioimmunoassay of a glycoprotein associated with malignancy

A glycoprotein associated with malignancy was purified from the 0.6M perchloric acid-soluble fraction of serum obtained from cancer patients. The purified glycoprotein contained sialic acid, which was responsible for binding to wheat-germ agglutinin-Sepharose. Gel electrophoresis showed one band with an apparent Mr of 50 000-55 000, and the isoelectric point was 4.4 +/- 0.1. The glycoprotein could be distinguished from carcinoembryonic antigen and alpha-fetoprotein. Iodination of this material with chloramine-T permitted development of a radioimmunoassay.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

PURIFICATION OF GONAD-STIMULATING SUBSTANCE OBTAINED FROM RADIAL NERVES OF THE STARFISH, ASTERIAS AMURENSIS

Purification of starfish gonad-stimulating substance (GSS), which induces shedding of gametes and oocyte maturation, was carried out using lyophilized radial nerves of Asterias amurensis as source material. In the first series of experiments, 1.3 mg of the purified GSS, which induced spawning at a concentration of 0.0096 μg/ml, was isolated from acetone powder of lyophilized radial nerves of 7,360 starfish through several steps of purification procedures consisting of gel-filtrations on Sephadex G–50 and G–25 columns of various sizes and ion-exchange chromatography on DEAE-Sephadex columns by gradient as well as step-wise elution. With this sample, the molecular weight and amino acid composition of GSS were estimated. Another series of experiments, conducted on a similar amount of material with purification procedures which were essentially the same as those of the first series except for employing 2 steps of partition chromatography instead of extensive gel-filtration, gave about 0.1 mg of purified sample which served as material for studies of the amino acid composition and electrophoretic properties of GSS.
The molecular weight of Asterias GSS was found to be about 2,100, as determined with the sedimentation equilibrium method. GSS seemed to consist of the following 22 amino acid residues: aspartic acid (2), threonine (1), serine (6), glutamic acid (1), proline (1), glycine (4), alanine (2), valine (1), isoleucine (1), leucine (1), histidine (1), and ornithine (1). The isoelectric point of GSS was found to be at about pH 4.5 as determined by the isoelectric focusing method.     

α1-Acute-phase globulins of rats. Microheterogeneity after isoelectric focusing

1. Improved resolution of mixtures of alpha(1)-globulins was obtained by the use of isoelectric focusing. 2. Because material recovered after isoelectric focusing in polyacrylamide gels behaved in a manner which suggested interaction with components derived from the gel, isoelectric focusing when used for preparative purposes was done in a matrix of Sephadex G-75. 3. By this means material from the individual bands formed by isoelectric focusing in 6m-urea could be isolated. The stability of these substances was examined by further isoelectric focusing. 4. Analysis of material that had been shown to be homogenous by isoelectric focusing in the absence of urea and of that from several individual bands derived from the same sample by isoelectric focusing in 6m-urea showed different proportions of sialic acid but no change in amino acid composition. 5. In the presence of 6m-urea the isoelectric points found were increased by 0.14-0.25 pH unit. After removal of most of the sialic acid with neuraminidase the increase was 0.36-0.72 pH unit. After treatment with 0.025m-H(2)SO(4) at 80 degrees C for 1h, which removed all the sialic acid, the increase was 0.40-0.87 pH unit. 6. Because removal of all the sialic acid did not decrease the number of bands formed by isoelectric focusing the observed heterogeneity could not be caused entirely by the presence of various proportions of sialic acid.     

Amino acid content of preimplantation rabbit embryos and fluids of the reproductive tract

As a companion to amino acid transport and protein synthetic studies, it was of interest to quantify the amino acid pools in embryos and reproductive tract fluids during preimplantation development. Primary amines in the acid-soluble extracts of embryo and fluid samples were separated by high-performance liquid chromatography, reacted with o-phthalaldehyde, and quantified by fluorescence emission. The amino acid compositions of embryos were like those of corresponding reproductive tract fluids. Taurine was high in eggs and fluids but declined with development, while glycine levels rose. Glycine was highest in concentration in all samples (except the egg), followed by glutamate and alanine, while most other amino acids were consistently of low abundance.     

Fast atom bombardment mass spectrometry and tandem mass spectrometry of biologically active peptidoglycan monomers from Neisseria gonorrhoeae

Fast atom bombardment mass spectrometry (FABMS) and tandem mass spectrometry (MS/MS) were employed to define the structures of Neisseria gonorrhoeae peptidoglycan monomers that were of interest because of their abilities to mediate diverse biological reactions ranging from arthritogenicity to somogenicity. FABMS-determined molecular weights of individual components present in several different enzymatically derived classes of gonococcal monomers revealed that each of these classes was a complex mixture of up to 13 distinct peptidoglycan fragments. These ranged from the predominant disaccharide tetrapeptides possessing reducing or nonreducing 1,6-anhydro-N-acetylmuramic acid ends to relatively minor constituents containing glycine or asparagine in addition to traditional peptidoglycan amino acids, i.e. alanine, glutamic acid, and diaminopimelic acid. FABMS of high performance liquid chromatography-purified monomers yielded some sequence information; however, analysis even of unfractionated peptidoglycan mixtures using a JEOL HX110/HX110 tandem mass spectrometer operating at 10 kV provided unambiguous primary sequence data for the peptidoglycan monomers and defined the position of glycine in four compounds as well as the location of O-acetyl substituents (present on some compounds) on C-6 of the N-acetylmuramic acid residue.     

Purification and Characterization of Chitinase from Bacillus circulans No.4.1

Bacillus circulans No.4.1 produced a high level of chitinase when cells were grown in tryptic soy broth supplemented with 0.3% colloidal chitin at 35°C for 5 days. Purification was carried out by protein precipitation with 80% saturation ammonium sulfate, anion-exchange chromatography with DEAE-Sephacel, and gel filtration with Sephadex G-100, sequentially. The purified enzyme could be demonstrated as a single band on SDS-PAGE, estimated to be 45 kDa. This enzyme could hydrolyze colloidal chitin, purified chitin, glycol chitin, carboxymethyl-chitin (CM-chitin), and 4-methylumbelliferyl-β-D-N,N′-diacetylchitobioside [4-MU-(GlcNAc)₂]. The optimal conditions for this chitinase were pH 8.0 and 40°C. The isoelectric point of the chitinase was 5.1. The amino acid composition of the purified chitinase was determined. The initial 20 amino acid residues of the N-terminal were found to be alanine (A), proline (P), tryptophan (W), asparagine (N), serine (S), lysine (K), glycine (G), asparagine (N), tyrosine (Y), alanine (A), leucine (L), proline (P), tyrosine (Y), tyrosine (Y), arginine (R), glycine (G), alanine (A), tryptophan (W), alanine (A), and valine (V). Knowledge of these properties of chitinase from B. circulans No. 4.1 should be useful in the development of genetically engineered Bacillus sp. as biopesticides. Received: 19 March 1999 / Accepted: 30 April 1999     

Mucins and molluscan calcification. Molecular characterization of mucoperlin, a novel mucin-like protein from the nacreous shell layer of the fan mussel Pinna nobilis (Bivalvia, pteriomorphia)

A cDNA expression library constructed from mantle tissue mRNA of the Mediterranean fan mussel Pinna nobilis was screened with antibodies raised against the acetic acid-soluble shell matrix of the same species. This resulted in the isolation of a 2138-base pair cDNA, containing 13 tandem repeats of 93 base pairs. The deduced protein has a molecular mass of 66.7 kDa and a isoelectric point of 4.8. This protein, which is enriched in serine and proline residues, was overexpressed, purified, and used for producing polyclonal antibodies. Immunological in situ and in vitro tests showed that the protein is localized in the nacreous aragonitic layer of P. nobilis, but not in the calcitic prisms. Because this protein of the nacre of P. nobilis exhibits some mucin-like characteristics, we propose the name mucoperlin. This is the first paper reporting the cloning of a molluscan mucin and the first molecular evidence for the involvement of a mucin in molluscan calcification. This finding corroborates our previous hypothesis that some of the proteinaceous constituents of the molluscan shell matrix would derive from mucins, common to many metazoan lineages of the late Precambrian (Marin, F., Smith, M., Isa, Y., Muyzer, G. and Westbroek, P. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 1554-1559). The adaptation of an ancestral mucin to a new function, the regulation of the mineralization process, may be one of the molecular events, among others, that would explain the simultaneous emergence of organized calcification in many metazoan lineages during the Cambrian explosion.     

The isolation of glycoproteins from bovine achilles tendon and their interaction with collagen

Two glycoprotein fractions, A and B, were isolated from bovine achilles tendon. Glycoprotein A was prepared from a 0.2m-sodium chloride extract and glycoprotein B was isolated from a 3m-magnesium chloride extract. They were free from serum proteins. Glycoprotein A was essentially free of collagen, but glycoprotein B contained about 8% collagen. Both glycoproteins gave several bands on isoelectric focusing. This technique was also used to demonstrate that both glycoprotein fractions interacted strongly with acid-soluble calf skin tropocollagen. It is concluded that these fractions are true components of tendon, and that they may have some function in the macromolecular organization of the tissue.