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Biomedical subjects

R Flengsrud

Publications and source records attributed to R Flengsrud.

16 recordsLinked to original sources
PubMed2000

Separation and characterization of basic barley seed proteins.

Basic proteins in barley starchy endosperm from developing seeds were separated by two-dimensional (2-D) nonequilibrium pH gel electrophoresis. Total as well as partial extracts were analyzed. Edman degradation sequencing and immunological detection were performed after transfer of separated proteins onto membranes. Only one protein could be analyzed by N-terminal sequencing of blotted and separated proteins from the total extract. Fractionation of extracts was done using cation exchange chromatography, concanavalin A and heparin affinity chromatography. Internal sequences were determined after in-gel cleavage of proteins using trypsin or cyanogen bromide and separation of the fragments by reversed-phase chromatography or in a gel electrophoresis system for peptide separation. This resulted in a new protocol for obtaining internal sequences from proteins separated by 2-D electrophoresis. A total of 16 sequences, including nine internal sequences, were analyzed, permitting the identification of ten proteins, including five that appeared to have a blocked N-terminus. An additional protein was identified using immunological detection. Three protein sequences remained unidentified. Separated proteins were also analyzed with a glycan detection method.

Acetates↗
PubMed2000

Salmon antithrombin has only three carbohydrate side chains, and shows functional similarities to human beta-antithrombin.

Antithrombin, a major coagulation inhibitor in mammals, has for the first time been cDNA cloned from a fish species. The predicted mature liver antithrombin of Atlantic salmon (Salmo salar) consists of 430 amino acids and shows about 67% sequence identity to mammalian and chicken antithrombins. Due to a single nucleotide replacement, Asn135 of the antithrombin in higher vertebrates is substituted by Asp in the salmon homolog. Hence, in contrast to the vertebrate antithrombins known so far, salmon antithrombin lacks the potential glycosylation site located close to the heparin binding site. The existence of only three N-linked side chains is evidenced by the sequential removal of three carbohydrate chains from salmon antithrombin during timed-digestion with N-glycosidase F. The high heparin binding affinity of the salmon inhibitor, Kd of 2.2 and 48 nM at I = 0.15 and 0.3, respectively, is very similar to that of the minor human isoform beta-antithrombin, which is not glycosylated at Asn135. Furthermore, the invariant third-position Ser137 at this glycosylation site of mammalian and chicken antithrombins is substituted by Thr in the salmon, a replacement that has been shown to induce full glycosylation in human antithrombin. Thus a rapidly reacting pool of antithrombin may have evolved in two different ways: absence of a glycosylation site in lower vertebrates vs. incomplete glycosylation of a part of the circulating antithrombin in higher vertebrates. Salmon antithrombin appears to have three complex oligosaccharide side chains containing sialic acid terminally linked alpha(2-3) to galactose, while trace amounts of Galbeta(1-4)GlcNAc suggest microheterogeneity due to partial loss of sialic acid.

Amino Acid Sequence↗
PubMed1995

Chymotrypsin isoenzymes in Atlantic cod; differences in kinetics and substrate specificity.

Two chymotrypsin isoenzymes, ChT1 and ChT2 from cod pyloric caeca showed different kinetics against both chromogenic peptide and proteinaceous substrates. The enzymes have similar kcat values but ChT1 had substantially lower values for KM compared to ChT2. The enzymes also differed in cleaving site specificity with the oxidised B-chain of bovine insulin as substrate. ChT1 exhibited broader cleavage specificity compared to ChT2. This difference is parallel to the difference between chymotrypsin C and the A and B isotypes in higher animals. Direct N-terminal amino acid sequence analysis of the isoenzymes revealed that the active forms consisted of two polypeptide chains. The A chains were 13 residues long and ChT1 and ChT2 differed in three positions. The Cod enzyme A-chains differed from the A-chains of bovine chymotrypsin in four and five positions, respectively. The N-terminal part of the B-chains of the cod enzymes were nearly identical to that of the bovine A and B isoenzymes.

Amino Acid Sequence↗
PubMed1994

Characterization of production and enzyme properties of an endo-beta-1,4- glucanase from Bacillus subtilis CK-2 isolated from compost soil.

Bacillus subtilis CK-2, isolated from garden organic waste compost, was found to have high hydrolytic activity against carboxymethylcellulose (CMC) due to the secretion of an endo-beta-1,4- glucanase. Enzyme production was related to the sporulation process, and was regulated by the concentration of readily metabolizable carbohydrate in growth medium. Enzyme production did not require CMC or other cellulose containing materials. The endo-beta-1,4-glucanase activity was optimal at pH 5.6-5.8 and at 65 degrees C, and achieved thermal stability up to 55 degrees C. The activity was inhibited by Hg2+. The purified enzyme gave a single band corresponding to a MW of 35.5 kDa on SDS-PAGE, while the Sephadex G-75 chromatography revealed a molecular weight of the active enzyme around 70 kDa, indicating a dimeric form of the active enzyme. The enzyme activity was irreversibly inhibited by SDS. Native PAGE and IEF revealed three different isoelectric forms of the enzyme, all with an identical N-terminal amino-acid sequence.

Amino Acid Sequence↗
PubMed1993

Separation of acidic barley endosperm proteins by two-dimensional electrophoresis.

Proteins in barley aleurone and starchy endosperm from developing seeds were separated by two-dimensional (2-D) electrophoresis according to O'Farrell's method. The effect of different extraction methods on the separation was compared. The two best extraction methods were compared by amino acid analysis of extracts and residues. Some tissue-specific proteins in both tissues are described. Different methods for transfer of separated proteins onto membranes were compared. Amino terminal and internal sequences were determined after transfer of separated proteins or their proteolytic products onto membranes. Amino acid sequence data allowed the identification of 12 different proteins, while 6 remain unidentified and 8 appeared to have a blocked amino terminal.

Acids↗
PubMed1989

A method for two-dimensional electrophoresis of proteins from green plant tissues.

A method of extraction of proteins from green plant tissues for two-dimensional electrophoresis is presented. The method is demonstrated on barley and potato leaves and on spruce needles for use in isoelectric focusing as well as in nonequilibrium pH gradient electrophoresis. The following three-step procedure was used: (i) The tissue was ground in liquid nitrogen and then in a pH 5.0 buffer with thiourea added to inhibit phenoloxidase and polyvinylpyrrolidone to bind phenolic compounds. (ii) The proteins were precipitated with acetone at -20 degrees C. (iii) The proteins were dialyzed. Protease activity was generally not a problem during this procedure.

Chromatography↗
PubMed1979

Purification and some characteristics of the human coagulation factor VII.

1. A purification procedure for factor VII (proconvertin) from human plasma is described. The procedure involves barium sulphate adsorption and elution. DEAE-Sephadex column chromatography, barium sulphate adsorption and elution, heparin-Sepharose column chromatography, preparative disc gel electrophoresis and finally adsorption with antiserum to prothrombin coupled to Sepharose and antiserum to albumin coupled to Sepharose. This procedure gave an approximately 8 . 10(5)-fold purification. 2. The factor VII obtained from the electrophoresis step was mainly a single-chain protein with an apparent molecular weight of 53000 +/- 2000. 3. After the final purification step, additional forms of factor VII, resulting from a fragmentation of the factor VII molecule were detected. 4. Amino acid composition data of the purified factor VII are given. 5. Antisera were raised in two different rabbits by injection of the purified factor VII. The antisera obtained gave a good titre against the factor VII activity and were not directed against any of the three other vitamin-K-dependent coagulation factors.

Amino Acids↗
PubMed1979

Quantitation of some amino-terminal residues in proteins using 3H-labelled dansyl chloride and 14C-labelled amino acids.

A method for quantitation of amino-terminal residues in proteins is presented. The method is a modification of a double isotope-labelling technique, using 3H-labelled dansyl chloride and 14C-labelled amino acids as internal standards. The method is demonstrated on human fibrinogen, horse myoglobin and on mouse myoloma IgA. A linear relationship between the ratio 3H/14C in the separated amino-terminal amino acid of the protein and the amount of protein added in the labelling mixture was obtained with standard deviations of +/- 7.4% +/- 3.4% and +/- 10.3%, respectively. An application of the method is demonstrated by measuring the increase in amino-terminal glycine in fibrinogen following the proteolytic action of thrombin. The method seems to be useful when 0.1 nmol or more of protein is used.

Amino Acids↗
PubMed1975

Purification and some characteristics of the coagulation factor IX from human plasma.

Non-activated coagulation factor IX was purified approx. 10,000-fold from human plasma. The final product was electrophoretically homogeneous and comprised a tingle polypeptide chain with a molecular weight of about 70,000 and a pI of 4.3-4.45. The N-terminal amino acid was glycine. The amino acid and the carbohydrate contents were analysed and a monospecific antiserum to the factor was raised in rabbits.

Amino Acids↗
PubMed1974

The localization of a vitamin K-induced modification in an N-terminal fragment of human prothrombin.

1. The N-terminal fragment (PF-I) split off from prothrombin during coagulation was purified to homogeneity from human serum. 2. The apparent molecular weight is 27000+/-2000 in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, whereas a value of about 19600 is obtained by calculation based on amino acid and carbohydrate analyses. The N-terminal sequence is an Ala-Asx bond. The fragment contains about 16% carbohydrate, binds phospholipids in the presence of Ca(2+) and is adsorbed to BaSO(4). The pK(a) of its BaSO(4)-binding group(s) is 3.1-3.5. 3. By CNBr cleavage of fragment PF-I two peptides (C-1 and C-2) were obtained with molecular weights of about 5900 (C-2) and 12400 (C-1) on the basis of amino acid and carbohydrate analyses. Only the smaller (N-terminal) peptide is adsorbed to BaSO(4) and, since the ability of the whole protein to bind to BaSO(4) is known to be absent in samples obtained from patients treated with vitamin K antagonists, this peptide probably contains the site of a modification to the structure of the protein which occurs during biosynthesis and depends on vitamin K. This peptide does not contain hexosamine or sialic acid.

Amino Acids↗
PubMed1972

Inhibition of the thrombin-fibrinogen reaction by a macromolecular factor from rat liver.

1. The supernatant obtained by centrifugation of a rat liver homogenate at 100000g for 1h contained a heat-labile macromolecular inhibitor of the thrombin-fibrinogen reaction. 2. The inhibitor was purified to electrophoretic homogeneity by repeated preparative polyacrylamide disc electrophoresis. Inhibition was observed with purified inhibitor equivalent to about 1mug of protein/ml. 3. The inhibitor had a pI of 3.50-3.75, a molecular weight (from sodium dodecyl sulphate-polyacrylamide-gel electrophoresis) of 72000+/-3000 and was inactivated by p-hydroxymercuribenzoate or 5,5'-dithiobis-(2-nitrobenzoic acid). 4. Kinetic studies revealed a non-competitive inhibition, with the inhibitor probably acting on the thrombin-fibrinogen complex.

Animals↗