scholarly journals The distribution and partial characterization of the serum apolipoproteins in the guinea pig

1975 ◽  
Vol 149 (2) ◽  
pp. 423-436 ◽  
Author(s):  
M J Chapman ◽  
G L Mills ◽  
J H Ledford
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Human Serum ◽  
Electrophoretic Mobility ◽  
Apolipoprotein B ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Low Density ◽  
Serum Lipoproteins ◽  
Soluble Components

1. Very-low-density (VLD), low-density (LD) and high-density (HD) lipoproteins were isolated by sequential ultracentrifugation from the serum of male guinea pigs fed on a diet containing 3–4% fat. The apoproteins of these lipoproteins (apo-VLD, apo-LD and apo-HD lipoproteins) were studied after delipidation with organic solvents or extraction with tetramethylurea. 2. The major apolipoprotein of LD lipoprotein isolated by gel filtration was found to closely resemble apolipoprotein B of human serum in its chemical and physical properties. Electrophoresis in sodium dodecyl sulphate-polyacrylamide gel showed that this apoprotein consisted of a number of polypeptides. 3. Tetramethylurea precipitated an apoprotein from guinea-pig serum lipoproteins that is probably the apolipoprotein B-like component. This apoprotein accounted for about 80% of the apo-LD lipoprotein, about 55% of the apo-VLD lipoprotein and about 50% of the apo-HD lipoprotein. 4. The distribution of apolipoproteins soluble in tetramethylurea was determined by densitometric scanning of stained polyacrylamide disc gels. 5. A glycine-rich component of high electrophoretic mobility (band I) and a triplet of soluble apolipoproteins (bands II-IV) were present in both VLD and LD lipoprotein classes. These components constituted a higher proportion of the tetramethylurea-soluble apoproteins of VLD lipoprotein (60–80%) than of LD lipoprotein (40–55%). 6. Small amounts (10–15%) of a component of intermediate mobility, which contained traces of half-cystine, were also present in both VLD and LD lipoproteins. 7. A group of soluble components of basic character (bands VI-X), present as minor components of VLD lipoprotein (10–20%), constituted a major proportion (30–45%) of the soluble apoproteins of LD lipoprotein. Two of these apoproteins were rich in lysine, and two of lower electrophoretic mobility were rich in arginine. 8. The pattern of tetramethylurea-soluble apoproteins in HD lipoprotein was distinguished by the presence of two polypeptides of low electrophoretic mobility as its predominant components. One of these components, band VI, resembled the A-I apolipoprotein of man in both its amino acid profile and in its electrophoretic mobility. The second major component, band VI-B, was rich in lysine and resembled the C-I apolipoprotein of man in amino acid composition. 9. The soluble components of bands I and IX were analogous in physicochemical properties to the R-X1 and R-X2 (high-arginine polypeptide) peptides of human serum lipoproteins respectively.

scholarly journals Separation and partial characterization of guinea-pig caseins

1978 ◽  
Vol 173 (2) ◽  
pp. 633-641 ◽  
Author(s):  
R K Craig ◽  
D McIlreavy ◽  
R L Hall
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Guinea Pig ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

scholarly journals Purification and properties of guinea-pig submandibular-gland kallikrein

1983 ◽  
Vol 209 (1) ◽  
pp. 125-134 ◽  
Author(s):  
F Fiedler ◽  
M J C Lemon ◽  
C Hirschauer ◽  
G Leysath ◽  
F Lottspeich ◽  
...  
Keyword(s):  
Amino Acid ◽  
Guinea Pig ◽  
Ethyl Ester ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Amino Acid Residues ◽  
Purification And Properties ◽  
Order Of Magnitude

Guinea-pig submandibular kallikrein has been purified from the glands to electrophoretic homogeneity by conventional procedures. The enzyme is active as a kininogenase, releasing kallidin at a rate of 462 micrograms/min per mg of protein from bovine kininogen, and proved potently hypotensive in the guinea pig and in the dog, properties which indicate its tissue kallikrein nature. The specific activity determined on the substrate N-alpha-benzoyl-L-arginine ethyl ester (11.1 mumol/min per mg of protein) is much lower than that measured with N-acetyl-L-phenylalanyl-L-arginine ethyl ester (483 mumol/min per mg of protein). The latter value is of an order of magnitude comparable with the specific activities of other tissue kallikreins determined with this sensitive kallikrein substrate. The enzyme is a glycoprotein consisting of 237 amino acid residues and containing three to four glucosamine molecules. Its amino acid composition is not identical with that reported for guinea-pig coagulating-gland kallikrein, but is remarkably similar to that of the porcine tissue kallikreins. Apparent Mr values are 29000 (sodium dodecyl sulphate/polyacrylamide-gel electrophoresis) or 34000 (gel filtration). The amino acid sequence of the first 31 N-terminal residues was determined and was found to be closely homologous with that of other tissue kallikreins.

scholarly journals Interactions of C-reactive protein with the complement system. III. Complement-dependent passive hemolysis initiated by CRP.

1975 ◽  
Vol 142 (5) ◽  
pp. 1065-1077 ◽  
Author(s):  
A.P. Osmand ◽  
R.F. Mortensen ◽  
Joan Siegel ◽  
H. Gewurz
Keyword(s):  
Guinea Pig ◽  
Human Serum ◽  
Complete System ◽  
Gel Filtration ◽  
C Reactive Protein ◽  
Inflammatory Reactions ◽  
Reactive Protein ◽  
Rabbit Igg ◽  
The Complement System ◽  
Pig Serum

Interactions of CRP with various substrates in the presence of human serum have been shown to result in efficient activation of C components C1-C5. We now report the ability of CRP to initiate C-dependent hemolysis. For this purpose CRP was isolated by affinity chromatography using pneumococcal CPS and gel filtration; its purity was established by several criteria. Erythrocytes were coated with CPS (E-CPS) and passively sensitized with CRP. C-dependent lysis of these cells was observed upon the addition of suitably absorbed human serum, and the efficiency of hemolysis compared favorably with that initiated by rabbit IgG anti-CPS antibody. CRP also sensitized E-CPS for lysis by guinea pig C; partial lysis was seen when C4-deficient guinea pig serum was used, suggesting that CRP also shares with antibody the ability of CRP to fully activate the C system and provide further evidence for a role for CRP similar to that of antibody in the initiation and modulation of inflammatory reactions via the complete system.

scholarly journals Amino acid composition of the proteins from chylomicrons and human serum lipoproteins

1967 ◽  
Vol 8 (5) ◽  
pp. 463-472 ◽  
Author(s):  
Robert S. Levy ◽  
Ann C. Lynch ◽  
Elizabeth D. McGee ◽  
John W. Mehl
Keyword(s):  
Amino Acid ◽  
Human Serum ◽  
Amino Acid Composition ◽  
Acid Composition ◽  
Serum Lipoproteins

scholarly journals Streptococcus salivarius Fimbriae Are Composed of a Glycoprotein Containing a Repeated Motif Assembled into a Filamentous Nondissociable Structure

2001 ◽  
Vol 183 (9) ◽  
pp. 2724-2732 ◽  
Author(s):  
Céline Lévesque ◽  
Christian Vadeboncoeur ◽  
Fatiha Chandad ◽  
Michel Frenette
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cell Surface ◽  
Polyclonal Antibodies ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Spontaneous Mutant ◽  
Streptococcus Salivarius ◽  
Streptococcal Species ◽  
Repeated Motif

ABSTRACT Streptococcus salivarius, a gram-positive bacterium found in the human oral cavity, expresses flexible peritrichous fimbriae. In this paper, we report purification and partial characterization of S. salivarius fimbriae. Fimbriae were extracted by shearing the cell surface of hyperfimbriated mutant A37 (a spontaneous mutant of S. salivarius ATCC 25975) with glass beads. Preliminary experiments showed that S. salivariusfimbriae did not dissociate when they were incubated at 100°C in the presence of sodium dodecyl sulfate. This characteristic was used to separate them from other cell surface components by successive gel filtration chromatography procedures. Fimbriae with molecular masses ranging from 20 × 106 to 40 × 106Da were purified. Examination of purified fimbriae by electron microscopy revealed the presence of filamentous structures up to 1 μm long and 3 to 4 nm in diameter. Biochemical studies of purified fimbriae and an amino acid sequence analysis of a fimbrial internal peptide revealed that S. salivarius fimbriae were composed of a glycoprotein assembled into a filamentous structure resistant to dissociation. The internal amino acid sequence was composed of a repeated motif of two amino acids alternating with two modified residues: A/X/T-E-Q-M/φ, where X represents a modified amino acid residue and φ represents a blank cycle. Immunolocalization experiments also revealed that the fimbriae were associated with a wheat germ agglutinin-reactive carbohydrate. Immunolabeling experiments with antifimbria polyclonal antibodies showed that antigenically related fimbria-like structures were expressed in two other human oral streptococcal species, Streptococcus mitis andStreptococcus constellatus.

scholarly journals Biochemical characterization including amino acid and carbohydrate compositions of canine and human brain Thy-1 antigen

1981 ◽  
Vol 197 (3) ◽  
pp. 629-636 ◽  
Author(s):  
J L McKenzie ◽  
A K Allen ◽  
J W Fabre
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Human Brain ◽  
Affinity Chromatography ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Antibody Affinity ◽  
Lentil Lectin

Human and canine brain Thy-1 antigens were solubilized in deoxycholate and antigen activity was followed both by conventional absorbed anti-brain xenosera of proven specificity and by mouse monoclonal antibodies to canine and human Thy-1. It is shown that greater than 80% of Thy-1 activity in the dog and man binds to lentil lectin, that the mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of canine and human Thy-1 is identical with that of rat Thy-1 and that the Stokes radius in deoxycholate of canine and human brain Thy-1 is 3.0 nm and 3.25 nm respectively. Both lentil lectin affinity chromatography followed by gel-filtration chromatography on the one hand and monoclonal antibody affinity chromatography on the other gave high degrees of purification of the brain Thy-1 molecule in the dog and man, resulting in single bands staining for both protein and carbohydrate on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis (except for a slight contaminant of higher molecular weight staining for protein but not carbohydrate with human Thy-1 purified by lentil lectin and gel-filtration chromatography). Analysis of canine and human brain Thy-1 purified by monoclonal antibody affinity chromatography with additional gel filtration through Sephadex G-200 showed that these molecules had respectively 38% and 36% carbohydrate. The amino acid and carbohydrate compositions were similar to those previously reported for Thy-1 of the rat and mouse, the main point of interest being the presence in canine and human brain Thy-1 of N-acetylgalactosamine, which has been reported in rat and mouse brain Thy-1 but not in Thy-1 from other tissues.

scholarly journals Biochemical and Genetic Characterization of an FK506-Sensitive Peptidyl Prolyl cis-trans Isomerase from a Thermophilic Archaeon, Methanococcus thermolithotrophicus

1998 ◽  
Vol 180 (2) ◽  
pp. 388-394 ◽  
Author(s):  
Masahiro Furutani ◽  
Toshii Iida ◽  
Shigeyuki Yamano ◽  
Kei Kamino ◽  
Tadashi Maruyama
Keyword(s):  
Amino Acid ◽  
Genomic Library ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ppiase Activity ◽  
Methanococcus Thermolithotrophicus ◽  
Fk506 Binding Protein ◽  
Thermophilic Archaeon

ABSTRACT A peptidyl prolyl cis-trans isomerase (PPIase) was purified from a thermophilic methanogen, Methanococcus thermolithotrophicus. The PPIase activity was inhibited by FK506 but not by cyclosporine. The molecular mass of the purified enzyme was estimated to be 16 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 42 kDa by gel filtration. The enzyme was thermostable, with the half-lives of its activity at 90 and 100°C being 90 and 30 min, respectively. The catalytic efficiencies (k cat/Km ) measured at 15°C for the peptidyl substrates,N-succinyl-Ala-Leu-Pro-Phe-p-nitroanilide andN-succinyl-Ala-Ala-Pro-Phe-p-nitroanilide, were 0.35 and 0.20 μM−1 s−1, respectively, in chymotrypsin-coupled assays. The purified enzyme was sensitive to FK506 and therefore was called MTFK (M. thermolithotrophicusFK506-binding protein). The MTFK gene (462 bp) was cloned from anM. thermolithotrophicus genomic library. The comparison of the amino acid sequence of MTFK with those of other FK506-binding PPIases revealed that MTFK has a 13-amino-acid insertion in the N-terminal region that is unique to thermophilic archaea. The relationship between the thermostable nature of MTFK and its structure is discussed.

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