scholarly journals Some molecular properties of NAD(P)H dehydrogenase from rat liver

1979 ◽  
Vol 181 (1) ◽  
pp. 127-135 ◽  
Author(s):  
Reidar Wallin
Keyword(s):  
Amino Acid ◽  
Rat Liver ◽  
Sodium Dodecyl Sulphate ◽  
Amino Acid Analysis ◽  
Sodium Dodecyl ◽  
Thiol Group ◽  
Native Enzyme ◽  
Sprague Dawley ◽  
Free Thiol Group ◽  
Km Value

NAD(P)H dehydrogenase (EC 1.6.99.2) purified from rat liver cytosol revealed three discrete bands, of mol.wts. about 27000, 18000 and 9000, when subjected to polyacrylamidegel electrophoresis in the presence of sodium dodecyl sulphate. Elution of the bands from the gel and individual re-electrophoresis on separate gels showed that the 27000-mol.wt. band yielded three bands similar to those obtained with the intact enzyme, whereas the 18000-mol.wt. band retained its characteristic mobility. Amino acid analysis of native enzyme and protein extracted from each of the three bands from sodium dodecyl sulphate/polyacrylamide gels suggests that the native enzyme is composed of two subunits and that each subunit consists of two dissimilar non-covalently bound polypeptides, so that altogether the enzyme is composed of four polypeptides, two of mol.wt. 18000 and two of mol.wt. 9000. NAD(P)H dehydrogenase was active over a wide pH range with no sharp optimum. The same Km value for NADH but different values for Vmax. were obtained for the enzyme purified from Sprague–Dawley and Wistar rats. In immunodiffusion, however, the enzymes from the two rat strains showed a reaction of complete identity. NAD(P)H dehydrogenase was effectively inhibited by thiol-blocking reagents, indicating that the activity is dependent on free thiol group(s). By amino acid analysis six cysteine residues were found per mol of enzyme. Guanidino-group- and amino-group-selective reagents had only moderate inactivating effects on the enzyme activity.

scholarly journals Isolation, characterization and N-terminal sequences of the CNBr-cleavage peptides from human complement Factor B. Localization of a free thiol group and a sequence defining the site cleaved by factor D

1982 ◽  
Vol 201 (3) ◽  
pp. 555-567 ◽  
Author(s):  
D L Christie ◽  
J Gagnon
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Alternative Pathway ◽  
Amino Sugar ◽  
Thiol Group ◽  
Complement Factor ◽  
Factor B ◽  
Free Thiol ◽  
Free Thiol Group ◽  
Cnbr Cleavage

Nine CNBr-cleavage peptides from Factor B (a component of the alternative pathway of complement) were isolated. Each was characterized by amino acid analysis and automated Edman degradation. One peptide contained a methionyl bond resistant to cleavage by CNBr. The number of CNBr-cleavage peptides is in agreement with the results of amino acid analysis of Factor B and the fragments Ba and Bb. A total of 358 unique residues were identified from the N-terminal sequences of the CNBr-cleavage peptides. These represent approx. 50% and 60% of the total residues of Factor B and fragment Bb respectively. Alignment of two CNBr-cleavage peptides (CB-VIc and CB-IV) provided a continuous segment of 140 residues. This sequence contained the site cleaved by Factor D to generate the Ba and Bb fragments during the activation of complement. Peptide CB-IV contained a free thiol group at a position corresponding to residue 33 of fragment Bb. Amino sugar analyses of Factor B and of fragments Bb and Ba indicated that all the carbohydrate structures of factor B are N-linked to asparagine through N-acetylglucosamine. The two carbohydrate-attachment sites of the Bb fragment were identified.

scholarly journals Structural and circular-dichroism studies on the interaction between human C̅1-esterase inhibitor and C̅1s

1983 ◽  
Vol 213 (3) ◽  
pp. 617-624 ◽  
Author(s):  
T Nilsson ◽  
I Sjöholm ◽  
B Wiman
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Amino Acid Analysis ◽  
Sodium Dodecyl ◽  
Complement Factor ◽  
Terminal Amino Acid ◽  
C1 Esterase Inhibitor ◽  
Terminal Amino ◽  
Esterase Inhibitor

The reaction between complement factor C1s and C1-esterase inhibitor has been investigated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, N-terminal amino acid analysis and c.d. studies. It is confirmed that a very stable stoichiometric 1:1 complex with a molecular weight of about 180000 is formed, involving the light chain of C1s. On the sodium dodecyl sulphate/polyacrylamide gels a small peptide with a molecular weight of about 5000 can be seen, which may be released from the C-terminal portion of the inhibitor moiety in a manner analogous to that occurring in other similar proteinase-inhibitor reactions. By N-terminal amino acid analysis, a newly formed threonine residue is found in the complex, suggesting that the inhibitor peptide chain is cleaved in the complex between C1s and C1-esterase inhibitor. The stabilizing bond may therefore be an ester bond. C.d. studies of the native C1-esterase inhibitor indicated the presence of about 38% alpha-helix, about 24% beta-structure and about 38% unordered structure. By gradual cleavage of the disulphide bridges under non-denaturating conditions, gradual changes in the c.d. spectra occurred, suggesting loss of ordered secondary structures. The c.d. spectra of the complex between C1s and C1-esterase inhibitor indicate that tryptophan residues are affected by the complex-formation.

scholarly journals The molybdenum–iron protein of Klebsiella pneumoniae nitrogenase. Evidence for non-identical subunits from peptide ‘mapping’

1976 ◽  
Vol 155 (2) ◽  
pp. 383-389 ◽  
Author(s):  
C Kennedy ◽  
R R. Eady ◽  
E Kondorosi ◽  
D K Rekosh
Keyword(s):  
Amino Acid ◽  
Klebsiella Pneumoniae ◽  
Sodium Dodecyl Sulphate ◽  
Azotobacter Vinelandii ◽  
Peptide Mapping ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Amino Acid Content ◽  
Rhizobium Japonicum ◽  
Fe Protein

The molybdenum- and iron-containing protein components of nitrogenase purified from Klebsiella pneumoniae, Azotobacter vinelandii, Azotobacter chroococcum and Rhizobium japonicum bacteroids all gave either one or two protein-staining bands after sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, depending on the commercial brand of sodium dodecyl sulphate used. The single band obtained with K. pneumoniae Mo-Fe protein when some commercial brands of sodium dodecyl sulphate were used in the preparation of the electrode buffer was resolved into two bands by the addition of 0.01% (v/v) dodecanol to the buffer. Protein extracted from the two bands obtained after electrophoresis of K. pneumoniae Mo-Fe protein gave unique and distinct peptide ‘maps’ after tryptic digestion. Undissociated Mo-Fe protein contained both sets of tryptic peptides. These data are consistent with Mo-Fe protein from K. pneumoniae being composed of non-identical subunits. Amino acid analyses of the subunit proteins revealed some clear differences in amino acid content, but the two subunits showed close compositional relatedness, with a different index [Metzer, H., Shapiro, M.B., Mosiman, J.E. & Vinton, J.G. (1968) Nature (London) 219, 1166-1168] of 4.7.

scholarly journals Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure

1983 ◽  
Vol 213 (1) ◽  
pp. 225-234 ◽  
Author(s):  
N Lambert ◽  
R B Freedman
Keyword(s):  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sedimentation Coefficient ◽  
Bovine Liver ◽  
Polyacrylamide Gel ◽  
Protein Disulphide Isomerase

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.

scholarly journals Mannitol-1-phosphate dehydrogenase of Escherichia coli Chemical properties and binding of substrates

1986 ◽  
Vol 239 (2) ◽  
pp. 435-443 ◽  
Author(s):  
T Chase
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Dissociation Constants ◽  
Ph Dependence ◽  
Covalent Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Thiol Group ◽  
Covalent Modification ◽  
Protective Effects ◽  
Phosphate Complex

Mannitol-1-phosphate dehydrogenase was purified to homogeneity, and some chemical and physical properties were examined. The isoelectric point is 4.19. Amino acid analysis and polyacrylamide-gel electrophoresis in presence of SDS indicate a subunit Mr of about 22,000, whereas gel filtration and electrophoresis of the native enzyme indicate an Mr of 45,000. Thus the enzyme is a dimer. Amino acid analysis showed cysteine, tyrosine, histidine and tryptophan to be present in low quantities, one, three, four and four residues per subunit respectively. The zinc content is not significant to activity. The enzyme is inactivated (greater than 99%) by reaction of 5,5′-dithiobis-(2-nitrobenzoate) with the single thiol group; the inactivation rate depends hyperbolically on reagent concentration, indicating non-covalent binding of the reagent before covalent modification. The pH-dependence indicated a pKa greater than 10.5 for the thiol group. Coenzymes (NAD+ and NADH) at saturating concentrations protect completely against reaction with 5,5′-dithiobis-(2-nitrobenzoate), and substrates (mannitol 1-phosphate, fructose 6-phosphate) protect strongly but not completely. These results suggest that the thiol group is near the catalytic site, and indicate that substrates as well as coenzymes bind to free enzyme. Dissociation constants were determined from these protective effects: 0.6 +/- 0.1 microM for NADH, 0.2 +/- 0.03 mM for NAD+, 9 +/- 3 microM for mannitol 1-phosphate, 0.06 +/- 0.03 mM for fructose 6-phosphate. The binding order for reaction thus may be random for mannitol 1-phosphate oxidation, though ordered for fructose 6-phosphate reduction. Coenzyme and substrate binding in the E X NADH-mannitol 1-phosphate complex is weaker than in the binary complexes, though in the E X NADH+-fructose 6-phosphate complex binding is stronger.

The purification and characterization of a phospholipase A2 activity from the 106 000 × g pellet (microsomal fraction) of bovine brain acting on phosphatidylinositol

1982 ◽  
Vol 60 (2) ◽  
pp. 108-117 ◽  
Author(s):  
N. C. C. Gray ◽  
K. P. Strickland
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Microsomal Fraction ◽  
Sodium Dodecyl ◽  
Fatty Acid Distribution ◽  
Phospholipase A ◽  
Strong Inhibition ◽  
Ph Optimum ◽  
Triton X

A phospholipase A2 acting on phosphatidylinositol (PI) has been purified from the 106 000 × g pellet (microsomal fraction) of bovine grey matter. The purification steps included extraction with Triton X-100 (0.05%), ammonium sulfate fractionation (20–50% fraction), consecutive column chromatographic runs on Sephadex G-200 and DEAE-Sephacel, and preparative gel electrophoresis (on 10.5% polyacrylamide gel). These steps achieved a purification of 1614 times. The purified enzyme ran as a single band on sodium dodecyl sulfate (SDS) gel electrophoresis. Molecular weight estimations gave values of 18 300 by SDS gel electrophoresis and 18 521 based on amino acid analysis. Amino acid analysis showed the presence of 173 residues with aspartic acid (46), glutamic acid (26) and glycine (21) being the most abundant. Single residues of cysteine, tyrosine, and arginine were measured. The remaining 11 amino acids were present in amounts ranging from 3 to 11 residues.The purified enzyme had a pH optimum of 7.4, was heat stable (to 70 °C), and was activated by Ca2+ (5 mM). Other divalent cations were either slightly inhibitory (Mg2+ and Mn2+) or strongly inhibitory (Zn2+). The nonionic detergents, Triton X-100 (0.02 to 0.03%) and octyl glucoside (30 mM) showed 70 and 25% stimulations, respectively. Other detergents showed no effect (Cutscum), slight inhibition (G3634A), or strong inhibition (cetyltrimethylammonium bromide). Determination of the apparent Km and Vmax by an Eisthenal–Cornish-Bowden plot gave values of 0.52 mM and 1440 nmol [1-14C]oleic acid min −1∙mg protein −1, respectively, for 1-acyl-2-[1-,14C]oleoyl-sn glycerol-3-phosphoinositol as substrate. The above plot confirmed the presence of a strong inhibition by substrate (i.e., PI) beyond 0.4 mM. The properties of this enzyme and its location (microsomal) make it uniquely different from other phospholipase A2 activities reported for brain. The microsomal location and preference for PI shown by this enzyme lend support to the view that it may function to form lyso-PI in a deacylation–reacylation cycle for altering the fatty acid distribution in PI.

scholarly journals Preparation of polyribosome aminoacyl-transfer ribonucleic acid from the muscle of chick embryos.

1975 ◽  
Vol 147 (3) ◽  
pp. 473-477 ◽  
Author(s):  
M Nwagwu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Sodium Dodecyl Sulphate ◽  
Ribonucleic Acid ◽  
Free Amino Acids ◽  
Free Amino ◽  
Sodium Dodecyl ◽  
Specific Radioactivity ◽  
Chick Embryos ◽  
Aminoacyl Transfer

A procedure for preparing polyribosome aminoacyl-tRNA free from contamination by supernatant aminoacyl-tRNA and free amino acids is described. Important features of the procedure are the use of acidic buffers to help protect the amino acid-tRNA linkage and the inclusion of sodium dodecyl sulphate, to inhibit ribonuclease activity. The specific radioactivity of polyribosome aminoacyl-tRNA is high within 30s and reaches a maximum in 2 1/2 min, well ahead of polyribosome peptides which, as described by Herrmann et al. (1971), attain maximum specific radioactivity in about 10 min.

scholarly journals The amino acid sequence and position of the free thiol group of a short-chain neurotoxin from common-death-adder (Acanthophis antarcticus) venom

1981 ◽  
Vol 199 (1) ◽  
pp. 211-218 ◽  
Author(s):  
H S Kim ◽  
N Tamiya
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Lethal Dose ◽  
Thiol Group ◽  
Cysteine Residue ◽  
Short Chain ◽  
Amino Acid Residues ◽  
The Family ◽  
Free Thiol Group ◽  
The Common

The amino acid sequence of a short-chain neurotoxin Acanthophis antarcticus c (toxin Aa c) from the venom of an Australian elapid snake, the common death adder (Acanthophis antarcticus, subfamily Acanthophiinae) was elucidated. Toxin Aa c is composed of 62 amino acid residues, including eight half-cystine residues and a cysteine residue. The amino acid sequence of toxin Aa c is homologous with those of other short-chain neurotoxins found in snakes of the family Elapidae, especially with those from snakes of the subfamily Hydrophiinae. The single cysteine residue was located in position 4. Toxin Aa c has a lethal dose (LD50) of 0.08 micrograms/g body weight of mouse on intramuscular injection.

Comparison of plasma membrane FABP and mitochondrial isoform of aspartate aminotransferase from rat liver

1993 ◽  
Vol 265 (5) ◽  
pp. G894-G902 ◽  
Author(s):  
D. D. Stump ◽  
S. L. Zhou ◽  
P. D. Berk
Keyword(s):  
Plasma Membrane ◽  
Fatty Acid ◽  
Amino Acid ◽  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Performance ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fatty Acid Binding ◽  
Sds Page

A relationship between plasma membrane fatty acid binding protein (FABPpm), a putative membrane transporter for long-chain fatty acids, and the mitochondrial isoform of aspartate aminotransferase (m-AspAT) has been reported. Accordingly, we have compared the chemical and immunological properties of rat liver m-AspAT with those of rat liver FABPpm isolated by two procedures: 1) detergent solubilization of the membranes followed by purification via fatty acid affinity chromatography (FABP-1) or 2) salt extraction of the membranes and subsequent purification by high-performance liquid chromatography (HPLC; FABP-2). Comparison of the three protein preparations revealed no differences with respect to NH2-terminal amino acid sequence, amino acid composition, peptides from tryptic digests, AspAT enzymatic activity, isoelectric point, mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), retention on five different HPLC columns, and immunoprecipitation and immunoblotting of SDS-PAGE separated proteins with polyclonal antisera. Examination of the proteins by nondenaturing PAGE showed a consistent second band in FABP-1 and FABP-2 not always present in m-AspAT. However, whenever present, this band was immunoreactive with antibodies to both m-AspAT and FABP-1. Hence, FABP-1 and FABP-2 are indistinguishable from one another. They are also at least closely related, if not identical, to m-AspAT.

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