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.

scholarly journals Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments

1969 ◽  
Vol 113 (3) ◽  
pp. 489-499 ◽  
Author(s):  
C. R. Parish ◽  
G. L. Ada
Keyword(s):  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Cyanogen Bromide ◽  
Degradation Products ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Gel Chromatography ◽  
Molecular Weights ◽  
Lysine Residues

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as ∈-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ‘C,D’ complex and a presumed ‘AB’ fragment. 4. The sum of the amino acid analyses of fragments A and B and the ‘C,D’ complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 ∈-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [125I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ‘C,D’ complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B–A–D–C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

scholarly journals The susceptibility of muscle phosphorylases a and b to digestion by a neutral proteinase from rat intestinal muscle. Comparison with the effects produced by pancreatic trypsin and chymotrypsin

1978 ◽  
Vol 175 (1) ◽  
pp. 105-113 ◽  
Author(s):  
Ian T. Carney ◽  
Robert J. Beynon ◽  
John Kay ◽  
Nigel Birket
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Pyridoxal Phosphate ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Covalent Modification ◽  
Protective Effects ◽  
Radioactive Label ◽  
Initial Event ◽  
Enzyme Degradation

1. Phosphorylase b was inactivated three times more rapidly than phosphorylase a by a neutral, trypsin-like proteinase from rat intestinal muscle. Digestion of phosphorylase a produced a modified form which was deactivated by AMP. Removal of the pyridoxal phosphate cofactor increased the rate of inactivation of the b form by about 3-fold but the subceptibility of apophosphorylase a was no different from the holo form. 2. The extent of proteolysis of both holoenzyme forms, as guaged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, was limited and similar digestion patterns were obtained in both cases. 3. With 32P-labelled phosphorylase a as substrate, the initial event in the inactivation was the release of a trichloroacetic acid-soluble peptide from the N-terminus of the enzyme, leaving the original 100000 subunit form essentially unchanged. Subsequent proteolysis was restricted, producing derivatives of mol.wt. 85000, 70000 and 65000, none of which contained any radioactive label. 4. By treatment of inactivated phosphorylase b with carboxypeptidase B, it was shown that the intestinal muscle proteinase had cleaved approximately 3 -Lys-X and 3 -Arg-X bonds in the polypeptide. 5. The protective effects of various allosteric modulators of phosphorylase on the inactivation of the a and b forms were generally in agreement with the known roles of the modifiers. Glucose increased the susceptibility of phosphorylase a. 6. Inactivation of phosphorylase b by trypsin and chymotrypsin also resulted in limited proteolysis but, in both cases, the digestion patterns obtained on sodium dodecyl sulphate/polyacrylamide gels were different from each other and from the pattern obtained with the intestinal muscle proteinase. 7. Inactivation of phosphorylase b by the muscle proteinase is about 100 times more rapid than the effects produced by trypsin or chymotrypsin when the activities are compared on an equimolar basis. 8. Consideration is given to regulation of the rate of enzyme degradation intracellularly by modulation of the conformation and susceptibility of the enzyme via factors such as covalent modification, allosteric ligands and state of aggregation.

scholarly journals Purification of aromatic l-amino acid decarboxylase from bovine brain with a monoclonal antibody

1988 ◽  
Vol 252 (2) ◽  
pp. 331-335 ◽  
Author(s):  
I Nishigaki ◽  
H Ichinose ◽  
K Tamai ◽  
T Nagatsu
Keyword(s):  
Monoclonal Antibody ◽  
Amino Acid ◽  
Adrenal Medulla ◽  
Ph Dependence ◽  
Polyacrylamide Gel Electrophoresis ◽  
Immunoblot Analysis ◽  
Bovine Brain ◽  
Acid Decarboxylase ◽  
Amino Acid Decarboxylase ◽  
Bovine Adrenal Medulla

Aromatic L-amino acid decarboxylase was purified from bovine brain for the first time by affinity chromatography using a monoclonal antibody to the enzyme, and it was compared with the decarboxylase purified from bovine adrenal medulla by the same procedure. The monoclonal antibody was produced from a hybridoma established for the enzyme highly purified from bovine adrenal medulla. The Mr values of brain and adrenal-medulla enzyme were both estimated to be approx. 100,000 by gel-permeation chromatography. SDS/polyacrylamide-gel electrophoresis revealed a single band with an apparent Mr of 50,000. Western immunoblot analysis showed that the antibody recognized each enzyme. With regard to substrate specificity, pH-dependence and effect of pyridoxal 5′-phosphate as a cofactor, both enzymes were similar.

scholarly journals A study of the subunit structure and the thiol reactivity of bovine liver uridine diphosphate glucose dehydrogenase

1972 ◽  
Vol 129 (4) ◽  
pp. 821-830 ◽  
Author(s):  
P. A. Gainey ◽  
T. C. Pestell ◽  
C. F. Phelps
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Peptide Mapping ◽  
Ph Dependence ◽  
Glucose Dehydrogenase ◽  
Group Analysis ◽  
Subunit Structure ◽  
Uridine Diphosphate ◽  
Thiol Groups ◽  
Thiol Reactivity

1. The amino acid analysis of UDP-glucose dehydrogenase is reported. 2. N-Terminal-group analysis indicates only one type of N-terminal amino acid, methionine, to be present. 3. Peptide ‘mapping’ in conjunction with the amino acid analysis indicates that the subunits of the enzyme are similar if not identical. 4. The various kinetic classes of thiol group were investigated by reaction with 5,5′-dithiobis-(2-nitrobenzoate). 5. NAD+, UDP-glucose and UDP-xylose protect the two rapidly reacting thiol groups of the hexameric enzyme. 6. Inactivation of the enzyme with 5,5′-dithiobis-(2-nitrobenzoate) indicates the involvement of six thiol groups in the maintenance of enzymic activity. 7. The pH-dependence of UDP-xylose inhibition of the enzyme was investigated. 8. The group involved in the binding of UDP-xylose to the protein has a heat of ionization of about 33kJ/mol and a pK of 8.4–8.6. 9. It is suggested that UDP-xylose has a cooperative homotropic effect on the enzyme.

scholarly journals 67 kDa calcimedin, a new Ca2+-binding protein

1986 ◽  
Vol 238 (1) ◽  
pp. 49-54 ◽  
Author(s):  
P B Moore
Keyword(s):  
Skeletal Muscle ◽  
Amino Acid ◽  
Binding Site ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Affinity Column ◽  
Binding Studies ◽  
High Affinity

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.

COVALENT BINDING OF CYCLOOXYGENASE AND LIPOXYGENASE PRODUCTS TO HUMAN PLATELET PROTEINS

1987 ◽  
Author(s):  
M Lecomte ◽  
J M Boeynaems
Keyword(s):  
Covalent Binding ◽  
Polyacrylamide Gel Electrophoresis ◽  
Human Platelets ◽  
Covalent Modification ◽  
Radioactive Material ◽  
Protein Pellet ◽  
Sds Page ◽  
Hydrolysis Of ◽  
Covalent Nature ◽  
Platelet Proteins

Several studies in acellular systems have shown a covalent binding of eicosanoids to proteins (1). We have therefore investigated whether eicosanoids bind covalently to proteins in intact platelets. After incubation of washed human platelets with 14C-arachidonic acid, ethanol precipitation followed by extractions, a small fraction of the radioactivity (0.3%) was tightly bound to the protein pellet. Four criteria suggest the covalent nature of this binding. The radioactivity remained bound after exhaustive extractions with solvents of various polarities, and was not removed by dialysis against SDS-buffer. 15 labelled protein bands could be separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Finally, exhaustive enzymatic hydrolysis of platelet proteins by several proteases liberated an amphipathic radioactive material which had a chromatographic behaviour similar to that of a known peptidolipid, leuko-triene c4 This covalent binding involved products of both cyclooxygenase and lipoxygenase pathways : it was partially inhibited by indomethacin (±40%) and completely abolished by eicosatetraynoic acid. The covalent binding was increased five-fold by dazoxiben, suggesting attachment of prostaglandin endoperoxydes , and by diamide (a glutathione depleting agent) (2) suggesting the involvement of 12-hydro-peroxyeicosatetraenoic acid. Dazoxiben and diamide intensified selectively the labelling of distinct protein bands, separated by SDS-PAGE. Further studies will be needed to evaluate the possible physiological significance of this covalent modification.(1) Wilson, A.G.E. et al. Prostaglandins 18: 409-422, 1979.(2) Bryant, R.W. et al., J. Biol. Chem. 257: 14937-14943, 1982

scholarly journals More Than One Disease Process in Chronic Sinusitis Based on Mucin Fragmentation Patterns and Amino Acid Analysis

2015 ◽  
Vol 2015 ◽  
pp. 1-8
Author(s):  
Mahmoud El-Sayed Ali ◽  
Jeffrey P. Pearson
Keyword(s):  
Amino Acid ◽  
Amino Acid Analysis ◽  
Chronic Sinusitis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Disease Process ◽  
Gel Chromatography ◽  
Sds Page ◽  
Amino Acid Contents ◽  
Fragmentation Patterns

Objective. To characterise fragmentation patterns and amino acid composition of MUC2 and MUC5AC in chronic sinusitis.Methods. Antigenic identity of purified sinus mucins was determined by ELISA. Fragmentation patterns of a MUC5AC rich sample mucin were analysed by Sepharose CL-2B gel chromatography. Samples, divided into one MUC2 rich and one MUC5AC rich group, were subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and their amino acid contents were analysed.Results. Reduction, trypsin digestion, and papain digestion produced progressively smaller mucin species. On SDS-PAGE, digested MUC5AC rich mucin produced four distinct products. Amino acid analysis was characteristic of mucins with high serine, threonine, and proline contents and reduction and proteolysis increased relative proportions of these amino acids. MUC5AC rich mucins contained more protein than MUC2 rich mucins.Conclusion. Sinus mucin fragmentation produced mucin subunits and glycopeptide units of smaller molecular sizes which are likely to have lower viscoelastic properties. Applying this in vivo could alter mucus physical properties and biologic functions. Amino acid contents of MUC2 and MUC5AC mucins are different. This could be contributing to biological properties and functions of sinus mucins. These data suggest that there may be different pathological processes occurring at the cellular level on chronic sinusitis.

scholarly journals Human liver alkaline phosphatase purified by affinity chromatography, ultracentrifugation and polyacrylamide-gel electrophoresis

1976 ◽  
Vol 159 (3) ◽  
pp. 697-705 ◽  
Author(s):  
A L Latner ◽  
A W Hodson
Keyword(s):  
Alkaline Phosphatase ◽  
Amino Acid ◽  
Gel Electrophoresis ◽  
Amino Acid Analysis ◽  
Human Liver ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Nitrophenyl Phosphate ◽  
Hydrolysis Of

A method is presented for the preparation of human liver alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1). The method gives a purification factor of 12.5 × 10(3) over the initial aq. butan-1-ol extract, a recovery of 6.0% and a specific activity for the preparation of 1450-1550 units/mg of protein, 1 unit being defined as the amount of enzyme catalysing the hydrolysis of 1mumol of p-nitrophenyl phosphate/min at 35 degrees C in 0.1 M-2-amino-2-methylpropan-1-ol/HCl buffer, pH 10.5, containing 10mM-p-nitrophenyl phosphate. Homogeneity was studied by ultracentrifugation, by immunoelectrophoresis and by polyacrylamide-gel electrophoresis. A single contaminating protein was present which was less than 5% of the total. Ultracentrifugation and equilibrium-gradient-pore electrophoresis techniques indicated a mol.wt. of 156000 and 160000 respectively. Equilibrium-gradient-pore electrophoresis indicated that the alkaline phosphatase molecule is possibly a dimer, comprising two subunits of about 80000 mol.wt. Amino acid analysis proved remarkably similar to that for alkaline phosphatase from other sources, regardless of species.

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.

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