scholarly journals Studies on the structure and activity of rabbit C1q (a subcomponent of the first component of complement)

1974 ◽  
Vol 143 (2) ◽  
pp. 265-272 ◽  
Author(s):  
Diane M. Lowe ◽  
Kenneth B. M. Reid
Keyword(s):  
Amino Acids ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Haemolytic Activity ◽  
Subunit Structure ◽  
Carboxypeptidase A ◽  
Rapid Loss ◽  
Collagenase Digestion ◽  
Polypeptide Chains ◽  
Structure And Activity

1. The subunit structure of rabbit subcomponent C1q was examined in a previous publication (Reid et al., 1972). The present paper describes some aspects of the structure of the polypeptide chains derived from the molecule. 2. The three polypeptide chains, produced by performic oxidation, of rabbit subcomponent C1q were isolated by ion-exchange chromatography in 8m-urea on DEAE-cellulose. 3. Each chain was found to contain 15–18% glycine and significant amounts of the amino acids hydroxyproline and hydroxylysine. 4. By means of collagenase digestion it was shown that all three chains of rabbit subcomponent C1q contain collagen-like sequences of amino acids which constitute about 40% of each chain. 5. By use of carboxypeptidase A it was established, indirectly, that the collagen-like sequences, in one of the chains, are probably located near, or at, the N-terminal end of the chain. 6. Collagenase digestion and heating at 52°C (but not at 49°C) caused rapid loss of native rabbit subcomponent C1q haemolytic activity.

Separation of Heparin into Subtractions by DEAE-Cellulose Chromatography

1979 ◽  
Vol 42 (05) ◽  
pp. 1452-1459 ◽  
Author(s):  
Robert H Yue ◽  
Toby Starr ◽  
Menard M Gertler
Keyword(s):  
High Activity ◽  
Uronic Acid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Net Charge ◽  
Uronic Acid Content ◽  
Successful Separation ◽  
Salt Gradient ◽  
Structure And Activity ◽  
Low Activity

SummaryCommercial porcine heparin can be separated into three distinct subtractions by using DEAE-cellulose chromatography and a stepped salt gradient. Gram quantities of heparin can be fractionated by this technique. All three heparin subtractions can accelerate the inhibition of thrombin by antithrombin III with different efficiency. The specific activities of the high activity heparin, intermediate activity heparin and low activity heparin are 228 units/mg, 142 units/mg and 95 units/mg, respectively. Both the uronic acid content and the quantity of N-SO4 for all three heparin subfractions have been evaluated. The high activity heparin has the lowest uronic acid and N-SO4 content. The successful separation of commercial heparin into three distinct subfractions by means of ion-exchange chromatography suggests that the net charge on these three heparin components will serve as a model system in the elucidation of the structure and activity relationship to the biological function of heparin.

scholarly journals Beta-Bungarotoxin. Separation of two discrete proteins with different synaptic actions

1978 ◽  
Vol 175 (1) ◽  
pp. 271-279 ◽  
Author(s):  
J MacDermot ◽  
R H Westgaard ◽  
E J Thompson
Keyword(s):  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Ion Exchanger ◽  
Phospholipase A ◽  
Major Protein ◽  
Subunit Structure ◽  
Crude Venom ◽  
Single Class ◽  
Physiological Properties ◽  
Polypeptide Chains

beta-Bungarotoxin, a specific presynaptic blocking agent, was prepared in two stages from the crude venom of Bungarus multicinctus by ion-exchange chromatography on the weakly acidic ion exchanger, CM-Sephadex, and on the strongly acidic ion exchanger, sulphopropyl-Sephadex. By these procedures it was purified to a single protein, which was shown by reduction to contain two polypeptide chains with mol.wts. of less than 15000. During purification of beta-bungarotoxin three other proteins were isolated. Two of these proteins have similar molecular weights, subunit structure and physiological properties to the major protein component. This latter is referred to as beta-bungarotoxin, since it has the same physiological properties as those described for unpurified beta-bungarotoxin by other workers. The first protein has very different physiological effects and biochemical properties from beta-bungarotoxin. This protein has a single class of polypeptide chains with an apparent molecular weight that is lower than the main beta-bungarotoxin protein, and appears to block synaptic transmission by a predominantly postsynaptic effect. It has been suggested [Oberg & Kelly (1976) J. Neurobiol. 7, 129-141] that the action of beta-bungarotoxin depends on its phospholipase A activity; however, in this preparation of the toxin less than 50 muunits of phospholipase A activity were detected (1 unit of activity is the amount of enzyme forming 1 mumol of L-alpha-phosphatidylcholine/min per mg of protein).

Digestive Enzymes of the American Lobster (Homarus americanus)

1970 ◽  
Vol 27 (8) ◽  
pp. 1357-1370 ◽  
Author(s):  
H. Brockerhoff ◽  
R. J. Hoyle ◽  
P. C. Hwang
Keyword(s):  
Gastric Juice ◽  
Proteolytic Activity ◽  
Deae Cellulose ◽  
Homarus Americanus ◽  
Enzymic Activity ◽  
Exchange Chromatography ◽  
Ribonuclease P ◽  
Carboxypeptidase A ◽  
Molecular Weights ◽  
Nitrophenyl Phosphate

Gastric juice of lobster hydrolyzed the following substrates: triolein (enzymic activity: lipase), tributyrin ("lipase"), Azocoll(R) (proteinase), TAME and BAEE ("trypsin"), ATEE ("chymotrypsin"), HPLA ("carboxypeptidase A"), DNA (deoxyribonuclease), RNA (ribonuclease), p-nitrophenyl-phosphate (phosphatase), and p-nitrophenyl-N-acetyl-β-glucosaminide (chitobiase). (The quotation marks signify that the substrate specificities are typical of the quoted enzymes of mammalian or microbial origin. The spectra of specificities, however, of these enzymes and the corresponding enzymes of lobster are not necessarily identical.) Very low activities were found against RBB-starch (amylase) p-nitro-phenyl-α-(and β)-glucopyranoside(α- and β-glucosidase), p-nitrophenyl-β-galactopyranoside (β-galactosidase), and chitin azure (chitinase). No activities corresponding to phospholipase (lecithin), carboxypeptidase B (benzoylglycyl-L-arginine), elastase, glycylglycine dipeptidase, or leucine aminopeptidase were found.Gel filtration of gastric juice proteins on Sephadex(R) G-100 yielded estimates of molecular weights, among them: chitobiase 100,000; phosphatase 80,000; lipase 43,000; DNase 33,000; "trypsin" and ribonuclease 25,000; and two proteinases with optima around pH 4 and 8 and the low molecular weight of 12,500; proteolytic activity at pH 4 was also associated with the molecular weights 25,000 and 50,000.In ion-exchange chromatography, enzymes were eluted from DEAE-cellulose in the following sequence of increasing anionic character: chitobiase, proteinase (pH 4), proteinase (pH 8), lipase, "carboxypeptidase A," DNase, phosphatase, and "trypsin" and "chymotrypsin."Of the principal enzymes, only one (chitobiase) had its optimum at the pH 5 normal for lobster gastric juice. The lipase had an optimal pH around 7, phosphatase near 9, and "trypsin" near 8; proteolytic activity shows a maximum at pH 7–8 and another maximum, unusual for Crustacea, at pH 4.Much of the protein of gastric juice could not be connected with any enzymic activity. The proportion of nonactive protein seemed to increase with starvation of the animals. We found no evidence for the occurrence of zymogens.

scholarly journals The isolation and partial characterization of the major glycoprotein (LGP-I) from the articular lubricating fraction from bovine synovial fluid

1977 ◽  
Vol 161 (3) ◽  
pp. 473-485 ◽  
Author(s):  
D A Swann ◽  
S Sotman ◽  
M Dixon ◽  
C Brooks
Keyword(s):  
Amino Acids ◽  
Synovial Fluid ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Galactose Oxidase ◽  
Sedimentation Equilibrium ◽  
Molar Ratio ◽  
Side Chains ◽  
Total Carbohydrate ◽  
Intact Molecule

The articular lubricating fraction from bovine synovial fluid was prepared by repeated fractionation in three consecutive CsCl density gradients to remove completely traces of hyaluronic acid. The major glycoprotein consituent (LGP-I) was then isolated by repeated gel-permeation chromatography. The yield of the LGP-I component was about 20 mg/litre of synovial fluid. Sedimentation-equilibrium measurements showed that this glycoprotein was homogeneous and the mol.wt. was calculated to be 227500. Amino acids represented 43% (w/w) and carbohydrate constituents 44% (w/w) of the molecule. Threonine, glutamic acid, proline and lysine (224, 127, 242 and 128 residues/1000 residues respectively) were the major amino acids. Galactosamine, galactose and N-acetylneuraminic acid (202, 162 and 114 residues/molecule of LGP-I component respectively) accounted for 98% of the total carbohydrate residues present. Small amounts of mannose and glucosamine (1 and 9mol respectively/mol of LGP-I component) were also present. After treatment of LGP-I component with alkali and NaB3H4 radioactivity was incorporated into alpha-aminobutyric acid and alanine in a molar ratio of 4:1, and radioactive galactosaminitol was isolated by ion-exchange chromatography from a cleaved oligosaccharide fraction. These data demonstrate the presence of threonine and serine -O-GalNAc linkages, but only 25% of the theoretical likages involving threonine were cleaved by a beta-elimination reaction. Digestion of LGP-I component with Pronase followed by chromatography on DEAE-cellulose yielded glycopeptide fractions with a similar amino acid and carbohydrate composition to the intact molecule. Treatment of desialylated and intact LGP-I component with galactose oxidase followed by reduction with NaB3H4 revealed the presence of 52mol of terminal galactose in the intact molecule and 153mol of galactose/mol of LGP-I component after treatment with neuraminidase. The data indicate the LGP-I component is composed of a single polypeptide chain containg more than 150 oligaosaccharide side chains composed of O-GaINAc-Gal distributed over the length of the peptide chain and that terminal sialic acid residues are linked to galactose in two-thirds of these side chains.

scholarly journals Molecular and structural characterization of NADPH-dependent d-glycerate dehydrogenase from the enteric parasitic protist Entamoeba histolytica

2003 ◽  
Vol 375 (3) ◽  
pp. 729-736 ◽  
Author(s):  
Vahab ALI ◽  
Yasuo SHIGETA ◽  
Tomoyoshi NOZAKI
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Entamoeba Histolytica ◽  
Reductase Activity ◽  
Phylogenetic Analyses ◽  
Kinetic Studies ◽  
Biochemical Properties ◽  
Exchange Chromatography ◽  
Subunit Structure ◽  
Protein Alignment

Putative NADPH-dependent GDH (d-glycerate dehydrogenase) of the protozoan parasite Entamoeba histolytica (EhGDH) has been characterized. The EhGDH gene encodes a protein of 318 amino acids with a calculated isoelectric point of 6.29 and a molecular mass of 35.8 kDa. EhGDH showed highest identities with GDH from ε-proteobacteria. This close kinship was also supported by phylogenetic analyses, suggesting possible lateral transfer of the gene from ε-proteobacteria to E. histolytica. In contrast with the implications from protein alignment and phylogenetic analysis, kinetic studies revealed that the amoebic GDH showed biochemical properties similar to those of mammalian GDH, i.e. a preference for NADPH as cofactor and higher affinities towards NADPH and β-hydroxypyruvate than towards NADP+ and d-glycerate. Whereas the amino acids involved in nucleotide binding and catalysis are totally conserved in EhGDH, substitution of a negatively charged amino acid with a non-charged hydroxy-group-containing amino acid is probably responsible for the observed high affinity of EhGDH for NADP+/NADPH. In addition, the amoebic GDH, dissimilar to the bacterial and mammalian GDHs, lacks glyoxylate reductase activity. Native and recombinant EhGDH showed comparable subunit structure, kinetic parameters and elution profiles on anion-exchange chromatography. We propose that the GDH enzyme is likely to be involved in regulation of the intracellular concentration of serine, and, thus, also in controlling cysteine biosynthesis located downstream of serine metabolic pathways in this protist.

Post-proline endopeptidase, further characterization of the enzyme from pig kidneys

1984 ◽  
Vol 49 (8) ◽  
pp. 1846-1853 ◽  
Author(s):  
Karel Hauzer ◽  
Tomislav Barth ◽  
Linda Servítová ◽  
Karel Jošt
Keyword(s):  
Amino Acids ◽  
Aspartic Acid ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Relative Molecular Mass ◽  
Enzyme Molecule ◽  
Thiol Compounds ◽  
Modified Method ◽  
N Terminus

A post-proline endopeptidase (EC 3.4.21.26) was isolated from pig kidneys using a modified method described earlier. The enzyme was further purified by ion exchange chromatography on DEAE-Sephacel. The final product contained about 95% of post-proline endopeptidase. The enzyme molecule consisted of one peptide chain with a relative molecular mass of 65 600 to 70 000, containing a large proportion of acidic and alifatic amino acids (glutamic acid, aspartic acid and leucine) and the N-terminus was formed by aspartic acid or asparagine. In order to prevent losses of enzyme activity, thiol compounds has to be added.

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