scholarly journals Purification and properties of human kidney-cortex hexosaminidases A and B

1977 ◽  
Vol 165 (1) ◽  
pp. 49-53 ◽  
Author(s):  
J E Wiktorowicz ◽  
Y C Awasthi ◽  
A Kurosky ◽  
S K Srivastava
Keyword(s):  
Amino Acid ◽  
Gel Filtration ◽  
Human Kidney ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Kidney Cortex ◽  
Enzyme Replacement ◽  
Purification And Properties ◽  
Hexosaminidase A ◽  
Specific Activities

Hexosaminidases (EC 3.2.1.30) A and B from human kidney cortex were purified to homogeneity by using concanavalin A affinity chromatography, ion-exchange chromatography and gel filtration. The yield of homogeneous isoenzymes improved approx. 20-fold, giving preparations of hexosaminidases A and B with specific activities of about 200 and 325 units/mg of protein respectively. The kinetic and structural properties of kidney hexosaminidase isoenzymes were studied and compared with the hexosaminidase isoenzymes from human placenta. The amino acid composition of hexosaminidase A was significantly different from that of hexosaminidase B. In the event of success in developing enzyme-replacement therapy for Tay-Sachs and Sandhoff's diseases, this modified procedure can furnish larger amounts of homogeneous isoenzymes.

Purification and Properties of Bovine Pituitary Renin

1982 ◽  
Vol 63 (s8) ◽  
pp. 179s-181s
Author(s):  
Tamiko Ohsawa ◽  
Shigehisa Hirose ◽  
Tadashi Inagami ◽  
Kazuo Murakami
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Anterior Pituitary ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Antigenic Properties ◽  
Hog Kidney

1. Renin was purified to homogeneity from bovine anterior pituitary by using batchwise DEAE-cellulose chromatography, pepstatin-aminohexyl-agarose affinity chromatography, Ultrogel AcA 44 gel filtration and DEAE-Sephacel and CM-cellulose ion exchange chromatography. 2. The enzyme has a molecular weight of 36 000 and an isoelectric point of 5.25, and exhibits optimum activity at a pH between 6.5 and 7.5. 3. The amino acid composition and antigenic properties of this purified renin are very similar to those of rat, dog and hog kidney renins.

scholarly journals Salivary apyrase of Rhodnius prolixus. Kinetics and purification

1986 ◽  
Vol 233 (3) ◽  
pp. 885-891 ◽  
Author(s):  
J J F Sarkis ◽  
J A Guimarães ◽  
J M C Ribeiro
Keyword(s):  
Atp Hydrolysis ◽  
Competitive Inhibition ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Rhodnius Prolixus ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Inorganic Pyrophosphatase ◽  
Blood Sucking ◽  
Specific Activities

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5′-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.

Isolation of a Crosslinked Peptide from Hitman Fibrin and Development of a Radioimmunoassay

1979 ◽  
Author(s):  
R. Canfield ◽  
B. Lahiri ◽  
R. D’Alisa ◽  
V. Butler ◽  
H. Nossel ◽  
...  
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Xiiia ◽  
Cross Linking ◽  
Edman Degradation ◽  
Cnbr Cleavage ◽  
Normal Hemostasis

Factor XIIIa introduces up to six crossllnklng bonds per molecule of fibrin; the bonds between the γ chains on adjacent fibrin molecules form most rapidly. Since cross linking is essential for normal hemostasis and is likely to be important in tests to detect thrombosis, we have attempted to develop a radioimmunoassay that exhibits specificity for the γ chain crosslinks. The immunogen consisted of a 54 amino acid, crosslinked peptide, isolated from purified human γ-γ chains following CNBr cleavage, gel filtration on Sephadex G-50 and ion-exchange chromatography on SP-Sephadex. Amino acid analysis and Edman degradation through step 24 confirmed the sequence of Chen and Doolittle (Biochemistry 10: i486, 1971), and the two degradation steps that failed to liberate the expected PTH-amino acids matched the reported location of the Gin-Lys crosslinks. Antisera were obtained against this immunogen coupled either to bovine thyroglobulin or bovine serum albumin. All antisera elicited bound immunogen that was covalently coupled to ribonuclease radiolabeled with 125I as a tracer. The unlabeled γ-γ, crosslinked peptide effectively inhibited binding (0.03-0.08 picomoles for 50% inhibition), while with some antisera up to 500 times more of the 27 amino acid γ monomer peptide was required for the same degree of inhibition. Fibrinogen and fragment D also were poor Inhibitors. The results Indicate that it is possible by radioimmunoassay to distinguish the COOH-termlnal region of the γ-γ dlmer from that of uncrosslinked molecules.

scholarly journals Isolation and a partial amino acid sequence of insulin from the islet tissue of cod (Gadus callarias)

1968 ◽  
Vol 106 (2) ◽  
pp. 531-541 ◽  
Author(s):  
P T Grant ◽  
K. B. M. Reid
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Bovine Insulin ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Performic Acid ◽  
Amino Acid Residues ◽  
Islet Tissue ◽  
A Chain

1. Insulin has been isolated by gel filtration and ion-exchange chromatography from extracts of the discrete islet tissue of cod. The final preparation yielded a single band on electrophoresis at two pH values. The biological potency was 11·5 international units/mg. in mouse-convulsion and other assay procedures. 2. Glycine and methionine were shown to be the N-terminal amino acids of the A and B chains respectively. An estimate of the molecular weight together with amino acid analyses indicated that cod insulin, like the bovine hormone, consists of 51 amino acid residues. In contrast, the amino acid composition differs markedly from bovine insulin. 3. Oxidation of insulin with performic acid yielded the A and B peptide chains, which were separated by ion-exchange chromatography. Sequence studies on smaller peptides isolated from enzymic digests or from dilute acetic acid hydrolysates of the two chains have established the sequential order of 14 of the 21 amino acid residues of the A chain and 25 of the 30 amino acid residues of the B chain.

scholarly journals Membrane-bound lactate dehydrogenases and mandelate dehydrogenases of Acinetobacter calcoaceticus. Purification and properties

1985 ◽  
Vol 231 (2) ◽  
pp. 407-416 ◽  
Author(s):  
N Allison ◽  
M J O'Donnell ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Purification And Properties ◽  
Molecular Masses ◽  
Membrane Bound ◽  
Lactate Dehydrogenases

Procedures were developed for the optimal solubilization of D-lactate dehydrogenase, D-mandelate dehydrogenase, L-lactate dehydrogenase and L-mandelate dehydrogenase from wall + membrane fractions of Acinetobacter calcoaceticus. D-Lactate dehydrogenase and D-mandelate dehydrogenase were co-eluted on gel filtration, as were L-lactate dehydrogenase and L-mandelate dehydrogenase. All four enzymes could be separated by ion-exchange chromatography. D-Lactate dehydrogenase and D-mandelate dehydrogenase were purified by cholate extraction, (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and chromatofocusing. The properties of D-lactate dehydrogenase and D-mandelate dehydrogenase were similar in several respects: they had relative molecular masses of 62 800 and 59 700 respectively, pI values of 5.8 and 5.5, considerable sensitivity to p-chloromercuribenzoate, little or no inhibition by chelating agents, and similar responses to pH. Both enzymes appeared to contain non-covalently bound FAD as cofactor.

The phosphatidyl-myo-inositol-4,5-bisphosphate phosphatase from Crithidia fasciculata

1981 ◽  
Vol 59 (7) ◽  
pp. 469-476 ◽  
Author(s):  
F. B. St. C. Palmer
Keyword(s):  
Cetyltrimethylammonium Bromide ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Phosphate Esters ◽  
Relative Mass ◽  
Crithidia Fasciculata ◽  
Specific Activities ◽  
Triton X

The phosphatase which specifically removes one phosphate group from phosphatidyl-myo-inositol 4,5-bisphosphate was purified up to 6000-fold from the cytosol of the protozoan Crithidia fasciculata. Lipoproteins which interfere with the purification were precipitated by reducing the pH to 4.5. The enzyme was isolated from the supernatant by ammonium sulfate fractionation, gel filtration (Sepharose CL-6B), ion–exchange chromatography (DEAE-Sepharose CL-6B), and hydrophobic chromatography on detergent-saturated phenyl-Sepha-rose CL-4B. The preparations had specific activities of 44–110 μmol∙min−1∙mg protein−1 with phosphatidyl-myo-inositol 4,5-bisphosphate, but were inactive with a variety of lipid and nonlipid phosphate esters. The enzyme was stable in the presence of salt and exhibited a relative mass of 117 000. It formed larger aggregates in the absence of salt and was dissociated into monomers of relative mass 57 000 by sodium dodecyl sulfate.Addition of Triton X-100 to the assay mixture reduced the dependence upon moderation of the charge of the substrate by cetyltrimethylammonium bromide. In the presence of both detergents the Mg2+ dependence of the enzyme was reduced (Km for Mg2+ = 40 μM) while the "apparent" Km for the substrate was unchanged at 240 μM. Substrate precipitation at higher Mg2+ concentrations was eliminated.

scholarly journals Purification and properties of l-mandelate dehydrogenase and comparison with other membrane-bound dehydrogenases from Acinetobacter calcoaceticus

1987 ◽  
Vol 248 (3) ◽  
pp. 871-876 ◽  
Author(s):  
M E Hoey ◽  
N Allison ◽  
A J Scott ◽  
C A Fewson
Keyword(s):  
Lactate Dehydrogenase ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Acinetobacter Calcoaceticus ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Ph Optimum ◽  
Purification And Properties ◽  
Potential Inhibitors ◽  
Triton X

L-Mandelate dehydrogenase was purified from Acinetobacter calcoaceticus by Triton X-100 extraction from a ‘wall + membrane’ fraction, ion-exchange chromatography on DEAE-Sephacel, (NH4)2SO4 fractionation and gel filtration followed by further ion-exchange chromatography. The purified enzyme was partially characterized with respect to its subunit Mr (44,000), pH optimum (7.5), pI value (4.2), substrate specificity and susceptibility to various potential inhibitors including thiol-blocking reagents. FMN was identified as the non-covalently bound cofactor. The properties of L-mandelate dehydrogenase are compared with those of D-mandelate dehydrogenase, D-lactate dehydrogenase and L-lactate dehydrogenase from A. calcoaceticus.

scholarly journals Studies on Marsupial Protein. II. Amino Acid Sequence of the -Chain of Haemoglobin from the Grey Kangaroo, Macropus Giganteus

1969 ◽  
Vol 22 (6) ◽  
pp. 1437 ◽  
Author(s):  
GM Air ◽  
EOP Thompson
Keyword(s):  
Amino Acid ◽  
Ion Exchange ◽  
Amino Acid Sequence ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Amino Acid Sequences ◽  
Tryptic Digestion ◽  
Grey Kangaroo

The amino acid sequence of the jS-chain of haemoglobin from M. giganteus has been determined. The soluble peptides formed by tryptic digestion were isolated by gel filtration, ion-exchange chromatography, and paper ionophoresis, and amino acid sequences determined by the "dansyl"-Edman procedure. Special procedures were necessary for three peptides which were insoluble.

Purification and Properties of Human Factor IXa

1976 ◽  
Vol 35 (03) ◽  
pp. 576-585 ◽  
Author(s):  
Katalin Váradi ◽  
Susan Elödi
Keyword(s):  
Molecular Weight ◽  
Human Factor ◽  
Gel Filtration ◽  
Heat Stability ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Factor Ix ◽  
Clotting Factors ◽  
Factor Ixa ◽  
Purification And Properties

SummaryHuman factor IXa was purified 5,000-fold from serum by ion exchange chromatography. The preparation was free from other clotting factors. Both pH sensitivity and heat stability of purified factor IXa appeared to be different from those of factor IX in the plasma. The molecular weight of human factor IXa is 80,000 as estimated from gel-filtration experiments. Modification of seryl or histidyl side chains abolished the activity of factor IXa.

Purification and properties of an exocellular β-glucosidase of Candida molischiana (Zikes) Meyer and Yarrow capable of hydrolyzing soluble cellodextrins

1985 ◽  
Vol 63 (11) ◽  
pp. 1160-1166 ◽  
Author(s):  
Pierre Gondé ◽  
Robert Ratomahenina ◽  
Alain Arnaud ◽  
Pierre Galzy
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Gel Filtration ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Optimum Temperature ◽  
Purification And Properties ◽  
Optimum Ph

The exocellular enzyme β-glucosidase of Candida molischiana was studied. This strain is able to ferment soluble cellodextrins. The enzyme was partially purified by ion-exchange chromatography and gel filtration. The molecular weight of this enzyme was 120 000; its optimum pH was between 4 and 4.5 and its optimum temperature was 60 °C. This enzyme was active against different soluble glucosides and was inhibited by p-chloromercuribenzoate, gluconolactone, and glucose. A "glucosyltransferase" activity appeared in the presence of ethanol. The biosynthesis of the enzyme was constitutive but repressed by glucose.

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