scholarly journals Purification and properties of 2-aminoadipate: 2-oxoglutarate aminotransferase from bovine kidney

1989 ◽  
Vol 261 (3) ◽  
pp. 761-768 ◽  
Author(s):  
D R Deshmukh ◽  
S M Mungre
Keyword(s):  
Rat Kidney ◽  
Deae Cellulose ◽  
Dicarboxylic Acids ◽  
Structural Similarity ◽  
Appreciable Amount ◽  
Kinetic Properties ◽  
Bovine Kidney ◽  
Purification And Properties ◽  
Kidney Enzyme ◽  
Structural Differences

Previous studies with rat kidney preparations indicated that 2-aminoadipate aminotransferase (AadAT) and kynurenine aminotransferase (KAT) activities are properties of a single protein. We found that bovine kidney contains an appreciable amount of AadAT activity, but lacks KAT activity. AadAT from bovine and rat kidney extracts were purified to electrophoretic homogeneity. The purification procedure included fractionation with (NH1)2SO1, heat treatment, DEAE-cellulose chromatography and hydroxyapatite chromatography. Physical and kinetic properties, such as pH optima, Km for substrates, Mr, electrophoretic mobility and inhibition by dicarboxylic acids of bovine kidney AadAT, were similar to those of the rat kidney enzyme. However, bovine kidney AadAT differed from rat kidney AadAT in substrate specificity, amino acid composition and stability when stored. The titration curve of bovine kidney AadAT was also different from that of the rat kidney enzyme. The results suggest that bovine kidney AadAT may have some structural similarity to rat kidney AadAT and that the structural differences observed between the two enzymes may explain the absence of KAT activity in bovine kidney.

scholarly journals Purification and properties of arginase of rat kidney

1973 ◽  
Vol 133 (4) ◽  
pp. 779-788 ◽  
Author(s):  
George A. Kaysen ◽  
Harold J. Strecker
Keyword(s):  
Rat Liver ◽  
Rat Kidney ◽  
Mitochondrial Fraction ◽  
Tissue Extracts ◽  
Low Concentrations ◽  
Purification And Properties ◽  
Hydrolysis Rates ◽  
Kidney Enzyme ◽  
Acetone Treatment ◽  
Unknown Factor

l-Arginase from rat kidney was partially purified and some properties were compared with those of l-arginase of rat liver. The kidney enzyme was firmly bound to the mitochondrial fraction and after solubilization required arginine or an unknown factor in tissue extracts for stabilization after dialysis. The two enzymes differed also in stability with respect to acetone treatment, heating or freezing. In further contrast with liver arginase, arginase from kidney was not adsorbed to CM-cellulose at pH7.5 and its activity was not increased by incubation with Mn2+. Other differences were seen in relative specificities for substrates, ratio of hydrolysis rates with high and low concentrations of arginine and effects of certain inhibitors. Antisera prepared to pure liver arginase did not cross-react with partially purified kidney arginase.

scholarly journals Purification and properties of kynurenine aminotransferase from rat kidney

1991 ◽  
Vol 279 (2) ◽  
pp. 595-599 ◽  
Author(s):  
M R Mawal ◽  
A Mukhopadhyay ◽  
D R Deshmukh
Keyword(s):  
Rat Kidney ◽  
Dicarboxylic Acids ◽  
Supernatant Fraction ◽  
Subunit Structure ◽  
Mitochondrial Enzyme ◽  
Kynurenine Aminotransferase ◽  
Kidney Mitochondria ◽  
Purification And Properties ◽  
Single Protein ◽  
Degree Of Purification

Previous reports indicated that a single protein exhibits kynurenine aminotransferase (KAT) and alpha-aminoadipate aminotransferase (AadAT) activities. However, recently we discovered that KAT and AadAT activities are associated with two different proteins. KAT from rat kidney supernatant fraction was purified to electrophoretic homogeneity by (NH4)2SO4 fractionation, DEAE-Sephacel and hydroxyapatite chromatography. This procedure separated KAT from AadAT and improved the overall yield and the degree of purification over previously published methods. Some of the properties of purified KAT, such as Mr, subunit structure and the inhibition by dicarboxylic acids, were identical with those reported previously. However, the substrate specificity and pI of purified KAT were different from earlier reports. The same procedure can also be used to purify KAT from rat kidney mitochondria. These results support our earlier observation that KAT and AadAT activities are associated with two proteins and suggest that cytosolic KAT may be structurally similar to the mitochondrial enzyme.

Chromatographic Isolation of the LDH-Isoenzymes of Human Blood Platelets and an Investigation of Their Enzyme Kinetics

1968 ◽  
Vol 20 (03/04) ◽  
pp. 301-313 ◽  
Author(s):  
W Schneider ◽  
K Schumacher ◽  
B Thiede ◽  
R Gross
Keyword(s):  
Human Blood ◽  
Blood Platelets ◽  
Deae Cellulose ◽  
Order Reaction ◽  
Kinetic Properties ◽  
Ldh Isoenzymes ◽  
Kinetic Investigations ◽  
Substrate Concentrations ◽  
Chromatographic Isolation ◽  
Human Blood Platelets

SummaryThe LDH-isoenzymes of human blood platelets show a distinct predominance of the isoenzymes 2 and 3 upon chromatography on DEAE-cellulose. Small amounts of LDH-1 are also present, while only traces of LDH-4 and -5 can be detected.Enzyme kinetic investigations of the principal isoenzymes LDH-1, -2 and -3 clearly show that the differences in inhibition constants with pyruvate as substrate which are demonstrable at 25° largely disappear at 37°. On the other hand, the differences among the isoenzymes in their affinity for pyruvate and lactate as substrate as well as in with respect to the optimal substrate concentrations of pyruvate are more marked at 37° than at 25°. Also, the type of inhibition found with lactate as substrate is increasingly the expression of a higher order reaction in going from LDH-1 to LDH-3. A dependence of the LDH distribution pattern upon the metabolism of the cell is discussed. A comparison of our results with thrombocytes with those of other workers with erythrocytes and leucocytes makes it unlikely that the LDH pattern is directly dependent upon the existence of an oxidative metabolism. Rather, the redox potential of the cell could be of importance for the nature of the pattern of isoenzymes and for their differing kinetic properties.

scholarly journals Purification and properties of two extracellular β-lactamases from Bacillus cereus 569/H

1970 ◽  
Vol 118 (3) ◽  
pp. 457-465 ◽  
Author(s):  
S. Kuwabara
Keyword(s):  
Molecular Weight ◽  
Bacillus Cereus ◽  
Zinc Sulphate ◽  
Deae Cellulose ◽  
Casein Hydrolysate ◽  
Crystalline Form ◽  
Casamino Acid ◽  
Fractional Precipitation ◽  
Purification And Properties ◽  
Rapid Production

1. When Bacillus cereus 569/H was grown in a casamino acid (casein-hydrolysate) medium containing zinc sulphate rapid production of extracellular β-lactamase II preceded that of β-lactamase I. 2. β-Lactamase I was separated from β-lactamase II by fractional precipitation with ammonium sulphate. 3. β-Lactamase I was purified by a process involving chromatography on Celite and DEAE-cellulose and β-lactamase II by chromatography on DEAE-cellulose after denaturation of β-lactamase I by heat. Both enzymes were obtained in crystalline form. 4. β-Lactamase II prepared in this way appeared to have a higher molecular weight than β-lactamase I and required Zn2+ as a cofactor for both cephalosporinase and penicillinase activities.

scholarly journals Purification and properties of α-mannosidase II from Golgi-like membranes of baculovirus-infected Spodoptera frugiperda (IPLB-SF-21AE) cells

1997 ◽  
Vol 324 (3) ◽  
pp. 951-956 ◽  
Author(s):  
Jianxin REN ◽  
Francis J. CASTELLINO ◽  
Roger K. BRETTHAUER
Keyword(s):  
Spodoptera Frugiperda ◽  
Reaction Pathway ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Mannose Residue ◽  
Lepidopteran Insect ◽  
Reducing Conditions ◽  
Apparent Homogeneity ◽  
Purification And Properties

An α-mannosidase II-like activity was identified in baculovirus-infected Spodoptera frugiperda (IPLB-SF21-AE) cells. The enzyme responsible was purified from Golgi-type membranes to apparent homogeneity by using a combination of steps including DEAE-cellulose, hydroxyapatite, concanavalin A–Sepharose and gel filtration chromatography. The molecular mass of this purified protein was approx. 120 kDa by SDS/PAGE under reducing conditions and approx. 240 kDa under non-reducing conditions, indicating that the enzyme is a disulphide-linked dimer. Substrates demonstrated to undergo hydrolysis with this enzyme were GlcNAc-Man5-GlcNAc-GlcNAc (non-reduced and reduced) and p-nitrophenyl α-d-mannopyranoside. The oligosaccharide substrate was converted into GlcNAc-Man3-GlcNAc-GlcNAc through an intermediate GlcNAc-Man4-GlcNAc-GlcNAc. Treatment of the isolated intermediate oligosaccharide with endoglycosidase H resulted in its conversion into GlcNAc-Man4-GlcNAc. This indicated that it contained the α-1,3-linked mannose residue on the α-1,6-linked mannose arm and showed that the α-1,6-linked mannose residue on the α-1,6-linked mannose arm had been preferentially hydrolysed by the mannosidase. The oligosaccharide lacking the β-1,2-linked GlcNAc residue on the α-1,3-linked mannose arm (Man5-GlcNAc-GlcNAc) was not hydrolysed in the presence of the enzyme. Metal ions were not required for enzymic activity on any of the substrates, but Cu2+ was strongly inhibitory. The activity of the enzyme was inhibited at low concentrations of swainsonine, but much higher concentrations of 1-deoxymannojirimycin were required to achieve inhibition. All of these properties are characteristic of mannosidase II enzymes from other eukaryotic tissues. The presence of mannosidase II in lepidopteran insect cells would allow entry of N-linked glycoproteins into the complex processing reaction pathway or into the terminal Man3-GlcNAc-GlcNAc pathway.

scholarly journals Metabolism of parathyroid hormone. Degradation of 125I-labelled hormone by kidney enzyme

1969 ◽  
Vol 111 (4) ◽  
pp. 509-514 ◽  
Author(s):  
T. J. Martin ◽  
R. A. Melick ◽  
M. de Luise
Keyword(s):  
Parathyroid Hormone ◽  
Trichloroacetic Acid ◽  
Rat Kidney ◽  
Gel Filtration ◽  
Void Volume ◽  
Control Medium ◽  
Ph Values ◽  
Kidney Enzyme ◽  
Rapid Destruction ◽  
Kidney Microsomes

A study was made of the enzymic degradation of 125I-labelled parathyroid hormone by rat kidney microsomes. Incubation with microsomes resulted in rapid destruction of the labelled hormone. The microsomal factor was not separable by dialysis, and the reaction was favoured by pH values in the physiological range. Velocity of the reaction varied directly as the substrate concentration, and additional crude parathyroid hormone (trichloroacetic acid-precipitated, 3·68mg./ml.) inhibited destruction of labelled hormone. There was much less inhibition with added trichloroacetic acid-precipitated calcitonin (3·92mg./ml.) and virtually none with added pig insulin (3·80mg./ml.). Gel filtration of control medium on P6 (Bio-Gel) yielded one radioactive peak at the void volume. After incubation with microsomes three further peaks were obtained on gel filtration. Only the void-volume peak contained intact 125I-labelled parathyroid hormone, indicating that the microsomal enzyme degraded labelled hormone to a number of smaller fragments.

scholarly journals THE STAGES IN CALCIFICATION OF THE RAT KIDNEY AFTER THE ADMINISTRATION OF URANIUM NITRATE

1953 ◽  
Vol 97 (5) ◽  
pp. 681-694 ◽  
Author(s):  
Lewis K. Dahl
Keyword(s):  
Ferrous Iron ◽  
Rat Kidney ◽  
Cellular Damage ◽  
Appreciable Amount ◽  
Wide Range ◽  
Uranium Nitrate ◽  
Speed Of Evolution ◽  
Dose Levels ◽  
Convoluted Tubules ◽  
Inner Cortex

The anatomical and histochemical alterations in rat kidneys after the parenteral administration of uranium nitrate [UO2(NO3)2·6H2O] have been studied. The histological effects produced by this agent over a wide range of dose levels were nearly identical in character and differed principally in their speed of evolution. The deposition of calcium always began at foci in the cytoplasm of the cells of the proximal convoluted tubules of the inner cortex. It remained intracellular until the cell boundaries were destroyed. Deposits of calcium could be found before any other cellular damage could be demonstrated by histological examination. Later, when degeneration and necrosis were present, the foci of calcification were imperfectly related to them in location or degree. In contrast, the amount of calcification was correlated with the dose of uranium nitrate, being greatest in the kidneys of rats that received 20 mg./kg., next greatest in the 30 mg./kg. rats, less in the 10 mg./kg. rats, and slight in those that received 2 mg./kg. Histochemical stains for ferric and ferrous iron, chondroitinsulfate, and polysaccharides gave results that were negative or unrelated to the deposits of calcium, thus making it unlikely that these substances held any appreciable amount of calcium in the tissue. Yet it is clear that some anion other than phosphate must be combined with part of the calcium; the results with the alizarin and von Kóssa stains confirmed an earlier result (1) in showing that the first deposits of calcium are formed without comparable accumulations of phosphate.

Structure and developmental tendency of the dorsal marginal zone in the early amphibian gastrula

Development ◽  
1971 ◽  
Vol 25 (2) ◽  
pp. 263-276
Author(s):  
Nobushige Ikushima ◽  
Setsuko Maruyama
Keyword(s):  
Outer Layer ◽  
Marginal Zone ◽  
Kinetic Properties ◽  
Electron Microscopic ◽  
Structural Differences ◽  
Peripheral Surface ◽  
Early Gastrula ◽  
A Share

The peripheral surface of a fertilized, uncleaved egg is subdivided through cleavage and is allotted to constituent cells. This is called the primary surface. In an early morula a constituent cell has two kinds of surfaces: the primary surface, and the secondary surface, which does not participate in forming the periphery of the embryo. Electron-microscopic observations showed structural differences between the two surfaces. When the dorsal marginal zone of an early gastrula of Hynobius nebulosus is excised and immersed in Feldman's solution, the piece can easily be separated into two layers: the outer layer, whose constituent cells are given a share of the primary surface, and the inner layer, whose constituent cells are completely covered only by the secondary surface. Both an explanted piece of the outer layer and an intact double-layered piece show three kinds of movement: spreading, convergence followed by stretching, and spherical thickening. The inner layer is kinetically very inert, showing slight spreading and thickening. An explanted piece of the outer layer differentiates into axial mesodermal structures, while the inner layer does not. When a piece of either the inner or the outer layer is implanted in the blastocoel of another gastrula, it induces deuterencephalic and spino-caudal structures and seems to differentiate into axial mesodermal structures. Differences of kinetic properties and differentiation are considered to result from the fact that the outer layer has the primary surface, while the inner layer does not. Functional effects of the primary surface on the movement of tissues and differentiation are discussed.

K depletion modifies the properties of Sch-28080-sensitive K-ATPase in rat collecting duct

1997 ◽  
Vol 272 (1) ◽  
pp. F124-F131 ◽  
Author(s):  
B. Buffin-Meyer ◽  
M. Younes-Ibrahim ◽  
C. Barlet-Bas ◽  
L. Cheval ◽  
S. Marsy ◽  
...  
Keyword(s):  
Collecting Duct ◽  
Rat Kidney ◽  
Lower Sensitivity ◽  
Kinetic Properties ◽  
Type I ◽  
Type Ii ◽  
Distribution Profile ◽  
Adenosine Triphosphatases ◽  
Collecting Ducts ◽  
K Depletion

Two distinct Sch-28080-sensitive K-adenosine triphosphatases (K-ATPases) were previously described in the rat nephron: a ouabain-resistant K-ATPase (type I) present in collecting ducts (CD) and a ouabain-sensitive from (type II) located in proximal tubules (PT) and thick ascending limbs (TAL). In K-depleted rats, K-ATPase activity is increased in CD, whereas it is reduced in PT and TAL. Because expression of colonic H-K-ATPase is restricted to the CD of K-depleted rats, we hypothesized that K-ATPase from the CD of K-depleted rats might be different from types I and II. Indeed, type III K-ATPase displays higher sensitivities to ouabain and to Sch-28080 than type II, a lower sensitivity to Sch-28080 than type I, and, conversely to types I and II, it can be stimulated by Na+. Pharmacological differences between types II and III K-ATPases were confirmed by [3H]ouabain binding experiments. Thus the rat kidney expresses three K-ATPases that differ by their pharmacological and kinetic properties, their distribution profile along the nephron and their behavior during K depletion.

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