scholarly journals The degradation of l-histidine in the rat. The formation of imidazolylpyruvate, imidazolyl-lactate and imidazolylpropionate

1973 ◽  
Vol 136 (3) ◽  
pp. 649-658 ◽  
Author(s):  
Alan V. Emes ◽  
Harold Hassall
Keyword(s):  
Deae Cellulose ◽  
Substrate Affinity ◽  
Significant Activity ◽  
Anaerobic Conditions ◽  
Ph Optimum ◽  
Oxidoreductase Activity ◽  
Molecular Weights ◽  
Optimum Stability ◽  
Nad Oxidoreductase

1. Soluble and mitochondrial forms of histidine–pyruvate aminotransferase were separated from rat liver preparations by chromatography on DEAE-cellulose. 2. These enzymes were characterized with respect to substrate specificity, substrate affinity, pH optimum, stability and molecular weight by chromatography on Sephadex G-200. 3. Each enzyme has a relatively broad specificity showing significant activity towards l-phenylalanine and l-tyrosine and catalysing transamination with a number of monocarboxylic 2-oxo acids. 2-Oxoglutarate is not a substrate for either enzyme. 4. The molecular weights of the two enzymes, by chromatography on Sephadex G-200, are in the range 130000–150000. 5. The formation in vitro of imidazolyl-lactate from imidazolylpyruvate and NADH was demonstrated by using liver preparations. 6. From a study of imidazolyl-lactate–NAD+oxidoreductase activity after electrophoresis of liver preparations on polyacrylamide gel, and from an examination of the activity of l-lactate–NAD+oxidoreductase (EC 1.1.1.27) towards imidazolylpyruvate, it is concluded that this latter enzyme is responsible for the formation of imidazolyl-lactate in the liver. 7. Preparations of bacteria obtained from rat faeces form imidazolylpropionate from l-histidine and urocanate without further subculture. The amount of imidazolylpropionate formed is increased under anaerobic conditions and more so in an atmosphere of H2. It is suggested that the gut flora of the rat contribute largely, if not exclusively, to the formation of imidazolylpropionate normally found in the urine.

scholarly journals Purification and characterization of two human erythrocyte arylamidases preferentially hydrolysing N-terminal arginine or lysine residues

1978 ◽  
Vol 175 (3) ◽  
pp. 1051-1067 ◽  
Author(s):  
K K Mäkinen ◽  
P L Mäkinen
Keyword(s):  
Substrate Inhibition ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Gel Permeation Chromatography ◽  
Ph Optimum ◽  
Preparative Scale ◽  
Molecular Weights ◽  
Gel Permeation ◽  
Lysine Residues ◽  
Enzyme I

Two arylamidases (I and II) were purified from human erythrocytes by a procedure that comprised removal of haemoglobin from disrupted cells with CM-Sephadex D-50, followed by treatment of the haemoglobin-free preparation subsequently with DEAE-cellulose, gel-permeation chromatography on Sephadex G-200, gradient solubilization on Celite, isoelectric focusing in a pH gradient from 4 to 6, gel-permeation chromatography on Sephadex G-100 (superfine), and finally affinity chromatography on Sepharose 4B covalently coupled to L-arginine. In preparative-scale purifications, enzymes I and II were separated at the second gel-permeation chromatography. Enzyme II was obtained as a homogeneous protein, as shown by several criteria. Enzyme I hydrolysed, with decreasing rates, the L-amino acid 2-naphtylamides of lysine, arginine, alanine, methionine, phenylalanine and leucine, and the reactions were slightly inhibited by 0.2 M-NaCl. Enzyme II hydrolysed most rapidly the corresponding derivatives of arginine, leucine, valine, methionine, proline and alanine, in that order, and the hydrolyses were strongly dependent on Cl-. The hydrolysis of these substrates proceeded rapidly at physiological Cl- concentration (0.15 M). The molecular weights (by gel filtration) of enzymes I and II were 85 000 and 52 500 respectively. The pH optimum was approx. 7.2 for both enzymes. The isoelectric point of enzyme II was approx. 4.8. Enzyme I was activated by Co2+, which did not affect enzyme II to any noticeable extent. The kinetics of reactions catalysed by enzyme I were characterized by strong substrate inhibition, but enzyme II was not inhibited by high substrate concentrations. The Cl- activated enzyme II also showed endopeptidase activity in hydrolysing bradykinin.

scholarly journals The nucleoside triphosphate–ribonucleic acid nucleotidyltransferase (EC 2.7.7.6) of Agrobacterium tumefaciens (Smith and Townsend) Conn. Purification and properties of the enzyme from the tumorigenic strain B6806

1974 ◽  
Vol 143 (3) ◽  
pp. 511-520 ◽  
Author(s):  
U. C. Knopf
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Gradient Centrifugation ◽  
Deae Cellulose ◽  
Nucleoside Triphosphate ◽  
Subunit Structure ◽  
Bacterial Cells ◽  
Protein Subunit ◽  
Ph Optimum ◽  
Protamine Sulphate

The RNA nucleotidyltransferase (RNA polymerase) of the plant-tumorigenic bacterium Agrobacterium tumefaciens was purified. The method involves the disruption of the bacterial cells with glass beads in a Waring Blendor, treatment with DEAE-cellulose, fractionation with (NH4)2SO4, protamine sulphate precipitation, DEAE-cellulose column chromatography and either glycerol-gradient centrifugation or phosphocellulose chromatography. The subunit structure of the highly purified enzyme is similar to, although not identical with, the RNA nucleotidyltransferase of Escherichia coli. It can be described as β′, β, χ1 and α (mol.wts. 160000, 150000, 98000, and 41000±10% respectively). χ1 is the temporary designation for a protein subunit, which might have the same functions as the σ subunit in E. coli. The enzyme of A. tumefaciens is rifampicin-sensitive, has a temperature optimum in vitro of 41±1°C and a pH optimum of 8.2±0.1. Mg2+ and Mn2+ are activators. The enzyme transcribes with different efficiencies artificial, viral, bacterial, plant and animal templates.

scholarly journals Mannosidosis in Angus cattle. The enzymic defect

1974 ◽  
Vol 137 (2) ◽  
pp. 363-371 ◽  
Author(s):  
N. C. Phillips ◽  
D. Robinson ◽  
B. G. Winchester ◽  
R. D. Jolly
Keyword(s):  
Gel Filtration ◽  
Residual Activity ◽  
Deae Cellulose ◽  
Exchange Chromatography ◽  
Starch Gel Electrophoresis ◽  
Ph Optimum ◽  
Molecular Weights ◽  
Angus Cattle ◽  
Starch Gel ◽  
Component C

Normal calf α-mannosidase activity exists in at least three forms separable by chromatography on DEAE-cellulose and by starch-gel electrophoresis. Two components, A and B, have optimum activity between pH3.75 and 4.75, but component C has an optimum of pH6.6. Components A and B are virtually absent from the tissues of a calf with mannosidosis and the residual activity is due to component C. The acidic and neutral forms of α-mannosidase differ in their molecular weights and sensitivity to EDTA, Zn2+, Co2+ and Mn2+. An acidic α-mannosidase component (pH optimum 4.0) accounts for most of the activity in normal plasma but it is absent from the plasma of a calf with mannosidosis. Although the acidic α-mannosidase component is probably related to tissue components A and B, it can be distinguished from them by ion-exchange chromatography and gel filtration. The optimum pH of the low residual activity in the plasma from a calf with mannosidosis is pH5.5–5.75. The results support the hypothesis that Angus-cattle mannosidosis is a storage disease caused by a deficiency of lysosomal acidic α-mannosidase activity.

Different Forms of Fructose 1,6-Bisphosphatase in Chlorella

1993 ◽  
Vol 48 (1-2) ◽  
pp. 22-27 ◽  
Author(s):  
N. Grotjohann ◽  
G. Schneider ◽  
W. Kowallik
Keyword(s):  
Deae Cellulose ◽  
Reducing Power ◽  
Exchange Chromatography ◽  
Substrate Affinity ◽  
Kinetic Properties ◽  
Cell Extracts ◽  
Fructose 1,6 Bisphosphatase ◽  
Chloroplast Stroma

In crude extracts of Chlorella kessleri two forms of fructosebisphosphatase can be separated by ion exchange chromatography or by acid precipitation. FBPase I is eluted from DEAE cellulose at 200mᴍ KC1 and precipitated at pH 4.5, FBPase II is correspondingly eluted at 310 mᴍ KC1 and soluble at pH 4.5. Both enzymes differ in substrate affinity and degree of cooperativity. Based on literature data, FBPase I is assumed to be of cytosolic and FBPase II of chloroplastic origin. The mole mass of FBPase I is identical (51-65 kDa) at pH 6.5 and pH 8.5. T hat of FBPase II is however, about four times larger at pH 6.5 (257 kDa) than at pH 8.5 (67 kDa). Other pH values have only been tried with crude cell extracts in which still larger mole masses of FBPase resulted at more acidic pH (1349 kD a at pH 6.0). The lower mole mass form of FBPase II shows three times higher catalytic activity. Reducing agents, such as DTT, also increase the activity of FBPase II in vitro. In vivo, alkalization and production of reducing power occurs in the chloroplast stroma during illumination. If the above alterations exist in vivo, they would be a means to activate FBPase in the light. Oligomerization of FBPase II to aggregates with altered catalytic activities and kinetic properties is discussed as result of the action of specific wavelengths and to be responsible for differences in carbohydrate metabolism of Chlorella exposed to red or blue light.

scholarly journals α-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro

1978 ◽  
Vol 175 (3) ◽  
pp. 1069-1077 ◽  
Author(s):  
J Williams ◽  
H Villarroya ◽  
F Petek
Keyword(s):  
Trifolium Repens ◽  
Gel Electrophoresis ◽  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Disc Electrophoresis ◽  
Polyacrylamide Gel ◽  
Ph Optimum

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).

scholarly journals Isolation and properties of α-D-mannosidase from human kidney

1976 ◽  
Vol 155 (2) ◽  
pp. 217-223 ◽  
Author(s):  
D V. Marinkovic ◽  
J N. Marinkovic
Keyword(s):  
Heavy Metals ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Human Kidney ◽  
Human Albumin ◽  
Homogeneous State ◽  
Thiol Groups ◽  
Ph Optimum ◽  
Molecular Weights

α-D-Mannosidase activity exists in three forms that can be separated by DEAE-cellulose chromatography, α-D-Mannosidase was isolated from human kidney in a homogeneous state, and was purified 2100-fold, with p-nitrophenyl α-D-mannoside as substrate. The purified α-D-mannosidase was practically free from all other glycosidases tested. The Km of the synthetic substrate with the enzyme was 1 × 10(-3) M and the pH optimum 4.5. It was inhibited by heavy metals, sodium dodecyl sulphate, urea and compounds that react with the thiol groups, and was activated by Zn2+, Na+, 2-mercaptoethanol, human albumin and γ-globulin. The mol. wt. of the enzyme was estimated to be 180 000 +/- 4500. After pretreatment with 2-mercaptoethanol and sodium dodecyl sulphate, α-D-mannosidase dissociated into subunits of mol. wts. of 58 000 +/- 600 and 30 000 +/- 380 respectively. Subunits of the same molecular weights were also obtained after the enzyme was heated at 100 degrees C.

scholarly journals Isolation and characterization of two polypeptides that form intermediate filaments in bovine esophageal epithelium.

1981 ◽  
Vol 88 (2) ◽  
pp. 317-322 ◽  
Author(s):  
L M Milstone
Keyword(s):  
Deae Cellulose ◽  
Esophageal Epithelium ◽  
X Ray Diffraction ◽  
Molecular Weights ◽  
Isolation And Characterization ◽  
Extractable Protein ◽  
Stratified Squamous Epithelium ◽  
Alpha Keratin

Cells in the stratified squamous epithelium of bovine esophagus contain abundant tonofilaments measuring 6-10 nm in diameter. Two polypeptides, extracted from esophageal epithelium with 0.05 M Tris, pH 7.4, containing 8 M urea and 25 mM beta-mercaptoethanol, comprise 35% of the total extractable protein. These polypeptides have apparent molecular weights of 46,000 and 56,000 daltons and are rich in glutamic acid-glutamine, glycine, and serine. Each polypeptide can be partially purified by DEAE-cellulose chromatography. Mixtures of the purified polypeptides from filaments in vitro that measured 6-10 nm in diameter. Neither polypeptide formed filaments by itself. Filaments formed in vitro give an alpha-keratin type x-ray diffraction pattern.. These data indicate that the tonofilaments in esophageal epithelium are formed primarily from these two polypeptides.

Soluble Thrombomodulin Purified from Human Urine Exhibits a Potent Anticoagulant Effect In Vitro and In Vivo

1995 ◽  
Vol 73 (05) ◽  
pp. 805-811 ◽  
Author(s):  
Yasuo Takahashi ◽  
Yoshitaka Hosaka ◽  
Hiromi Niina ◽  
Katsuaki Nagasawa ◽  
Masaaki Naotsuka ◽  
...  
Keyword(s):  
Human Urine ◽  
Specific Activity ◽  
Anticoagulant Activity ◽  
Rabbit Lung ◽  
Soluble Thrombomodulin ◽  
Molecular Weights ◽  
Dose Dependent Fashion ◽  
Dose Dependent

SummaryWe examined the anticoagulant activity of two major molecules of soluble thrombomodulin purified from human urine. The apparent molecular weights of these urinary thrombomodulins (UTMs) were 72,000 and 79,000, respectively. Both UTMs showed more potent cofactor activity for protein C activation [specific activity >5,000 thrombomodulin units (TMU)/mg] than human placental thrombomodulin (2,180 TMU/mg) and rabbit lung thrombomodulin (1,980 TMU/mg). The UTMs prolonged thrombin-induced fibrinogen clotting time (>1 TMU/ml), APTT (>5 TMU/ml), TT (>5 TMU/ml) and PT (>40 TMU/ml) in a dose-dependent fashion. These effects appeared in the concentration range of soluble thrombomodulins present in human plasma and urine. In the rat DIC model induced by thromboplastin, administration of UTMs by infusion (300-3,000 TMU/kg) restored the hematological abnormalities derived from DIC in a dose-dependent fashion. These results demonstrate that UTMs exhibit potent anticoagulant and antithrombotic activities, and could play a physiologically important role in microcirculation.

Toxicity of Heparinoids, with Special Reference to the Precipitation of Fibrinogen

1964 ◽  
Vol 12 (01) ◽  
pp. 232-261 ◽  
Author(s):  
S Sasaki ◽  
T Takemoto ◽  
S Oka
Keyword(s):  
Molecular Weight ◽  
Dextran Sulfate ◽  
Anticoagulant Activity ◽  
Molecular Structures ◽  
Severe Reaction ◽  
Clinical Use ◽  
Molecular Weights ◽  
Close Relationship

SummaryTo demonstrate whether the intravascular precipitation of fibrinogen is responsible for the toxicity of heparinoid, the relation between the toxicity of heparinoid in vivo and the precipitation of fibrinogen in vitro was investigated, using dextran sulfate of various molecular weights and various heparinoids.1. There are close relationships between the molecular weight of dextran sulfate, its toxicity, and the quantity of fibrinogen precipitated.2. The close relationship between the toxicity and the precipitation of fibrinogen found for dextran sulfate holds good for other heparinoids regardless of their molecular structures.3. Histological findings suggest strongly that the pathological changes produced with dextran sulfate are caused primarily by the intravascular precipitates with occlusion of the capillaries.From these facts, it is concluded that the precipitates of fibrinogen with heparinoid may be the cause or at least the major cause of the toxicity of heparinoid.4. The most suitable molecular weight of dextran sulfate for clinical use was found to be 5,300 ~ 6,700, from the maximum value of the product (LD50 · Anticoagulant activity). This product (LD50 · Anticoagulant activity) can be employed generally to assess the comparative merits of various heparinoids.5. Clinical use of the dextran sulfate prepared on this basis gave satisfactory results. No severe reaction was observed. However, two delayed reactions, alopecia and thrombocytopenia, were observed. These two reactions seem to come from the cause other than intravascular precipitation.

Platelet Microtubule Subunit Proteins

1979 ◽  
Vol 42 (05) ◽  
pp. 1630-1633 ◽  
Author(s):  
A G Castle ◽  
N Crawford
Keyword(s):  
Epithelial Cells ◽  
Gel Electrophoresis ◽  
Blood Platelets ◽  
Polyacrylamide Gel Electrophoresis ◽  
Polyacrylamide Gel ◽  
Lens Epithelial Cells ◽  
Molecular Weights ◽  
Kinetics Of ◽  
Microtubular Structures

SummaryBlood platelets contain microtubule proteins (tubulin and HMWs) which can be polymerised “in vitro” to form structures which resemble the microtubules seen in the intact platelet. Platelet tubulin is composed of two non-identical subunits a and p tubulin which have molecular weights around 55,000 but can be resolved in alkaline SDS-polyacrylamide gel electrophoresis. These subunits associate as dimers with sedimentation coefficients of about 5.7 S although it is not known whether the dimer protein is a homo- or hetero-dimer. The dimer tubulin binds the anti-mitotic drug colchicine and the kinetics of this binding are similar to those reported for neurotubulins. Platelet microtubules also contain two HMW proteins which appear to be essential and integral components of the fully assembled microtubule. These proteins have molecular weights greater than 200,000 daltons. Fluorescent labelled antibodies to platelet and brain tubulins stain long filamentous microtubular structures in bovine lens epithelial cells and this pattern of staining is prevented by exposing the cells to conditions known to cause depolymerisation of cell microtubules.

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