Partial purification of phosphodiesterase I from microsomes of rat intestinal mucosa

1970 ◽  
Vol 48 (10) ◽  
pp. 1141-1150 ◽  
Author(s):  
I. Hynie ◽  
S. H. Zbarsky
Keyword(s):  
Aqueous Phase ◽  
Intestinal Mucosa ◽  
Phosphate Buffer ◽  
High Speed ◽  
Deae Cellulose ◽  
Amyl Alcohol ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Enzyme Solution ◽  
Alkaline Phosphomonoesterase

Intestinal mucosa was homogenized in Krebs–Ringer phosphate buffer, pH 7.4, containing 6% Dextran. After centrifugation, the sediment was rehomogenized in 0.24 M sucrose and the homogenate centrifuged. The supernatant material from both steps was combined and centrifuged at high speed through a layer of 25% sucrose to yield a pellet of microsomes. This pellet was suspended in Tris–HCl buffer, pH 8.4, made isotonic in KCl, and the suspension was sonicated and centrifuged. A suspension of the sediment in Tris–HCl buffer was shaken with t-amyl alcohol to yield the soluble enzyme in the aqueous phase. The enzyme was purified further by chromatography on DEAE-cellulose using elution with a KCl gradient in Tris–HCl buffer, pH 8, made 6 M in urea. Phosphodiesterase I and alkaline phosphomonoesterase were eluted in peaks which overlapped only partially enabling the collection of phosphodiesterase I free of Phosphomonoesterase. The enzyme solution was concentrated and freed of urea by pressure dialysis.

scholarly journals Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 377-388 ◽  
Author(s):  
BL Evatt ◽  
J Levin ◽  
KM Algazy
Keyword(s):  
Ammonium Sulfate ◽  
Phosphate Buffer ◽  
Carboxymethyl Cellulose ◽  
Room Temperature ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Elution Volume ◽  
Ammonium Sulfate Saturation ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Abstract Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.

scholarly journals THE ENZYMATIC HYDROLYSIS OF AMINO ACID β-NAPHTHYLAMIDES II. PARTIAL PURIFICATION AND PROPERTIES OF A PARTICLE-BOUND COBALT-ACTIVATED RAT KIDNEY AMINOPEPTIDASE

1966 ◽  
Vol 14 (5) ◽  
pp. 401-413 ◽  
Author(s):  
K. FELGENHAUER ◽  
G. G. GLENNER
Keyword(s):  
Amino Acid ◽  
Rat Kidney ◽  
Deae Cellulose ◽  
Enzymic Activity ◽  
Disc Electrophoresis ◽  
Partial Purification ◽  
Soluble Enzyme ◽  
Solvent Fractionation ◽  
Biochemical Systems ◽  
Hydrolysis Of

In order to determine the relationship and characteristics of the soluble and tissue-bound (so-called "lyo" and "desmo") components of enzymes in histochemical and biochemical systems, a biochemical investigation of the hydrolysis of l-leucyl β-naphthylamide in rat kidney was undertaken. By ultracentrifugation, autolysis, salt and solvent fractionation procedures and DEAE-cellulose column chromatography primary soluble and tissuue-bound enzymes were separated and partially purified. The soluble enzyme was found to be inhibited by p-chloromercuribenzoate and reactivated by cysteine. The tissue-bound enzyme was found to be inhibited by o-phenanthroline and reactivated by Co++, and hydrolyzed peptides and amino acid β-naphthylamides at rates differing markedly from those of the soluble enzyme and commercial leucine aminopeptidase. Disc electrophoresis of the tissue-bound enzyme preparation demonstrated two protein bands, both revealing enzymic activity with almost identical hydrolytic characteristics, i.e., isozymes. This evidence established the identity of the tissue-bound Co++-activated enzyme with that found histochemically in the brush border of the proximal tubuli, and indicated its functional capacity as that of an aminopolypeptidase. It was also concluded that in the system investigated the "lyo" and "desmo" components represented, in reality, distinct enzymes with unique characteristics.

scholarly journals Partial purification of thrombopoietin from the plasma of thrombocytopenic rabbits

Blood ◽  
1979 ◽  
Vol 54 (2) ◽  
pp. 377-388
Author(s):  
BL Evatt ◽  
J Levin ◽  
KM Algazy
Keyword(s):  
Ammonium Sulfate ◽  
Phosphate Buffer ◽  
Carboxymethyl Cellulose ◽  
Room Temperature ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Elution Volume ◽  
Ammonium Sulfate Saturation ◽  
Citrate Phosphate ◽  
Citrate Phosphate Buffer

Partially purified thrombopoiesis-stimulating activity was prepared from the plasma of thrombocytopenic rabbits using ammonium sulfate precipitation and DEAE cellulose, Sephadex, and carboxymethyl cellulose chromatography. The protein fraction precipitated by an ammonium sulfate saturation of 60%-80%, previously shown to contain thrombopoiesis-stimulating activity, was used as starting material. Column chromatography was carried out at room temperature at pH 5.6. Under these conditions, thrombopoiesis-stimulating activity (thrombopoietin) was retained by DEAE cellulose (0/03 M citrate- phosphate buffer) and carboxymethyl cellulose (0/003 M citrate- phosphate buffer), and eluted with 0.4 M NaCl. Thrombopoietin was retarded by Sephadex G-100; the ratio of the elution volume to the void volume was 1.32:1. Immunoelectrophoretic analysis of partially purified thrombopoietin indicated that following removal of most of the albumin by DEAE chromatography, only proteins with the mobilities of beta- globulins and albumin and traces of other anodally migrating proteins were detectable in the fractions that contained thrombopoiesis- stimulating activity. Thrombopoietin was not dialyzable and was stable from at least pH 5.6 to 7.5. It was approximately 1000-fold purified following sequential chromatography with DEAE and carboxymethyl cellulose. Although the three fractions described reproducibly stimulated thrombopoiesis, as measured by increased levels of selenomethionine-75Se (75SeM) in the circulating platelets, platelet counts did not increase.

Partial Purification and Some Properties of a Hydroxycinnamoyl Glucosyltransferase from Tomato Fruits

1980 ◽  
Vol 35 (11-12) ◽  
pp. 967-972 ◽  
Author(s):  
A. Fleuriet ◽  
J. J. Macheix ◽  
R. Suen ◽  
R. K. Ibrahim
Keyword(s):  
Ferulic Acid ◽  
Metal Ions ◽  
Deae Cellulose ◽  
Divalent Metal ◽  
Partial Purification ◽  
Divalent Metal Ions ◽  
Tomato Fruits ◽  
Ammonium Sulphate Precipitation ◽  
Sh Group ◽  
Cherry Tomatoes

A glucosyltransferase was isolated from immature “cherry” tomatoes and was partially purified (200-fold) by ammonium sulphate precipitation and successive chromatography on Sephadex G-100 and DEAE-cellulose columns. The enzyme utilised the free hydroxycinnamic acids and UDP-glucose in the formation of their respective glucosides (pH 8.0) and glucose esters (pH 7.0); but did not accept the CoA thiolesters of HCAs in the presence of glucose-1-phosphate. The constant glucoside/glucose ester ratio observed during purification suggests that both reactions are catalysed by the same enzyme. The Km values for ρ-coumaric, caffeic, ferulic and sinapic acids were 0.8, 1.5, 1.4 and 2.5 μᴍ, respectively. With ferulic acid as substrate, the Km value for UDPG was 10 μᴍ. The enzyme required an -SH group for activity and the reaction was strongly inhibited by EDTA, divalent metal ions and UDP.

Roles of ET-1 in endotoxin-induced microcirculatory disturbance in rat small intestine

1996 ◽  
Vol 271 (3) ◽  
pp. G461-G469 ◽  
Author(s):  
S. Miura ◽  
D. Fukumura ◽  
I. Kurose ◽  
H. Higuchi ◽  
H. Kimura ◽  
...  
Keyword(s):  
Nitric Oxide ◽  
Intestinal Mucosa ◽  
High Speed ◽  
Platelet Activating Factor ◽  
Video Camera ◽  
Mucosal Damage ◽  
Dependent Manner ◽  
Camera System ◽  
Eta Receptor Antagonist ◽  
Leukocyte Sticking

The major objective of this study was to investigate whether endothelin-1 (ET-1) plays a significant role in endotoxin-induced microcirculatory disturbances of the intestinal mucosa. Submucosal microvessels of the rat ileum were observed by intravital microscopy with a high-speed video camera system. Preceding the apparent intestinal mucosal damage, red blood cell (RBC) velocity was significantly decreased 30 min after endotoxin treatment in both arterioles and venules. The number of leukocytes sticking to submucosal venules was significantly increased at 30 min. BQ-123, an ETA-receptor antagonist, significantly attenuated the decrease in RBC velocity and also prevented an increase in leukocyte sticking as well as the subsequent mucosal damage induced by endotoxin. The ET-1 concentrations began to be elevated in plasma at 15 min and in the mucosa at 30 min and subsequently further increased in a time-dependent manner. A significant decrease in calcium-dependent nitric oxide synthase activity and significant increases in the concentration of platelet-activating factor (PAF) were demonstrated in the intestinal mucosa after endotoxin treatment. BQ-123 also significantly attenuated these changes. We concluded that the increased ET-1 production in intestinal mucosa induced by endotoxin stimulation could lead to leukocyte sticking and decreased RBC velocity in the intestinal microcirculatory beds via ETA receptors, which are closely related to increased production of PAF and decreased synthesis of constitutive nitric oxide.

Guanosine 3’:5’-Cyclic Monophosphate -Dependent Particulate Protein Kinase Activity from Yeast (Saccharomyces cerevisiae)

1999 ◽  
Vol 54 (1-2) ◽  
pp. 84-93 ◽  
Author(s):  
Hans Eckstein ◽  
Birgit Flügge
Keyword(s):  
Protein Kinase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Deae Cellulose ◽  
Temperature Tolerance ◽  
Maximum Activity ◽  
Partial Purification ◽  
Protein Kinase Activity ◽  
Ph Optimum ◽  
Yeast Saccharomyces Cerevisiae ◽  
High Concentrations

Continuing our studies on cGMP in growing yeast we detected a particulate cGMPdependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 °C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 °C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G’s, but also of Pk-A’s

scholarly journals Purification of phosphodiesterase II from rat and guinea-pig intestinal mucosa

1976 ◽  
Vol 155 (3) ◽  
pp. 607-613 ◽  
Author(s):  
P R Flanagan ◽  
S H Zbarsky
Keyword(s):  
Guinea Pig ◽  
Intestinal Mucosa ◽  
Deae Cellulose ◽  
Large Loss ◽  
Arrhenius Plots ◽  
The Poor ◽  
Km Value ◽  
Soluble Material

Phosphodiesterase II from extracts of intestinal mucosa of rat and guinea pig was purified by chromatography on DEAE-cellulose, CM-cellulose and agarose. The rat enzyme was purified 350-550-fold, with recoveries ranging up to 46%. The best purification of the guinea-pig enzyme was 15-fold, and the recovery was only 2.6%, the large loss occurring during chromatography on DEAE-cellulose and agarose. The poor results with the guinea-pig enzyme reflect the difficulty in obtaining a truly soluble material. Repeated sonication of the crude guinea-pig preparations yielded material that was initially soluble but tended to re-aggregate quickly. Purification of the rat phosphodiesterase II increased its thermostability, the temperature of half-inactivation being increased from 54degrees to 60degreesC. Both enzymes had a Km value of 4 × 10(-5) M with thymidine 3′-(2,4-dinitrophenyl) phosphate as substrate and showed similar pH optima for activity. Both enzymes were inhibited slightly in 0.1 M-MgC12 or 2M-urea and much more strongly in 2M-(NH4)2SO4 or 6M-NaC1. The guinea-pig enzyme was usually inhibited more than the rat enzyme. The Arrhenius plots of the two enzymes differed slightly in slope, but both were biphasic, showing breaks between 30degrees and 40degreesC. It was concluded that the two enzymes were markedly similar in behaviour and that the differences found were related to the different degrees of purification attained by the procedures described.

Isolation and some properties of extracellular heat-stable lipases fromPseudomonas fluorescensstrain AFT 36

1983 ◽  
Vol 50 (1) ◽  
pp. 77-89 ◽  
Author(s):  
Patrick F. Fox ◽  
Leszek Stepaniak
Keyword(s):  
Phosphate Buffer ◽  
Lipolytic Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Lipase Production ◽  
Maximum Activity ◽  
Temperature Range ◽  
Milk Serum ◽  
Heat Stable ◽  
Ph Range

SummaryAeration increased the growth and lipase production in milk byPseudomonas fluorescensstrain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented ∼ 71 % of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8·0 and 35 °C; it had aKmon tributyrin of 3·65 mM. and was inhibited by concentrations of substrate > ∼ 17 mM. The enzyme was very stable over the pH range 6–9; it was relatively heat-labile in phosphate buffer in the temperature range 60–80 °C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100–150 °C: theDvalues at 150 °C were ∼ 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the correspondingZvalues in the temperature range 100–150 °C were ∼ 40 and ∼ 42 °C and theEafor inactivation were 7·65 × 104J mol-1and 6·97 × 104J mol-1respectively.

The Separation of two Deoxyribonucleases from Extracts of Intestinal Mucosa of the Rat

1972 ◽  
Vol 50 (6) ◽  
pp. 697-703 ◽  
Author(s):  
C. Y. Lee ◽  
Sheilia Lawrence ◽  
S. H. Zbarsky
Keyword(s):  
Intestinal Mucosa ◽  
Ammonium Acetate ◽  
Divalent Cations ◽  
Deae Cellulose ◽  
Sodium Formate ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Dnase Ii ◽  
Ammonium Acetate Buffer ◽  
Optimum Activity

Two deoxyribonucleases have been demonstrated in cell-free extracts of rat intestinal mucosa. The enzymes were separated by ion-exchange chromatography on DEAE-cellulose and further purified by partition on hydroxyapatite. One DNase had optimum activity at pH 6.8–7.0 in ammonium acetate buffer, required Mn2+ or Mg2+ for activity, and was inhibited by EDTA. This enzyme is a DNase I by Laskowski's criteria Advan. Enzymol. 29, 165 (1967). The second enzyme was a DNase II with optimum activity at pH 3.5–4.0 in sodium formate buffer, was not inhibited by EDTA, and showed no requirement for divalent cations. Both enzymes were active only with native DNA and had no action on heated DNA or purified yeast transfer RNA.

Type I nitroreductases of Escherichia coli

1981 ◽  
Vol 27 (1) ◽  
pp. 81-86 ◽  
Author(s):  
D. W. Bryant ◽  
D. R. McCalla ◽  
M. Leeksma ◽  
P. Laneuville
Keyword(s):  
Escherichia Coli ◽  
Reductase Activity ◽  
Deae Cellulose ◽  
Partial Purification ◽  
Type I ◽  
Activity Type ◽  
Purification Step ◽  
The Past ◽  
Type Ia ◽  
Resistant Strains

Analysis of partially purified crude extract of Escherichia coli K12 by chromatography and gel electrophoresis has resulted in the separation of three distinct activities which catalyse the reduction of nitrofurazone (semicarbazone of 5-nitro-2-furaldehyde) in the presence of oxygen (type I nitroreductases). The major enzymatic activity (type IA), which was dependent solely on NADPH as a cofactor, was absent from nitrofurazone-resistant strains NFR 402 and NFR 502, but present in SIL 41, a strain which is only marginally resistant to the nitrofuran. The remaining nitroreductase activities (IB1 and IB2) utilize either NADH or NADPH as a cofactor. These activities coelute from DEAE-cellulose at pH 7.2, but may be differentiated by their behaviour on CM-cellulose at pH 5.8. The reductase activity missing in SIL 41 was observed in extracts of strain NFR 402 but not NFR 502. This enzyme (IB1) though retained by DEAE-cellulose had no affinity for CM-cellulose. The only reductase present in extracts of NFR 502 (a nitrofuran-resistant strain selected after two mutational events) was type IB2. This activity, also detectable in SIL 41 and NFR 402, has not been mapped genetically. An interesting feature of the type IB2 enzyme is its apparent inactivation by MnCl2 which has been routinely used as a partial purification step in the past.

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