scholarly journals Guanine deaminase in rat liver and mouse liver and brain

1972 ◽  
Vol 128 (5) ◽  
pp. 1079-1088 ◽  
Author(s):  
K. S Kumar ◽  
A. Sitaramayya ◽  
P. S. Krishnan
Keyword(s):  
Rat Brain ◽  
Rat Liver ◽  
Inhibitory Activity ◽  
Substrate Concentration ◽  
Mouse Liver ◽  
Deae Cellulose ◽  
The Other ◽  
Guanine Deaminase ◽  
Sigmoidal Kinetics ◽  
Km Value

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis–Menten kinetics. The Km value of enzyme A for guanine was 5.3μm and that of enzyme B 20μm. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the Km values for the reaction with guanine were respectively 5 and 66μm. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.

scholarly journals Modulation of guanine deaminase

1973 ◽  
Vol 131 (4) ◽  
pp. 683-687 ◽  
Author(s):  
K. Sree Kumar ◽  
A. Sitaramayya ◽  
P. S. Krishnan
Keyword(s):  
Rat Brain ◽  
Substrate Concentration ◽  
Mouse Liver ◽  
Deae Cellulose ◽  
Supernatant Fraction ◽  
Saturation Curve ◽  
Guanine Deaminase ◽  
Sigmoidal Kinetics ◽  
Substrate Saturation

1. Guanine deaminases purified from the 15000g supernatant fraction of iso-osmotic sucrose homogenates of rat and mouse liver and brain were tested for the influence of GTP and allantoin. 2. The suffixes A and B were assigned to the isoenzyme fractions eluted from DEAE-cellulose with the lower and the higher molarity of eluent respectively. Isoenzyme A from rat liver, the activity of which showed a sigmoid dependence on substrate saturation, was activated by GTP and inhibited by allantoin. Isoenzyme B, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin. 3. Isoenzyme A from rat brain, the activity of which had a sigmoid dependence on substrate concentration, was stimulated by GTP. Isoenzyme B, which showed classical Michaelis–Menten kinetics, was inhibited by allantoin. 4. Mouse liver guanine deaminase was not influenced by either GTP or allantoin. 5. Isoenzyme A from mouse brain, which had a hyperbolic substrate-saturation curve, was not influenced by GTP or allantoin but isoenzyme B, with sigmoidal kinetics, was inhibited by allantoin. 6. Mg2+ activated, or inhibited or did not have an effect on guanine deaminase, depending on the source of the enzyme. 7. The bearing of the above findings on the possible regulation of guanine deaminase activity in vivo is discussed.

scholarly journals The fractionation of nuclei from mammalian cells by zonal centrifugation

1968 ◽  
Vol 109 (1) ◽  
pp. 127-135 ◽  
Author(s):  
I R Johnston ◽  
A P Mathias ◽  
F. Pennington ◽  
D. Ridge
Keyword(s):  
Rat Liver ◽  
Mammalian Cells ◽  
Mouse Liver ◽  
The Other ◽  
Slow Speed ◽  
Adult Rats ◽  
Sucrose Solutions ◽  
Liver Nuclei ◽  
Effect Of Age ◽  
Zonal Rotor

1. Purified liver nuclei from adult rats separate into two main zones when centrifuged in the slow-speed zonal rotor. One zone contains diploid nuclei, the other tetraploid. 2. The effect of age on the pattern of rat liver ploidy was examined. Tetraploid nuclei are virtually absent from young animals. They increase in proportion steadily with age. Partial hepatectomy disturbs the pattern of ploidy. 3. The zonal centrifuge permits the separation of diploid, tetraploid, octaploid and hexadecaploid nuclei from mouse liver. 4. Rat liver nuclei are isopycnic with sucrose solutions of density 1·35 at 5°.

scholarly journals Guanine deaminase inhibitor from rat liver. Isolation and characterization

1974 ◽  
Vol 137 (1) ◽  
pp. 85-92 ◽  
Author(s):  
Shahid Ali ◽  
A. Sitaramayya ◽  
K. Sree Kumar ◽  
Padmanabhan S. Krishnan
Keyword(s):  
Outer Membrane ◽  
Rat Liver ◽  
Active Form ◽  
Deae Cellulose ◽  
Heat Inactivation ◽  
Rat Liver Mitochondria ◽  
Inhibitor Activity ◽  
Isolation And Characterization ◽  
Guanine Deaminase ◽  
Triton X

1. An inhibitor of cytoplasmic guanine deaminase of rat liver was isolated from liver ‘heavy mitochondrial’ fraction after freezing and thawing and treatment with Triton X-100. 2. Submitochondrial fractionation revealed that the inhibitor was localized in the outer-membrane fraction. 3. The method of purification of inhibitor, involving precipitation with (NH4)2SO4 and chromatography on DEAE-cellulose, its precipitability by trichloroacetic acid and the pattern of absorption in the u.v. indicated that the inhibitor was a protein. In confirmation, tryptic digestion of the isolated material resulted in destruction of the inhibitor activity. The inhibitor was stable to acid, but labile to heat. 4. The isolated inhibitor required phosphatidylcholine (lecithin) for activity. Phosphatidylcholine also partially protected the inhibitor against heat inactivation. 5. When detergent treatment was omitted, the inhibitor activity of frozen mitochondria was precipitated by (NH4)2SO4 in a fully active form without supplementation with phosphatidylcholine, indicating that Triton X-100 ruptured the linkage between inhibitor and lipid. 6. A reconstituted sample of inhibitor–phosphatidylcholine complex was precipitated in a fully active form by dialysis against 2-mercaptoethanol, but treatment of the precipitate with NaCl yielded an extract which was inactive unless supplemented with fresh phosphatidylcholine. 7. We interpret the results as evidence that the inhibitor was present in vivo as a lipoprotein and that once the complex was dissociated by the action of detergent and the protein precipitated, there was an absolute need for exogenous phosphatidylcholine for its activity. The manner in which inhibitor associated with the outer membrane of rat liver mitochondria might regulate the activity of the enzyme in the supernatant has been suggested.

scholarly journals Comparative studies on the 25-hydroxylations of cholecalciferol and 1α-hydroxycholecalciferol in perfused rat liver

1978 ◽  
Vol 170 (3) ◽  
pp. 495-502 ◽  
Author(s):  
Masafumi Fukushima ◽  
Yasuho Nishii ◽  
Michiko Suzuki ◽  
Tatsuo Suda
Keyword(s):  
Vitamin D ◽  
Rat Liver ◽  
Linear Relation ◽  
Comparative Studies ◽  
Similar Rate ◽  
The Other ◽  
Perfused Rat Liver ◽  
Km Value ◽  
Derivatives Of ◽  
25 Hydroxycholecalciferol

The 25-hydroxylations of [3H]cholecalciferol and 1α-hydroxy[3H]cholecalciferol in perfused rat liver were compared. Results showed that about twice as much 1α(OH)D3 (1α-hydroxycholecalciferol) was incorporated into the liver as cholecalciferol. 25-Hydroxy[3H]cholecalciferol and 1α-25-dihydroxy[3H]cholecalciferol were not incorporated significantly. Livers isolated from vitamin D-deficient rats formed the 25-hydroxy derivatives of cholecalciferol and 1α(OH)D3 respectively linearly with time for at least 120min. The rate of 1α,25(OH)2D3 (1α,25-dihydroxycholecalciferol) production increased exactly 10-fold on successive 10-fold increases in the dose of 1α(OH)D3, suggesting that hepatic 25-hydroxylation of 1α(OH)D3 is not under metabolic control. On the other hand, the rate of conversion of cholecalciferol into 25(OH)D3 (25-hydroxycholecalciferol) did not increase linearly with increase in the amount of cholecalciferol in the perfusate. The 25-hydroxylation of cholecalciferol seemed to proceed at a similar rate to that of 1α(OH)D3 at doses of less than 1nmol, but with doses of more than 2.5nmol, the conversion of cholecalciferol into 25(OH)D3 became much less efficient, though the linear relation between the amounts of substrate and product was maintained. A reciprocal plot of data on the 25-hydroxylation of cholecalciferol gave two Km values of about 5.6nm and 1.0μm, whereas that for the 25-hydroxylation of 1α(OH)D3 gave a single Km value of about 2.0μm. These results suggest that there are two modes of 25-hydroxylation of cholecalciferol in the liver, which seem to be closely related to the mechanism of control of 25(OH)D3 production by the liver.

Properties of 1-phosphofructokinase from Pseudomonas piutida

1977 ◽  
Vol 23 (6) ◽  
pp. 721-725 ◽  
Author(s):  
Sookie S. Bang ◽  
Paul Baumann ◽  
Mark H. Sawyer
Keyword(s):  
Pseudomonas Putida ◽  
Adenosine Triphosphate ◽  
Column Chromatography ◽  
Inhibitory Activity ◽  
Reaction Rate ◽  
Adenosine Monophosphate ◽  
The Other ◽  
Kinetic Properties ◽  
Sephadex Column ◽  
Sigmoidal Kinetics

The 1-phosphofructokinase (1-PFK, EC 2.7.1.56) from Pseudomonas putida was partially purified by a combination of (NH4)2SO4 fractionation and DEAE-Sephadex column chromatography. In its kinetic properties, this enzyme resembled the 1-PFK's from other bacteria. With the substrates fructose-1-phosphate (F-1-P) and adenosine triphosphate (ATP) Michaelis–Menten kinetics were observed, the Km for one substrate being unaffected by a variation in the concentration of the other substrate. At pH 8.0, the Km values for F-1-P and ATP were 1.64 × 10−4 M and 4.08 × 10−4 M, respectively. At fixed concentrations of F-1-P and ATP, an increase in the Mg2+ resulted in sigmoidal kinetics. Activity was inhibited by ATP when the ratio of ATP:Mg2+ was greater than 0.5 suggesting that ATP:2 Mg2+ was the substrate and free ATP was inhibitory. Activity of 1-PFK was stimulated by K+ and to a lesser extent by NH4+ and Na+. The reaction rate was unaffected by 2 mM K2HPO4, pyruvate, phosphoenolpyruvate, adenosine monophosphate, adenosine 3′,5′-cyclic monophosphate, fructose-6-phosphate, glucose-6-phosphate, 6-phosphogluconate, 2-keto-3-deoxy-6-phosphogluconate, or citrate. The results indicated that the 1-PFK from P. putida was not allosterically regulated by a number of metabolites which may play an important role in the catabolism of D-fructose.

scholarly journals Purification from rat liver of an enzyme that catalyses the sulphurylation of phenols

1971 ◽  
Vol 123 (5) ◽  
pp. 901-906 ◽  
Author(s):  
F. A. McEvoy ◽  
J. Carroll
Keyword(s):  
Enzyme Activity ◽  
Methyl Ester ◽  
Rat Liver ◽  
The Other ◽  
Male Rat ◽  
Thiol Compounds ◽  
Km Value ◽  
Rat Livers

1. An enzyme (EC 2.8.2.1) that catalyses the transfer of sulphate from adenosine 3′-phosphate 5′-sulphatophosphate to phenols was purified approx. 2000-fold from male rat livers. 2. The purified preparation did not catalyse the sulphurylation of dehydroepiandrosterone, butan-1-ol, l-tyrosine methyl ester, 1-naphthylamine or serotonin. 3. At pH8.0 and 37°C the Km values of the enzyme for p-nitrophenol and adenosine 3′-phosphate 5′-sulphatophosphate are 51 and 14μm respectively. The Km value for either substrate is independent of the concentration of the other. 4. The sulphurylation of phenol is inhibited by thiol compounds and glutathione at a concentration of 3mm caused an approx. 50% decrease in enzyme activity. 5. The Km of the enzyme for adenosine 3′-phosphate 5′-sulphatophosphate is unaffected by the presence of added glutathione but at a concentration of 5mm-glutathione the Km of the enzyme for its phenolic substrate is decreased.

scholarly journals Induction of guanine deaminase and its inhibitor in rodent liver and brain

1974 ◽  
Vol 138 (2) ◽  
pp. 143-146 ◽  
Author(s):  
A. Sitaramayya ◽  
Shahid Ali ◽  
K. Sree Kumar ◽  
P. S. Krishnan
Keyword(s):  
Inhibitory Activity ◽  
Mouse Liver ◽  
Actinomycin D ◽  
Rat Tissues ◽  
Maximum Induction ◽  
Guanine Deaminase ◽  
Mouse Tissues

1. Guanine deaminase activities in homogenates and supernatant fractions of liver and brain of rat and mouse were elevated by administration of guanine to the animals. The maximum induction in mouse tissues occurred within 24h and in rat tissues within 48h. 2. Mitochondria of rat (but not mouse) liver and brain contain an inhibitor of supernatant guanine deaminase, and this was also increased by guanine treatment. 3. Administration of ethionine, cycloheximide or actinomycin D prevented the guanine-dependent increase in deaminase activity and also the increase in mitochondrial inhibitory activity; chloramphenicol suppressed only the latter.

The Effects of Some Synthetic Compounds on In Vitro Fibrinolytic Activity Measured by Different Methods and the Relevance to Activity In Vivo

1972 ◽  
Vol 28 (03) ◽  
pp. 351-358
Author(s):  
A.J Baillie ◽  
A. K Sim
Keyword(s):  
Inhibitory Activity ◽  
Substrate Concentration ◽  
Fibrinolytic Activity ◽  
Synthetic Compounds ◽  
In Vitro Methods ◽  
Narrow Concentration Range

SummaryThe activity of several synthetic compounds, rated from good to poor (or inactive) fibrinolytic activators, has been assessed by two different commonly-used in vitro methods. Compounds shown to be active over a narrow concentration range in the hanging clot test were shown to be inhibitors of plasmin and trypsin in the casein-olytic test. The inhibitory activity of these compounds was shown to increase with increasing substrate concentration and apparent activity in the hanging clot test. Possible explanations and relevance of these observations are discussed.

scholarly journals Ganglioside incorporation and release by the isolated perfused rat liver

1991 ◽  
Vol 274 (2) ◽  
pp. 581-585 ◽  
Author(s):  
S C Kivatinitz ◽  
A Miglio ◽  
R Ghidoni
Keyword(s):  
Rat Liver ◽  
The Other ◽  
Regulatory Role ◽  
Isolated Perfused Rat Liver ◽  
Order Kinetic ◽  
Perfused Rat Liver ◽  
Ganglioside Gm1 ◽  
First Order ◽  
Pseudo First Order

The fate of exogenous ganglioside GM1 labelled in the sphingosine moiety, [Sph-3H]GM1, administered as a pulse, in the isolated perfused rat liver was investigated. When a non-recirculating protocol was employed, the amount of radioactivity in the liver and perfusates was found to be dependent on the presence of BSA in the perfusion liquid and on the time elapsed after the administration of the ganglioside. When BSA was added to the perfusion liquid, less radioactivity was found in the liver and more in the perfusate at each time tested, for up to 1 h. The recovery of radioactivity in the perfusates followed a complex course which can be described by three pseudo-first-order kinetic constants. The constants, in order of decreasing velocity, are interpreted as: (a) the dilution of the labelled GM1 by the constant influx of perfusion liquid; (b) the washing off of GM1 loosely bound to the surface of liver cells; (c) the release of gangliosides from the liver. Process (b) was found to be faster in the presence of BSA, probably owing to the ability of BSA to bind gangliosides. The [Sph-3H]GM1 in the liver underwent metabolism, leading to the appearance of products of anabolic (GD1a, GD1b) and catabolic (GM2, GM3) origin; GD1a appeared before GM2 and GM3 but, at times longer than 10 min, GM2 and GM3 showed more radioactivity than GD1a. At a given time the distribution of the radioactivity in the perfusates was quite different from that of the liver. In fact, after 60 min GD1a was the only metabolite present in any amount, the other being GM3, the quantity of which was small. This indicates that the liver is able to release newly synthesized gangliosides quite specifically. When a recirculating protocol was used, there were more catabolites and less GD1a than with the non-recirculating protocol. A possible regulatory role of ganglioside re-internalization on their own metabolism in the liver is postulated.

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