scholarly journals CYTOCHEMICAL DEMONSTRATION OF ORNITHINE CARBAMOYLTRANSFERASE ACTIVITY IN LIVER MITOCHONDRIA OF RAT AND MOUSE

1968 ◽  
Vol 16 (3) ◽  
pp. 172-180 ◽  
Author(s):  
AKIRA MIZUTANI
Keyword(s):  
Lead Nitrate ◽  
Liver Mitochondria ◽  
Ornithine Carbamoyltransferase ◽  
Carbamoyl Phosphate ◽  
Acid And Alkaline Phosphatase ◽  
Cytochemical Demonstration ◽  
Brush Borders ◽  
Liver And Kidney ◽  
Cold Acetone ◽  
Tris Maleate

In order to demonstrate ornithine carbamoyltransferase activity cytochemically, thin slices of liver and kidney of rat and mouse were fixed in cold acetone or formol-calcium, and incubated in a medium containing l-ornithine, carbamoyl phosphate, Tris-maleate buffer (pH 7.2), lead nitrate and sucrose. The specific reaction product occurred in the mitochondria of the hepatocytes only, and not in other cells of the liver or kidney. The specificity of the reaction was supported by the following observations. (1) The mitochondrial reaction was not obtained in sections incubated in a medium from which ornithine was omitted. (2) Other amino acids gave no reaction. (3) p-Chloromercuribenzoate suppressed the reaction. (4) The hepatocytes of chick (uricotelic) did not give the reaction. A nonspecific reaction in lysosomes and brush borders is caused probably by acid and alkaline phosphatase activities.

scholarly journals Altered enzyme activities and citrulline synthesis in liver mitochondria from ornithine carbamoyltransferase-deficient sparse-furash mice

1989 ◽  
Vol 257 (1) ◽  
pp. 251-257 ◽  
Author(s):  
N S Cohen ◽  
C W Cheung ◽  
L Raijman
Keyword(s):  
Protein Interactions ◽  
Mitochondrial Protein ◽  
Normal Liver ◽  
Liver Mitochondria ◽  
Synthetase Activity ◽  
Single Mutation ◽  
Ornithine Carbamoyltransferase ◽  
Carbamoyl Phosphate Synthetase ◽  
Carbamoyl Phosphate ◽  
Citrulline Synthesis

Male mice carrying the spfash mutation have 5-10% of the normal activity of ornithine carbamoyltransferase, yet are only slightly hyperammonaemic and develop quite well. A study of liver mitochondria from normal and spfash males showed that they differ in important ways. (1) The spfash liver contains about 33% more mitochondrial protein per g than does normal liver. (2) The specific activities of carbamoyl-phosphate synthetase (ammonia) and glutamate dehydrogenase are about 15% lower than normal in mitochondria from spfash mice, whereas those of beta-hydroxybutyrate dehydrogenase and cytochrome oxidase are 22% higher and 30% lower respectively. (3) In the presence of 10 mM-ornithine and the substrates for carbamoyl phosphate synthesis, coupled and uncoupled mitochondria from spfash mice synthesize citrulline at unexpectedly high rates, about 25 and 44 nmol/min per mg respectively. Though these are somewhat lower than the corresponding rates obtained with normal mitochondria, the difference does not arise from the deficiency in ornithine carbamoyltransferase, but from the lower carbamoyl-phosphate synthetase activity of the mutant mitochondria. (4) At lower external [ornithine] (less than 2 mM), a smaller fraction of the carbamoyl phosphate synthesized is converted into citrulline in spfash than in normal mitochondria. These studies show that what appears to be a single mutation brings about major adaptations in the mitochondrial component of liver. In addition, they clarify the role of ornithine transport and of protein-protein interactions in citrulline synthesis in normal mitochondria.

scholarly journals Kinetic properties of carbamoyl-phosphate synthase (ammonia) and ornithine carbamoyltransferase in permeabilized mitochondria

1992 ◽  
Vol 282 (1) ◽  
pp. 173-180 ◽  
Author(s):  
N S Cohen ◽  
C W Cheung ◽  
E Sijuwade ◽  
L Raijman
Keyword(s):  
De Novo ◽  
Functional Organization ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Kinetic Properties ◽  
Ornithine Carbamoyltransferase ◽  
Carbamoyl Phosphate ◽  
The Matrix ◽  
Phosphate Synthase

Previous studies using intact rat liver mitochondria have shown that the soluble matrix enzymes carbamoyl-phosphate synthase (ammonia) (CPS) and ornithine carbamoyltransferase (OCT) display some kinetic properties which would not be observed if they were homogeneously distributed in the matrix. In the present work we have extended these studies, using toluene-treated mitochondria which are fully permeable to substrates and inhibitors, yet retain 90% of their soluble enzymes. The results provide evidence of functional organization of CPS and OCT in situ. The major findings are as follows. (1) The apparent Km values of matrix OCT for carbamoyl phosphate and ornithine are respectively 8 and 2 times those measured for the soluble enzyme. delta-N-Phosphonacetyl-L-ornithine inhibits OCT in situ less than in solution, especially when carbamoyl phosphate is synthesized in the mitochondria rather than added to the medium. (2) During citrulline synthesis from endogenously generated carbamoyl phosphate, the concentration of the latter in permeabilized mitochondria is more than 10 times that in the medium, although the mitochondria are freely permeable to added molecules of this size. (3) Endogenously formed carbamoyl phosphate is used preferentially by OCT in situ; addition of a 200-fold excess of unlabelled carbamoyl phosphate has little effect on the conversion of labelled endogenously formed carbamoyl phosphate into citrulline by matrix OCT. (4) The synthesis de novo of carbamoyl phosphate from NH3, HCO3- and ATPMg is the same in the presence and absence of ornithine. (5) Studies with co-immobilized CPS and OCT gave results concordant with some of the above observations and with previous ones with intact mitochondria.

scholarly journals HISTOCHEMICAL DEMONSTRATION OF ACID PHOSPHATASE ACTIVITY IN OSTEOCLASTS

1959 ◽  
Vol 7 (1) ◽  
pp. 39-41 ◽  
Author(s):  
M. S. BURSTONE
Keyword(s):  
Alkaline Phosphatase ◽  
Acid Phosphatase ◽  
Phosphatase Activity ◽  
Comparative Studies ◽  
Acid Phosphatase Activity ◽  
Azo Dye ◽  
Histochemical Demonstration ◽  
Accurate Localization ◽  
Acid And Alkaline Phosphatase ◽  
Cold Acetone

High acid phosphatase activity was observed in osteoclasts of several species using a reproducible azo-dye technique. High activity of two distinct enzymes, acid and alkaline phosphatase, are associated with osteoclasts and osteoblasts respectivey. Althouth frozen-dried tissues are recommended for definitive studies, the enzyme techniques used give satisfactory results with cold acetone-fixed tissues. The most accurate localization of acid phosphatase in osteoclasts in controlled comparative studies is obtained with double-embedded frozen-dried undecalcified tissues in conjunction with naphthol AS-phosphates.

Effect of Garlic Oil on the Levels of Various Enzymes in the Serum and Tissue of Streptozotocin Diabetic Rats

2001 ◽  
Vol 21 (1) ◽  
pp. 19-24 ◽  
Author(s):  
O. C. Ohaeri
Keyword(s):  
Serum Amylase ◽  
Diabetic Control ◽  
Diabetic Rats ◽  
Red Cell ◽  
Alkaline Phosphatases ◽  
Garlic Oil ◽  
Acid And Alkaline Phosphatase ◽  
Liver And Kidney ◽  
Streptozotocin Diabetic Rats ◽  
Aspartate Transferase

Levels of red cell, serum acid, and alkaline phosphatases, serum amylase, alanine and aspartate transferase and bilirubin were examined in streptozotocin-induced diabetic rats treated with garlic oil and compared with the corresponding levels in diabetic control rats, normal rats and normal rats on garlic oil. Values of tissue amylase and total protein were also assessed from the pancreas, liver, and kidney. Treatment of diabetic rats with garlic oil significantly decreased the red cell phosphatase (p<0.01), serum acid and alkaline phosphatase (p<0.001) when compared to diabetic control rats. Serum alanine and asparate transferases were significantly (p<0.001) decreased as well as serum amylase (p<0.002) in garlic oil treated diabetic rats as compared with diabetic control rats. When treated with garlic oil, however, diabetic and normal rats showed significant increase (p<0.05) in the amylase levels of the pancrease, liver, and kidney.

A More Sensitive Automated Method for Determination of Ornithine Carbamoyltransferase Activity in Human Serum

1975 ◽  
Vol 21 (6) ◽  
pp. 754-757 ◽  
Author(s):  
Kathleen J Clayson ◽  
James S Fine ◽  
Paul E Strandjord
Keyword(s):  
Kinetic Studies ◽  
Sample Volume ◽  
Ornithine Carbamoyltransferase ◽  
Hepatocellular Injury ◽  
Carbamoyl Phosphate ◽  
Automated Method ◽  
Upper Limit ◽  
Threefold Increase ◽  
Assay Conditions

Abstract Ornithine carbamoyltransferase (EC 2.1.3.3) activity is a sensitive, specific indicator of hepatocellular injury. This paper describes development of an improved automated procedure for measurement of this activity. Triethanolamine—ethylenedlaminetetraacetate is used as a buffer, and activity is determined by measuring the concentration of the product, citrulline. Kinetic studies have been performed to determine optimal pH and L-ornithine and carbamoyl phosphate concentrations. Recovery of citrulline was studied. The upper limit of normal obtained in a study of 106 blood-bank donors was 6 U/liter. The automated procedure developed as a result of these studies, in which optimal assay conditions are used, produces a threefold increase in sensitivity and permits use of a sample volume of 1 ml.

scholarly journals Two forms of aspartate aminotransferase in rat liver and kidney mitochondria

1968 ◽  
Vol 108 (4) ◽  
pp. 619-624 ◽  
Author(s):  
M. M. Bhargava ◽  
A. Sreenivasan
Keyword(s):  
Rat Liver ◽  
Aspartate Aminotransferase ◽  
High Speed ◽  
Rat Kidney ◽  
Liver Mitochondria ◽  
Supernatant Fraction ◽  
Rat Liver Mitochondria ◽  
Aminotransferase Activity ◽  
Kidney Mitochondria ◽  
Liver And Kidney

1. Butan-1-ol solubilizes that portion of rat liver mitochondrial aspartate aminotransferase (EC 2.6.1.1) that cannot be solubilized by ultrasonics and other treatments. 2. A difference in electrophoretic mobilities, chromatographic behaviour and solubility characteristics between the enzymes solubilized by ultrasonic treatment and by butan-1-ol was observed, suggesting the occurrence of two forms of this enzyme in rat liver mitochondria. 3. Half the aspartate aminotransferase activity of rat kidney homogenate was present in a high-speed supernatant fraction, the remainder being in the mitochondria. 4. A considerable increase in aspartate aminotransferase activity was observed when kidney mitochondrial suspensions were treated with ultrasonics or detergents. 5. All the activity after maximum activation was recoverable in the supernatant after centrifugation at 105000g for 1hr. 6. The electrophoretic mobility of the kidney mitochondrial enzyme was cathodic and that of the supernatant enzyme anodic. 7. Cortisone administration increased the activities of both mitochondrial and supernatant aspartate aminotransferases of liver, but only that of the supernatant enzyme of kidney.

scholarly journals The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria

1976 ◽  
Vol 154 (2) ◽  
pp. 415-421 ◽  
Author(s):  
J D. McGivan ◽  
N M. Bradford ◽  
J Mendes-Mourão
Keyword(s):  
Protein Content ◽  
Nitrogen Source ◽  
Rat Liver ◽  
Liver Mitochondria ◽  
Rat Liver Mitochondria ◽  
Urea Synthesis ◽  
Carbamoyl Phosphate ◽  
Citrulline Synthesis ◽  
Ornithine Transcarbamoylase ◽  
Phosphate Synthase

The rate at which isolated rat liver mitochondria synthesized citrulline with NH4C1 as nitrogen source was markedly dependent on the protein content of the diet. 2. Citrulline synthesis was not rate-limited by substrate concentration, substrate transport or ornithine transcarbamoylase activity under the conditions used. 3. The intramitochondrial content of an activator of carbamoyl phosphate synthase, assumed to be N-acetyl-glutamate, varied markedly with dietary protein content. The variation in the concentration of this activator was sufficient to account for the observed variation in the rates of citrulline synthesis if this synthesis were rate-limited by the activity of carbamoyl phosphate synthase. 4. The rates of urea formation from NH4Cl as nitrogen source in isolated liver cells showed variations in response to diet that closely paralleled the variations in the rates of citrulline synthesis observed in isolated mitochondria. 5. These results are consistent with the postulate that when NH4Cl plus ornithine are present in an excess, the rate of urea synthesis is regulated at the level of carbamoyl phosphate synthase activity.

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