Vitamin B6 metabolism in Morris hepatomas

The enzymes involved in the metabolism of vitamin B6 were measured in Morris hepatomas and livers of female Buffalo rats fed pyridoxine-sufficient and deficient diets. Pyridoxal phosphate levels in plasmas hepatomas, and livers were also determined. Nontumor-bearing animals were maintained as controls. Regardless of the B6 nutritional status, the concentration of pyridoxal phosphate was lower in the hepatomas than in the livers of the host animals. The apoenzyme levels of ornithine decarboxylase, a pyridoxal phosphate-dependent enzyme, were higher in the hepatomas from animals fed the B6-deficient diet. Liver pyridoxine kinase activity was higher in B6-sufficient animals. In contrast, tumor pyridoxine kinase activity was influenced by B6 intake and was significantly lower than that in host liver. Liver pyridoxine phosphate oxidase activity was not significantly affected by B6 intake or by the presence of tumor. In contrast, hepatomas had little or no pyridoxine phosphate oxidase activity. Pyridoxine phosphate phosphatase activity was elevated in tumors relative to livers. These data indicate that the metabolism of vitamin B6 is markedly different in the hepatomas than in host or control livers and suggest that the tumor is apparently incapable of the complete synthesis of co-enzymatically active pyridoxal phosphate from inactive precursor forms such as pyridoxine.     

Antioxidant and oxidative status in tissues of manganese superoxide dismutase transgenic mice

Manganese superoxide dismutase (Mn-SOD) plays an important role in attenuating free radical-induced oxidative damage. The purpose of this research was to determine if increased expression of Mn-SOD gene alters intracellular redox status. Twelve week old male B6C3 mice, engineered to express human Mn-SOD in multiple organs, and their nontransgenic littermates were assessed for oxidative stress and antioxidant status in heart, brain, lung, skeletal muscle, liver, and kidney. Relative to their nontransgenic littermates, transgenic mice had significantly (p <.01) higher activity of Mn-SOD in heart, skeletal muscle, lung, and brain. Copper, zinc (Cu,Zn)-SOD activity was significantly higher in kidney, whereas catalase activity was lower in brain and liver. The activities of selenium (Se)-GSH peroxidase and non-Se-GSH peroxidase, and levels of vitamin E, ascorbic acid and GSH were not significantly different in any tissues measured between Mn-SOD transgenic mice and their nontransgenic controls. The levels of malondialdehyde were significantly lower in the muscle and heart of Mn-SOD mice, and conjugated dienes and protein carbonyls were not altered in any tissues measured. The results obtained showed that expression of human SOD gene did not systematical alter antioxidant systems or adversely affect the redox state of the transgenic mice. The results also suggest that expression of human SOD gene confers protection against peroxidative damage to membrane lipids.     

Effect of ruthenium complexes on the activities of succinate dehydrogenase and cytochrome oxidase

In this article, we report the effects of acute administration of ruthenium complexes, trans-[RuCl(2)(nic)(4)] (nic=3-pyridinecarboxylic acid) 180.7 micromol/kg (complex I), trans-[RuCl(2)(i-nic)(4)] (i-nic=4-pyridinecarboxylic acid) 13.6 micromol/kg (complex II), trans-[RuCl(2)(dinic)(4)] (dinic=3,5-pyridinedicarboxylic acid) 180.7 micromol/kg (complex III) and trans-[RuCl(2)(i-dinic)(4)]Cl (i-dinic=3,4-pyridinedicarboxylic acid) 180.7 micromol/kg (complex IV) on succinate dehydrogenase (SDH) and cytochrome oxidase (COX) activities in brain (hippocampus, striatum and cerebral cortex), heart, skeletal muscle, liver and kidney of rats. Our results showed that complex I inhibited SDH activity in hippocampus, cerebral cortex, heart and liver; and inhibited COX in heart and kidney. Complex II inhibited SDH in heart and hippocampus; COX was inhibited in hippocampus, heart, liver and kidney. SDH activity was inhibited by complex III in heart, muscle, liver and kidney. However, COX activity was increased in hippocampus, striatum, cerebral cortex and kidney. Complex IV inhibited SDH activity in muscle and liver; COX activity was inhibited in kidney and increased in hippocampus, striatum and cerebral cortex. In a general manner, the complexes tested in this work decrease the activities of SDH and COX in heart, skeletal muscle, liver and kidney. In brain, complexes I and II were shown to be inhibitors and complexes III and IV activators of these enzymes. In vitro studies showed that the ruthenium complexes III and IV did not alter COX activity in kidney, but activated the enzyme in hippocampus, striatum and cerebral cortex, suggesting that these complexes present a direct action on COX in brain.     

Tissue metabolism and enzyme activities in the rodent Heterocephalus glaber, a poor temperature regulator.

1. Tissue oxygen uptake and enzyme activities were investigated in the naked mole rat, Heterocephalus glaber, a mammal notable for its low body temperature and metabolism and poor temperature regulating ability. 2. Q10 for O2 uptake of Heterocephalus crude liver homogenates ranged from 1.91 for the temperature interval 25-30 degrees C to 1.76 within the range 30-38 degrees C, values similar to those reported for typical homoiotherms. 3. Km pyruvate of lactate dehydrogenase in heart muscle had the same temperature dependence in the mole rat and mouse. 4. O2 uptake and cytochrome oxidase activity of skeletal muscle were higher for mole rat than mouse. The reverse was true for heart muscle. Brain and liver O2 uptake showed similar values for both species, while kidney O2 uptake was highest in the mouse. 5. Pyruvate kinase activity in heart and skeletal muscle was higher in mouse than mole rat, suggesting a greater reliance on glycolysis in the former. 6. Na+, K+ -ATPase activity of liver and kidney was 60% higher in mouse than mole rat, while brain was 30% higher in mouse. 7. The results indicate that the effects of temperature on tissue metabolism in the mole rat conform to those in typical homoiotherms. The low body temperature and O2 uptake in the mole rat find no expression in the tissue respiratory capacity.     

Effect of starvation and exercise on actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues.

Starvation does not change the actual activity per g of tissue of the branched-chain 2-oxo acid dehydrogenase in skeletal muscles, but affects the total activity to a different extent, depending on the muscle type. The activity state (proportion of the enzyme present in the active state) does not change in diaphragm and decreases in quadriceps muscle. Liver and kidney show an increase of both activities, without a change of the activity state. In heart and brain no changes were observed. Related to organ wet weights, the actual activity present in the whole-body muscle mass decreases on starvation, whereas the activities present in liver and kidney do not change, or increase slightly. Exercise (treadmill-running) of untrained rats for 15 and 60 min causes a small increase of the actual activity and the activity state of the branched-chain 2-oxo acid dehydrogenase complex in heart and skeletal muscle. Exercise for 1 h, furthermore, increased the actual and the total activity in liver and kidney, without a change of the activity state. In brain no changes were observed. The actual activity per g of tissue in skeletal muscle was less than 2% of that in liver and kidney, both before and after exercise and starvation. Our data indicate that the degradation of branched-chain 2-oxo acids predominantly occurs in liver and to a smaller extent in kidney and skeletal muscle in fed, starved and exercised rats.     

Isolation and properties of cytochrome c oxidase from rat liver and quantification of immunological differences between isozymes from various rat tissues with subunit-specific antisera

Cytochrome c oxidase was isolated from rat liver either by affinity chromatography on cytochrome-c--Sepharose 4B or by chromatography on DEAE-Sepharose. Dodecyl sulfate gel electrophoresis of both preparations showed the same subunit pattern consisting of 13 different polypeptides. Kinetic analysis of the two preparations gave a higher Vmax for the enzyme isolated by chromatography on DEAE-Sephacel. Specific antisera were raised in rabbits against nine of the ten nuclear endoded subunits. A monospecific reaction of each antiserum with its corresponding subunit was obtained by Western blot analysis, thus excluding artificial bands in the gel electrophoretic pattern of the isolated enzyme due to proteolysis, aggregation or conformational modification of subunits. With an antiserum against rat liver holocytochrome c oxidase a different reactivity was found by Western blot analysis for subunits VIa and VIII between isolated cytochrome c oxidases from pig liver or kidney and heart or skeletal muscle. For a quantitative analysis of immunological differences a nitrocellulose enzyme-linked immunosorbent assay was developed. Monospecific antisera against 12 of the 13 subunits of rat liver cytochrome c oxidase were titrated with increasing amounts of total mitochondrial proteins from different rat tissues dissolved in dodecyl sulfate and dotted on nitrocellulose. The absorbance of a soluble dye developed by the second peroxidase-conjugated antibody was measured. From the data the following conclusions were obtained: (a) The mitochondrial encoded catalytic subunits I-III of cytochrome c oxidase are probably identical in all rat tissues. (b) All nine investigated nuclear encoded subunits of cytochrome c oxidase showed immunological differences between two or more tissues. Large immunological differences were found between liver, kidney or brain and heart or skeletal muscle. Minor but significant differences were observed for some subunits between heart and skeletal muscle and between liver, kidney and brain. (c) Between corresponding nuclear encoded subunits of cytochrome c oxidase from fetal and adult tissues of liver, heart and skeletal muscle apparent immunological differences were observed. The data could explain cases of fatal infantile myopathy due to cytochrome c oxidase deficiency.     

Investigating the role of the physiological isoform switch of cytochrome c oxidase subunits in reversible mitochondrial disease

Reversible infantile respiratory chain deficiency is characterised by spontaneous recovery of mitochondrial myopathy in infants. We studied whether a physiological isoform switch of nuclear cytochrome c oxidase subunits contributes to the age-dependent manifestation and spontaneous recovery in reversible mitochondrial disease. Some nuclear-encoded subunits of cytochrome c oxidase are present as tissue-specific isoforms. Isoforms of subunits COX6A and COX7A expressed in heart and skeletal muscle are different from isoforms expressed in the liver, kidney and brain. Furthermore, in skeletal muscle both the heart and liver isoforms of subunit COX7A have been demonstrated with variable levels, indicating that the tissue-specific expression of nuclear-encoded subunits could provide a basis for the fine-tuning of cytochrome c oxidase activity to the specific metabolic needs of the different tissues.We demonstrate a developmental isoform switch of COX6A and COX7A subunits in human and mouse skeletal muscle. While the liver type isoforms are more present soon after birth, the heart/muscle isoforms gradually increase around 3 months of age in infants, 4 weeks of age in mice, and these isoforms persist in muscle throughout life. Our data in follow-up biopsies of patients with reversible infantile respiratory chain deficiency indicate that the physiological isoform switch does not contribute to the clinical manifestation and to the spontaneous recovery of this disease. However, understanding developmental changes of the different cytochrome c oxidase isoforms may have implications for other mitochondrial diseases.This article is part of a Directed Issue entitled: Energy Metabolism Disorders and Therapies.     

Tissue distribution of cytochrome c oxidase isoforms in mammals. Characterization with monoclonal and polyclonal antibodies

Monoclonal and polyclonal antibodies specific to the two isoforms of subunit VIa of bovine cytochrome c oxidase were generated and used to study the tissue distribution of this subunit pair in beef, human and rat. The so-called H-(heart) form was found exclusively in heart and skeletal muscle, whereas the so-called L-(liver) form was the only isoform present in brain, kidney, liver and smooth muscle. Little or no L-form was detected in skeletal muscle. In bovine heart no subunit VIa-L was detected, while in human heart the subunit VIa-H and VIa-L isoforms were present in roughly equal proportions. These results imply that, in humans, the deficiency of a subunit VIa isoform may have a different effect on the physiology of heart then on the physiology of skeletal muscle.     

Postnatal development of the actual and total activity of the branched-chain 2-oxo acid dehydrogenase complex in rat tissues

Actual and total activities of the branched-chain 2-oxo acid dehydrogenase complex were determined in homogenates of quadriceps muscle, heart, liver, kidney and brain from rats of 0-70 days age. All rat tissues except quadriceps muscle showed a marked increase of total activity between 0 and 21 days, heart and kidney also after weaning. The actual activity rose after birth in liver, kidney and brain and after weaning in liver, kidney and heart. The activity state was always about 100% in liver and varied between 40-60% in kidney and brain, 10-23% in heart and 6-12% in quadriceps muscle. The actual activities measured indicate, that the degradation of branched-chain 2-oxo acids mainly takes place in the liver of the newborn, suckling and young-adult rat.     

Immunochemical characteristics of the peroxidases of several mouse tissues]

Enzymes catalyzing peroxidase reaction of a lysosomal fraction in bone marrow, leucocytes, spleen, thyroid gland, stomach, kidney, heart, lungs, brain and skeletal muscle of mice were investigated by immunochemical methods. A high level of peroxidase activity was discovered in leucocytes, bone marrow, spleen, heart and lung, a lower activity appeared to be characteristic of liver, thyroid gland and kidney. The peroxidase activities in brain, skeletal muscle and stomach were low. The reaction of immunoprecipitation with myeloperoxidase-specific antiserum revealed considerable antigenic distinctions between the enzymes catalysing peroxidase reaction in various tissues of mice.     

Comparisons of copper deficiency states in the murine mutants blotchy and brindled. Changes in copper-dependent enzyme activity in 13-day-old mice.

The activity of two copper-dependent enzymes, cytochrome c oxidase and copper, zinc-superoxide dismutase, was determined in six tissues of age-matched (13-day-old) copper-deficient mutant and normal mice. In the two mutants 'brindled' and 'blotchy', brain, heart and skeletal muscle had significant enzyme deficiencies. Cytochrome c oxidase was more severely affected than was superoxide dismutase. In these three tissues the degree of deficiency could be correlated with decreased copper concentration; however, enzyme activity was normal in liver, kidney and lung, despite abnormal copper concentrations in these tissues. In nutritionally copper-deficient mice, all six tissues showed decreased enzyme activity, which was most marked in brain, heart and skeletal muscle, the tissues which showed enzyme deficiencies in the mutants. Analysis in vitro of cytochrome c oxidase (temperature coefficient = 2) at a single temperature was found to underestimate the deficiency of this enzyme in hypothermic copper-deficient animals. Cytochrome c oxidase deficiency may therefore be sufficiently severe in vivo to account for the clinical manifestations of copper deficiency. An injection of copper (50 micrograms of Cu+) at 7 days increased cytochrome c oxidase activity by 13 days in all deficient tissues of brindled mice, and in brain and heart from blotchy mice. However, skeletal-muscle cytochrome c oxidase in blotchy mutants did not respond to copper injection. Cytochrome c oxidase activity increased to normal in all tissues of nutritionally copper-deficient mice after copper injection, except in the liver. Hepatic enzyme activity remained severely deficient despite a liver copper concentration three times that found in copper-replete controls. Superoxide dismutase activity did not increase with treatment in either mutant, but its activity was higher than control levels in nutritionally deficient mice after injection. This difference is probably due to sequestration of copper in mutant tissue such as kidney, but a defect in the copper transport pathway to superoxide dismutase cannot be excluded.     

Possible influence of gonadotrophins on formation in vivo of pyridoxal phosphate

FSH administered to normal rats increased the activity of pyridoxine phosphate oxidase of both liver and kidney and, consequently, pyridoxal phosphate levels in these tissues were elevated. LH administration, on the other hand, decreased the activity of pyridoxine phosphate oxidase, resulting in diminished pyridoxal phosphate level in the tissues. The stimulatory effect of FSH on the activity of liver and kidney pyridoxine phosphate oxidase was not observed in castrated-adrenalectomised rats unless supplemented with cortisone and testosterone, respectively. Puromycin treatment prevented the FSH-induced rise in the activity of liver and kidney pyridoxine phosphate oxidases. It is suggested that FSH stimulates the activity of liver and kidney pyridoxine phosphate oxidase by increasing the synthesis of apoproteins of the enzyme, and the effect of FSH on liver is dependent on the presence of adrenal corticoids while the presence of testosterone is a prerequisite for the FSH to have its effect on kidney pyridoxine phosphate oxidase.     

Glucose dehydrogenase in teleosts: tissue distribution and proposed function

Tissue extracts of skeletal muscle, heart, eye, brain, liver, kidney, gill and stomach were electrophoretically examined for glucose dehydrogenase (EC 1.1.1.47) activity in 21 species of marine teleost fishes. Glucose dehydrogenase expression was detected only in liver extracts. Considerable interordinal variation was found in levels of enzymatic activity. Available data support the hypothesis that glucose dehydrogenase provides NADPH for the mixed-function oxidase system in teleosts.     

Immunochemical analysis of the peroxisomal beta-oxidation enzymes in rat and human heart and skeletal muscle and in skeletal muscle of Zellweger patients

Immunoblot analyses with antibodies against the peroxisomal beta-oxidation enzymes from rat liver showed the presence of these enzymes in rat and human liver and kidney and rat adrenal gland. The bifunctional protein could not be detected in muscle tissues or cultured muscle cells. Acyl-CoA oxidase was detected in rat heart and cultured human muscle cells. 3-Ketoacyl-CoA thiolase was also detected in human and rat heart and skeletal muscle; however, this enzyme was not detectable in skeletal muscle of Zellweger patients, in agreement with the absence of peroxisomal fatty acid oxidation.     

The intracellular distribution and activities of phosphoenolpyruvate carboxykinase isozymes in various tissues of several mammals and birds.

1. The intracellular distribution and/or activities of phosphoenolpyruvate carboxykinase isozymes were determined in liver, kidney, gastrointestinal mucosa, adipose, skeletal muscle, brain, spleen, lung and heart of fed and fasted rabbits, guinea pigs, rats, chickens and pigeons. 2. Liver and kidney of all species contained the highest enzyme activity/g. 3. Carboxykinase activity/g gastrointestinal mucosa of rabbits was quite high compared to the low activity in guinea pig and rat mucosa and essentially undetectable activity in chicken and pigeon mucosa. 4. Activity/g was high in rat brown adipose. 5. Low carboxykinase activity/g was found in skeletal muscle of all species and in white adipose of guinea pig, rabbit and rat although activity was undetectable in white adipose of chicken and pigeon. 6. Carboxykinase activity was essentially undetectable in brain, spleen, lung and heart of all species.     

Hormone- and fluoride-sensitive adenylate cyclases in human fetal tissues

Adenylate cyclase activities have been assayed in the human fetal adrenal, heart ventricle, brain, liver, testis, kidney, skeletal muscle and lung during the first trimester of pregnancy. The requirements for adenylate cyclases are similar to those reported in all adult tissues. Of all tissues studied, heart ventricle had the highest level of enzymatic activity, and this tissue was most responsive to hormonal stimulation. Although adenylate cyclases from all of these tissues were stimulated by F^?in vitro, hormonal stimulation was observed only in the liver, adrenal and heart ventricle. The presence of hormone-responsive adenylate cyclase in human fetal tissues suggests that cyclic AMP may be involved in gene expression.     

Bovine cytochrome c oxidases,purified from heart,skeletal muscle,liver and kidney,differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.     

Serum and organ creatine phosphokinase alterations in exercise.

Rats that swam for 3 h showed a 6-fold increase in serum creatine phosphokinase (SCPK) activity which declined to control values within 7 h after swimming. Of the excess SCPK, 77% was BB isoenzyme; the remainder was mainly MM with traces of MB. Kidney, liver, brain and lung contain mainly BB (50-80%) and only a trace of MB (0-7%). Heart CPK was composed of little BB (8%) and more MB (28%) and MM (64%). Skeletal muscle CPK was almost entirely MM. CPK activity is highest in skeletal muscle, intermediate in heart and brain and lowest in kidney, liver and lung. It is suggested that skeletal muscle and heart are not involved in CPK release in swimming, and kidney, liver and brain may be sites of release.     

A study of iodothyronine 5'-monodeiodinase activities in normal and pathological tissues in man and their comparison with activities in rat tissues

The present study was undertaken to investigate the peripheral iodothyronine 5'-monodeiodination in different human and rat tissues. We studied iodothyronine 5'-monodeiodinase type I (5'-DI) activity in liver, kidney, intestine, right cardiac atrium and skeletal muscle and we compared the results with those in rat tissues. Lodothyronine 5'- monodeiodinase type II (5'-DII) activity was studied in normal and ischemic human heart and in rat normal myocardium and brain. The 5'-DI activity (fmol/min x mg protein) in liver and kidney was significantly higher (p < 0.001, ANOVA) in normal rat tissue than in human. However, no significant differences were observed in 5'-DI activity between normal and tumoral human intestine or between intestinal tissue of man and rat. 5'-DI activity in normal human skeletal muscle was significantly higher than that in rat skeletal muscle (p < 0.05). The 5'-DI activity was lower in human ischemic myocardium when compared to normal myocardium either in humans (p < 0.05) or rat (p < 0.001). The Km of 5'-DI was significantly lower in rat than in human kidney and liver (p < 0.05). We conclude that 1) 5'-DI is distributed widely among extrathyroidal human and rat tissues and 5'-DII activity is detectable both in human and rat heart; 2) 5'-DI activity in liver and kidney is lower in man than in rat; 3) 5'-DI activity in the skeletal muscle is higher in man than in the rat; 4) 5'-DI activity is decreased in tumoral tissues of human liver and kidney and in ischemic myocardium, while no significant difference was found between human and rat cardiac 5'-DII activity.     

Comparative studies on lipogenic enzyme activities in the liver of human and some animal species

1. The activities of enzymes involved in fatty acid synthesis in the human liver (sample taken during abdominal surgery) and in the livers of some animals were studied. 2. Fatty acid synthase, ATP-citrate lyase and malic enzyme activities were found to be from 4 to 70-fold lower in human liver than in rat or bird livers. 3. The activities of hexose monophosphate shunt dehydrogenases in human liver were from half to almost equal to the corresponding activities in birds, but much lower than in rat liver. 4. The activities of all enzymes listed above in human and beef liver were very similar (except fatty acid synthase which was undetectable in the beef liver). 5. Very high activity of NADP-linked isocitrate dehydrogenase was found in livers of all species tested. 6. These results are discussed in relation to the role of the human liver in lipogenesis. 7. The activities of the enzymes generating NADPH in human liver taken during abdominal surgery were similar to the activities observed in the tissue obtained post mortem. 8. This suggested that post mortem tissue may be used as a reliable human material for some enzyme assays. 9. Thus we also examined the activity of malic enzyme in post mortem human kidney cortex, heart, skeletal muscle and brain. 10. Relatively high activity of NADP-linked malic enzyme has been observed in human brain.