Studies on the intermediary carbohydrate metabolism of aquatic animals; the distribution of acid-soluble phosphorus and certain enzymes in dolphin tissues

1. Liver, kidney, brain, skeletal muscle, and cardiac muscle from one newborn and three adult long-snouted dolphins (Stenella plagiodon) were obtained for enzyme studies. 2. All of the dolphin tissues exhibited cytochrome oxidase, succinic dehydrogenase, and malic dehydrogenase activity. Considerable differences in the enzyme activities of the various tissues were noted, with cardiac muscle exhibiting the highest respiratory enzyme activity. The enzyme activities of dolphin tissues were lower than those of the corresponding rat tissues. 3. All of the dolphin tissues exhibited adenosine triphosphatase activity which was accelerated by magnesium and manganese but, in contrast to rat tissues, was only slightly activated by calcium. 4. Measurements of the distribution of acid-soluble phosphorus in dolphin tissues indicated that glycolysis in all of the tissues examined proceeded through the Emden-Meyerhof phosphorylation scheme. 5. The average glycogen content of dolphin skeletal muscle was 0.98 per cent as compared with 0.16 to 0.20 per cent for rat skeletal muscle. The high glycogen content of dolphin skeletal muscle indicates a ready source of substrate for glycolysis even during submergence when the blood supply may be differentially shunted to other organs. 6. Measurements of the organ weights of dolphins showed that the lungs occupy over three times and the liver one-half as much of the total body weight as do these organs in the rat. The heart and the thyroid gland of the dolphin are also larger in proportion to the total body weight than in the rat while the relative weights of the other tissues in the two species are about the same.     

Effect of hibernation, thyroid hormones and dexamethasone on cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase from jerboa (Jaculus orientalis)

Tissue distribution of the cytosolic and mitochondrial glycerol-3-phosphate dehydrogenase (cGPDH and mGPDH) activities in jerboa (Jaculus orientalis), a hibernator, shows the highest level of enzyme activity in skeletal muscle and brown adipose tissue, respectively. The effect of hibernation on cGPDH indicates an increase of activity in all tissues examined. In contrast, hibernation decreases mGPDH activity in all tissues, except skeletal muscle. The effect of thyroid hormones on GPDH activity was tissue specific: in kidneys, cGPDH activity doubled in euthermic jerboas treated with T4. In contrast, 6-n-propyl-2-thiouracil treatment provokes an increase of enzyme activity in brown adipose tissue, liver and brain. T4 treatment leads to a 2.7-fold increase in liver mGPDH activity. 6-n-propyl-2-thiouracil treatment decreases mGPDH activity in the skeletal muscle whereas the opposite effect was observed in brain. Dexamethasone stimulates cGPDH in all tissues examined, except skeletal muscle and kidneys. In the case of mGPDH activity, this increase was observed only for brown adipose tissue and brain. Our results suggest that hibernation, thyroid hormones and dexamethasone probably play a role in the regulation of cGPDH and mGPDH activities in jerboa. Our findings confirm that these enzymes are involved in metabolic adaptation to thermal stress in Jaculus orientalis.     

Electrophoretic analyses of alcohol dehydrogenase, aldehyde dehydrogenase, aldehyde oxidase, sorbitol dehydrogenase and xanthine oxidase from mouse tissues.

1. Cellulose acetate zymograms of alcohol dehydrogenase (ADH), aldehyde dehydrogenase, sorbitol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase extracted from tissues of inbred mice were examined. 2. ADH isozymes were differentially distributed in mouse tissues: A2--liver, kidney, adrenals and intestine; B2--all tissues examined; C2--stomach, adrenals, epididymis, ovary, uterus, lung. 3. Two NAD+-specific aldehyde dehydrogenase isozymes were observed in liver and kidney and differentially distributed in other tissues. Alcohol dehydrogenase, aldehyde oxidase, "phenazine" oxidase and xanthine oxidase were also stained when aldehyde dehydrogenase was being examined. 4. Two aldehyde oxidase isozymes exhibited highest activities in liver. 5. "Phenazine oxidase" was widely distributed in mouse tissues whereas xanthine oxidase exhibited highest activity in intestine and liver extracts. 6. Genetic variants for ADH-C2 established its identity with a second form of sorbitol dehydrogenase observed in stomach and other tissues. The major sorbitol dehydrogenase was found in high activity in liver, kidney, pancreas and male reproductive tissues.     

Partial structure and hormonal regulation of rabbit liver inhibitor-1; distribution of inhibitor-1 and inhibitor-2 in rabbit and rat tissues

Inhibitor-1 purified from rabbit liver could not be distinguished from the skeletal muscle protein by chromatographic, electrophoretic and immunological criteria. Amino acid sequences comprising 68% of rabbit liver inhibitor-1 were identical to the skeletal muscle protein indicating that they are products of a single gene. Total inhibitor-1 activity in heat-treated rabbit liver extracts was similar to that in skeletal muscle extracts, and the phosphorylation state of inhibitor-1 increased from 14% to 42% in rabbit liver in vivo after an intravenous injection of glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-1 recognised a single major protein of identical electrophoretic mobility (26 kDa) in each rabbit tissue examined (skeletal muscle, liver, brain, heart, kidney, uterus and adipose). The antibodies also recognised a single major (30 kDa) protein in the same rat tissues, except liver. The results show that while there are interspecies differences in apparent molecular mass, inhibitor-1 is likely to be the same gene product in each mammalian tissue. Inhibitor-1 was not detected in rat liver, either by activity measurements or immunoblotting, irrespective of the age, sex or strain of the animals. Immunoblotting also failed to detect inhibitor-1 in mouse liver, although it was present in guinea pig, porcine and sheep liver. The absence of inhibitor-1 in rat liver indicates that phosphorylation of this protein cannot underlie the increased phosphorylation of hydroxymethylglutaryl-CoA reductase observed after stimulation by glucagon. Monospecific antibodies to rabbit skeletal muscle inhibitor-2 recognised a 31 kDa protein in each rabbit tissue, and a 33 kDa protein in all rat tissues including liver. The results suggest that inhibitor-2 is the same gene product in each mammalian tissue.     

A comparative electrophoretic analysis of mammalian carbonic anhydrase isozymes: evidence for a third isozyme in red skeletal muscles.

1. Starch gel electrophoretic patterns of carbonic anhydrase (CA) isozymes were examined from tissue extracts of cats, sheep, rabbits and mice. 2. In addition to the widely distributed and extensively studied B and C isozymes, an additional isozyme (called CA-A) was observed. 3. Tissue distribution studies showed the A isozyme to be predominantly localized in red skeletal muscles, although this activity was also observed in white and "mixed" skeletal muscles of the cat, sheep and rabbit, as well as sheep lung and rabbit liver. 4. A, B and C isozymes of carbonic anhydrase from cat, sheep and mice exhibited independent variations in nett surface charge. In terms of decreasing anodal migration, the following results are reported: cat A greater than C greater than B; sheep C greater than B greater than A; and mouse B greater than C greater than A. 5. These results are consistent with the existence of 3 genetic loci encoding carbonic anhydrase in mammalian tissues.     

Different Thermostability of Skeletal Muscle Glyceraldehyde-3-phosphate Dehydrogenase from Hibernating and Euthermic Jerboa （Jaculus orientalis）

D Glyceraldehyde 3 phosphatedehydrogenase(GAPDH ,EC 1.2 .1.12 )isakeyenzymeoftheglycolyticpathwaythatispresentinthecytosolofallorganismssofarstudied[1] .TheglycolyticGAPDHhasbeenremarkablyconservedduringevolution ,havingahomotetramericstructurewithsubunitsof 35 - 37kD[1] .GAPDHhasbeenisolatedfromavarietyofspecies[2 ] ,includingmesophilic ,moderatelythermophilicandhyperthermophilicmicroorganisms[3 ] .Theseenzymes ,whichdifferinthermalstability ,havebeenshowntobehighlysimilarinaminoacidse…     

ENZYMES CATALYSING ETHANOL METABOLISM IN NEURAL AND SOMATIC TISSUES OF THE RAT

Abstract— The enzymes catalysing ethanol metabolism, alcohol dehydrogenase (EC 1.l.1.1) and aldehyde dehydrogenase (EC 1.2.1.3), were assayed in a variety of neural and somatic tissues of the rat, the human counterparts of which are known to be vulnerable to excessive ethanol. The activity of alcohol dehydrogenase was assayed by the coupled oxidation of ethanol and reduction of lactaldehyde, a method which we have recently found to be sufficiently sensitive and specific to measure the relatively low levels of activity in whole brain. Detectable activities of these enzymes were found in peripheral nerve, skeletal muscle, retina, optic nerve and various regions of brain, as well as in a variety of non-neural tissues. The levels of the enzymic activities in all tissues were markedly lower than those of liver, but probably sufficient to perform a local function in the metabolism of ethanol or other endogenous substrates. The activity of alcohol dehydrogenase in the various tissues, like that of liver, was confined to the cytosol and exhibited kinetic properties and responses to inhibitors almost identical to those of the liver enzyme. We consider the results to be consistent with the hypothesis that the pathological effects of alcohol may be related, at least in part, to local mechanisms for the metabolism of alcohol.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

Mild Hyperbaric Oxygen Improves Decreased Oxidative Capacity of Spinal Motoneurons Innervating the Soleus Muscle of Rats with Type 2 Diabetes

Rats with type 2 diabetes exhibit decreased oxidative capacity, such as reduced oxidative enzyme activity, low-intensity staining for oxidative enzymes in fibers, and no high-oxidative type IIA fibers, in the skeletal muscle, especially in the soleus muscle. In contrast, there are no data available concerning the oxidative capacity of spinal motoneurons innervating skeletal muscle of rats with type 2 diabetes. This study examined the oxidative capacity of motoneurons innervating the soleus muscle of non-obese rats with type 2 diabetes. In addition, this study examined the effects of mild hyperbaric oxygen at 1.25 atmospheres absolute with 36 % oxygen for 10 weeks on the oxidative capacity of motoneurons innervating the soleus muscle because mild hyperbaric oxygen improves the decreased oxidative capacity of the soleus muscle in non-obese rats with type 2 diabetes. Spinal motoneurons innervating the soleus muscle were identified using nuclear yellow, a retrograde fluorescent neuronal tracer. Thereafter, the cell body sizes and succinate dehydrogenase activity of identified motoneurons were analyzed. Decreased succinate dehydrogenase activity of small-sized alpha motoneurons innervating the soleus muscle was observed in rats with type 2 diabetes. The decreased succinate dehydrogenase activity of these motoneurons was improved by mild hyperbaric oxygen. Therefore, we concluded that rats with type 2 diabetes have decreased oxidative capacity in motoneurons innervating the soleus muscle and this decreased oxidative capacity is improved by mild hyperbaric oxygen.     

Changes of the NADP-linked malic enzyme in the developing rat skeletal muscle

The activities of NADP-linked malic enzyme, hexose monophosphate shunt dehydrogenases and NADP-linked isocitrate dehydrogenase were studied during development of skeletal muscle and compared with those in the liver. The variation patterns of malic enzyme activity in the liver and in the skeletal muscle were very similar, however the amplitude of the changes was different. The enzyme activity increased approx 16-fold in the liver and about 2-fold in skeletal muscle at the same stage of development. In skeletal muscle the increase of the malic enzyme activity was only slightly higher than of lactic dehydrogenase and citrate synthase. Studies on the intracellular distribution of malic enzyme in skeletal muscle showed that both mitochondrial and extramitochondrial enzymes increased between 20th and 37th day of life, the increase of the extramitochondrial enzyme being more pronounced. The hexose monophosphate shunt dehydrogenases activity showed an increase in the liver but no change was observed in the skeletal muscle at the weaning time. Changes in the activity of the liver and skeletal muscle isocitrate dehydrogenase were not significant between 10th and 80th day of life. The results suggest that the malic enzyme in the liver is playing a different physiological role than in the skeletal muscle.     

Characterization of a new continuous cell line from the flood water mosquito,Aedes vexans

A new cell line, UM-AVE1, was established from embryos of the mosquito Aedes vexans. Banding patterns for the isozymes lactate dehydrogenase (LDH), malate dehydrogenase (MDH), isocitrate dehydrogenase (IDH), xanthine dehydrogenase (XDH), and esterases were compared with those of larval Aedes vexans tissues as well as those of four other mosquito cell lines and one moth cell line. Karyotype analyses confirmed that the dipteran cell lines were not contaminated with lepidopteran cells, because in all mosquito lines the modal number of chromosomes was 6 (=2n) or 7. Isozyme electrophoresis established a specific profile for each cell line. Two isozymes present in UM-AVE1 (LDH, IDH) were not detected in larvae; this could be a reflection of the different stages used for cell line isolation and enzyme analysis, or lability of sample preparations. It is significant that extracts from UM-AVE1 cells and Aedes vexans larvae had an identical double band for XDH, while all other cell lines examined exhibited only a single band.     

Tissue distribution of immunoreactive mouse extracellular superoxide dismutase

Protein content and mRNA expression ofextracellular superoxide dismutase (EC-SOD) were investigated in 16 mouse tissues. We developed a double-antibody sandwich ELISA using theaffinity-purified IgG against native mouse EC-SOD. EC-SOD could bedetected in all of the tissues examined (lung, kidney, testis, brownfat, liver, adrenal gland, pancreas, colon, white fat, thymus, stomach,spleen, heart, skeletal muscle, ileum, and brain, in decreasing order of content measured as µg/g wet tissue). Lung showed a markedly higher value of EC-SOD than other tissues. Interestingly, white fat hada high content of EC-SOD in terms of micrograms per milligram protein,which corresponded to that of lung. Kidney showed the strongestexpression of EC-SOD mRNA. Relatively strong expression of the mRNA wasobserved in lung, white fat, adrenal gland, brown fat, and testis.Heart and brain showed only weak signals, and no such expression couldbe detected in either digestive organs or skeletal muscle.Immunohistochemically, EC-SOD was localized mainly to connectivetissues and vascular walls in the tissues examined. Deep staining inthe cytosol was observed in the cortical tubular cells of kidney. Theseresults suggest that EC-SOD is distributed systemically inmice and that the physiological importance of this enzyme may be acompensatory adaptation to oxidative stress, particularly in lung andkidney.

     

Chinook salmon NADP+-dependent cytosolic isocitrate dehydrogenase: Electrophoretic and genetic dissection of a complex isozyme system and geographic patterns of variation

Species in the genus Oncorhynchus express complicated isocitrate dehydrogenase (IDHP) isozyme patterns in many tissues. Subcellular localization experiments show that the electrophoretically distinct isozymes of low anodal mobility expressed predominantly in skeletal and heart muscle are mitochondrial forms (mIDHP), while the more anodal, complex isolocus isozyme system predominant in liver and eye is cytosolic (sIDHP). The two loci encoding sIDHP isozymes are considered isoloci because the most common allele at one of these loci cannot be separated electrophoretically from the most common allele of the other. Over 12 electrophoretically detectable alleles are segregating at the two sIDHP* loci in chinook salmon. Careful electrophoretic comparisons of the sIDHP isozyme patterns of muscle, eye, and liver extracts of heterozygotes reveal marked differences between the tissues with regard to both relative isozyme staining and the expression of several common alleles. Presumed single-dose heterozygotes at the sIDHP isolocus isozyme system exhibit approximate 9:6:1 ratios of staining intensity in liver and eye, while they exhibit approximate 1:2:1 ratios in skeletal muscle. The former proportions are consistent with the equal expression of two loci (isolocus expression), while the latter are consistent with the expression of a single locus. Screening of over 10,000 fish from spawning populations and mixed-stock fishery samples revealed that certain variant alleles (*127, *50) are detectable only in liver and eye, while other alleles (*129, *94, and *74) are strongly expressed in muscle, eye, and liver. The simplest explanation for these observations is that the "isolocus" sIDHP system of chinook salmon (and that of steelhead and rainbow trout) results from the expression of two distinct loci (sIDHP-1* and sIDHP-2*) that have the same common allele (as defined by electrophoretic mobility). IDHP expression in skeletal muscle is due to the nearly exclusive expression of the sIDHP-1* locus, while IDHP expression in eye and liver tissues is due to high levels of expression of both sIDHP-1* and sIDHP-2*--giving rise to the isolocus situation in these latter tissues. Direct inheritance studies confirm this model of two genetically independent (disomic) loci encoding sIDHP in chinook salmon. Extensive geographic surveys of chinook salmon populations from California to British Columbia reveal marked differences in allele frequencies at both sIDHP-1* and sIDHP-2* and considerably more interpopulation differentiation than was recognized previously when sIDHP was treated as an isolocus system with only five recognized alleles.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lactate downregulates the glycolytic enzymes hexokinase and phosphofructokinase in diverse tissues from mice

We examined the effects of lactate on the enzymatic activity of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK) in various mouse tissues. Our results showed that lactate inhibited PFK activity in all the analyzed tissues. This inhibitory effect was observed in skeletal muscle even in the presence of insulin. Lactate directly inhibited the phosphorylation of PFK tyrosine residues in skeletal muscle, an important mechanism of the enzyme activation. Moreover, lactate indirectly inhibited HK activity, which resulted from its cellular redistribution, here attributed to alterations of HK structure. PK activity was not affected by lactate. The activity of HK and PFK is directly related to glucose metabolism. Thus, it is conceivable that lactate exposure can induce inhibition of glucose consumption in tissues.     

Isozymes of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle

The distribution of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle tissues was examined. With DEAE-cellulose chromatography, two peaks (I and II) of Fru 6-P,2-kinase activity were detected in all tissue extracts. Peak I was the predominant form both in rat and bovine heart tissue, while peak II was the major form in liver and skeletal muscle. Antibodies to heart enzyme reacted specifically with peak I, and antibodies to liver enzyme reacted with peak II from both liver and skeletal muscle. All the isozymes were bifunctional. All the tissues examined contained other isozymes in minor amounts.     

The value of enzyme histochemical techniques in the classification of fibre types of human skeletal muscle

Summary Classification of human skeletal muscle into type I and type II fibres is frequently based on their weak or strong staining with the myosin adenosine triphosphatase reaction. In order to evaluate the reliability of this screening technique a combined histochemical and biochemical study was performed on normal and diseased skeletal muscle of human subjects. In the present investigation activities of enzymes which play a role in the aerobic and anaerobic pathways and which can characterize fibre type, were examined in human muscle specimens with disease of the neuromuscular system.Special attention is given to the maximal activities of phosphofructokinase and fructose-1,6-diphosphatase, the rate limiting enzymes for the regulation of the glycolysis and gluconeogenesis respectively. Moreover the activities of enzymes of the pentose phosphate pathway are determined.A most important feature of the biochemical findings is that the constancy of activity ratios of the examined enzymes, as is found apparently normal human skeletal muscle, was frequently not present in diseased human skeletal muscle. From these results and from the histochemical results it can be concluded that for fibre classification in diseased human skeletal muscle the histochemical demonstration of myosin ATPase activity exclusively is not sufficient, but that it is necessary to apply other enzyme histochemical techniques too.Moreover it was found that in diseased human skeletal muscles the activity of the NADPH regenerating enzymes glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase was strongly increased. A third observation was the relative decrease of the activity of the examined aerobic enzymes in affected muscle fibres of neurogenic muscle diseases.     

50 Hz magnetic fields activate mussel immunocyte p38 MAP kinase and induce HSP70 and 90

We studied the effects of a sublethal concentration of pyrethroid insecticide fenvalerate on metabolic enzymes, RNA and protein of brain, liver and skeletal muscle of the freshwater catfish, Clarias batrachus. Exposure to fenvalerate gradually decreased the activity of citrate synthase (CS), glucose 6-phosphate dehydrogenase (G6-PDH) and lactate dehydrogenase (LDH) in brain, liver and skeletal muscle up to 21 days. The maximum decrease in enzyme activity was 23-47%. Withdrawal of fenvalerate from the medium for 21 days restored enzyme activity to their control level in all three tissues. RNA and protein content in brain, liver and skeletal muscle decreased significantly with exposure of fenvalerate up to 21 days. The maximum decrease in RNA and protein was 22-32%. Withdrawal of fenvalerate from the medium for 21 days restored the RNA and protein contents to control levels. The present study suggests that fenvalerate impairs cellular metabolism and its biochemical effects are reversible after withdrawal of fenvalerate.     

Dolichol kinase in rat sarcoplasmic reticulum membrane preparations

A dolichol kinase (EC 2.7.1.108) was found in sarcoplasmic reticulum membrane fractions from rat leg muscle. This enzyme specifically required CTP as a phosphoryl donor and relatively little activity was found in the absence of exogenous detergent-suspended dolichol. Unlike other reported dolichol kinases, the kinase from skeletal muscle was activated almost equally well by Ca2+, Zn2+, or Mg2+, but not Mn2+. No effect of calmodulin was seen. The kinase exhibited a single pH optimum at pH 7-8 in contrast to kinases from certain other tissues. Despite the low level of dolichol present in skeletal muscle, the kinase in the sarcoplasmic reticulum fraction had an activity comparable to that of microsomal preparations from tissues such as brain and liver, which may indicate that skeletal muscle has a high capacity for dolichol phosphorylation and protein glycosylation.     

Glyceraldehyde-3-phosphate dehydrogenase from the newt Pleurodeles waltl. Protein purification and characterization of a GapC gene

The NAD(+)-dependent cytosolic glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) has been purified to homogeneity from skeletal muscle of the newt Pleurodeles waltl (Amphibia, Urodela). The purification procedure including ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulted in a 24-fold increase in specific activity and a final yield of approximately 46%. The native protein exhibited an apparent molecular weight of approximately 146 kDa with absolute specificity for NAD(+). Only one GAPDH isoform (pI 7.57) was obtained by chromatofocusing. The enzyme is an homotetrameric protein composed of identical subunits with an apparent molecular weight of approximately 37 kDa. Monospecific polyclonal antibodies raised in rabbits against the purified newt GAPDH immunostained a single 37-kDa GAPDH band in extracts from different tissues blotted onto nitrocellulose. A 510-bp cDNA fragment that corresponds to an internal region of a GapC gene was obtained by RT-PCR amplification using degenerate primers. The deduced amino acid sequence has been used to establish the phylogenetic relationships of the Pleurodeles enzyme--the first GAPDH from an amphibian of the Caudata group studied so far--with other GAPDHs of major vertebrate phyla.