Skeletal Muscle Enzyme Research Articles

79-Year-Old Woman With Blue Toes

79-Year-Old Woman With Blue Toes

Activity of Dehydrogenases and Enzymes of Nitrogen Metabolism in Cardiac Tissue and Skeletal Muscle of Steers Fed Monensin

Kala~njuk G. I., M. Marounek, L.G.Kala~njuk, M. G. Gerasymiv, O. G. S a v k a : Activity of Dehydrogenases and Enzymes of Nitrogen Metabolism in Cardiac Tissue and Skeletal Muscle of Steers fed Monensin. Acta vet. Brno 1995, 64: 157-161. Activites of lactate dehydrogenase (E.C. 1.1.1.27), malate dehydrogenase (E.C. 1. 1. 1.40), 2-oxoglutarate dehydrogenase, (E.C.1.2.4.2) glutamate dehydrogenase (E.C.1.4.1.3), glutamin synthetase (E.C.6.3.1.2), arginase (E.C.3.5.3.1), ornithine carbamoyltransferase (E.C.2.1.3.3), aspartate aminotransferase (E.C.2.6.1.1) and alanine aminotransferase (E.C.2.6.1.2) were measured in cardic and skeletal muscles of steers fed ration with or without monensin. Steers, 9 months old at the beginning, were fed concentrate, molasses, grass, lucerne and maize chaff. Five steers received monensin in amount of 0.5 mg per 1 kg of live body mass per day. Control ration, without monensin was fed to the other five steers. After 10 months steers were slaughtered and activity of enzymes assayed in the mitochondrial and cytoplasmic fraction of cells. Monensin increased activity of 2-oxoglutarate dehydrogenase and decreased activity of glutamate dehydrogenase in both fractions of the skeletal muscle tissue. Enzymatic activities found in heart mitochondria were higher in monensin-fed steers than in control steers. Steers given monensin gained 8.23 % more than control steers (263 vs 243 kg). Monensin, enzyme, heart, muscle Feed antibiotics, including monensin, have the ability to improve perfonnance and feed efficiency in ruminants. It is known that monensin is absorbed from the alimentary tract (D a vis 0 n 1983; Don 0 h 01984) and several authors demonstrated its influence on intermediary metabolism of ruminants (Armstrong and Spears 1988; Benz et al.1989;Marounek et al. 1989). Little is known about the effect of monensin on activity of tissue enzymes in ruminants. K a I a c n j uk et al. (1993) measured activity of various enzymes in rumen mucosa and liver of steers fed monensin at recommended level. Authors found no consistent effect of monensin on enzymes of both tissues. In this paper we report data on activity of nine enzymes in cardiac and skeletal muscles of steers fed ration with or without monensin for ten months. Cardiac and skeletal muscles are primary target tissues at high intake of monensin (T 0 d d et al. 1984; Van V lee t et al. 1985) and, presumably, also during long-term supplementation of rations under normal feeding conditions. ' Materials and Methods Ten crossbred steers, 9 months old at the beginning of experiment, were divided into two groups, according to feed additive treatment. Steers were individually housed and kept on a diet consisting of concentrate (I kg per 100 kg of the live weight), molasses (0.5 1.0 kg) and grass, lucerne and maize chaff ad libitum. In winter, the roughage portion of the diet consisted of maize silage and beet ad libitum. Concentrate contained ground barley (63%), dried poultry litter from broilers which did not receive a ionophore in their diet (20%), grass meal (15%) and zeolite (2%).

Use of a selective inhibitor of liver carnitine palmitoyltransferase I (CPT I) allows quantification of its contribution to total CPT I activity in rat heart. Evidence that the dominant cardiac CPT I isoform is identical to the skeletal muscle enzyme

It has recently been established that rat heart mitochondria contain two isoforms of carnitine palmitoyltransferase I (CPT I), the minor 88-kDa variant being identical to liver CPT I (L-CPT I) and the dominant 82-kDa form resembling the skeletal muscle enzyme (M-CPT I) (Weis, B. C., Esser, V., Foster, D. W., and McGarry, J. D. (1994) J. Biol. Chem. 269, 18712-18715). To quantify the functional contribution of L-CPT I to overall CPT I activity in heart mitochondria a selective inhibitor of the former was needed. The dinitrophenol analog of 2[6-(4-chlorophenoxy)hexyl]oxirane-2-carboxylic acid (etomoxir) (DNP-Et) was found to have this property. When liver and skeletal muscle mitochondria were exposed to DNP-Et in the presence of ATP and CoASH, the DNP-Et-CoA formed completely inhibited liver CPT I while leaving the muscle enzyme unaffected. Similar treatment of heart mitochondria blocked only the L-CPT I component. This had the effect of shifting the apparent Km for carnitine from approximately 200 to approximately 500 microM and the I50 value for malonyl-CoA (the concentration needed to suppress enzyme activity by 50%) from approximately 0.18 to approximately 0.06 microM, i.e. the heart system now behaved exactly the same as that from skeletal muscle. Taking the Km for carnitine of L-CPT I and M-CPT I to be 30 and 500 microM, respectively, it could be calculated that the former contributes approximately 2% to the total CPT I in heart. When the 82-kDa CPT I isoforms of heart and skeletal muscle were labeled with [3H]etomoxir and then exposed to trypsin, the fragmentation patterns obtained were identical and quite distinct from that given by CPT I from liver. We conclude that (i) DNP-Et, unlike other agents of the oxirane carboxylic acid class, has remarkable inhibitory selectivity for L-CPT I over M-CPT I; (ii) the previously puzzling observation that rat heart CPT I displays kinetic characteristics intermediate between those of the enzymes from liver and skeletal muscle is entirely accounted for by the low level expression of L-CPT I in the cardiac myocyte; and (iii) the dominant 82-kDa CPT I isoform in heart is identical to the muscle enzyme. The data reaffirm that, in contrast to CPT II, CPT I exists in at least two isoforms and that both are present in rat heart.

Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. The minor component is identical to the liver enzyme.

To begin to explore the basis for the tissue-specific expression of mitochondrial carnitine palmitoyltransferase I (CPT I), we focused on three rat tissues (liver, heart, and skeletal muscle) in which the enzyme was known to display very different properties. In Northern blot analysis, a cDNA probe corresponding to liver CPT I readily hybridized to a 4.5-kilobase species of mRNA in liver and heart, but not in skeletal muscle. Using the same probe to screen a neonatal rat heart cDNA library, a full-length clone, surprisingly having 100% sequence identity to the liver CPT I cDNA, was isolated. The paradox was resolved by two additional experiments. First, in Western blots of mitochondrial membranes, an antibody raised against liver CPT I recognized the 88-kDa protein in heart, as well as in liver, but not in skeletal muscle. Second, high specific activity [3H]deschloroetomoxir (a covalent ligand for CPT I) reacted with a single form of CPT I in liver (approximately 88 kDa) and skeletal muscle (approximately 82 kDa), while proteins of both sizes were labeled in the cardiac myocyte. Tritiated ligand binding to the two heart proteins was blocked by excess unlabeled malonyl-CoA. It is concluded that liver and skeletal muscle each contains a single and distinct isoform of CPT I with monomeric size of approximately 88 and 82 kDa, respectively. The heart contains a CPT I protein of approximately 82 kDa in size (probably identical to the skeletal muscle protein) but, importantly, also expresses the liver-type enzyme. The results likely explain why previous studies of heart CPT I yielded an apparent Km for carnitine and I50 value for malonyl-CoA inhibition that were intermediate between those of the liver and skeletal muscle enzymes.

Concomitant Branching Enzyme and Phosphorylase Deficiencies. An Unusual Glycogenosis with Extensive Neuronal Polyglucosan Storage

A baby girl was born hypotonic and was respirator-dependent until death at 43 days of age. A muscle biopsy revealed PAS-positive, diastase-resistant sarcoplasmic inclusions with a vaguely fibrillar structure by electron microscopy. Biochemical studies at autopsy disclosed complete absence of branching enzyme in skeletal muscle and heart, and a deficiency of phosphorylase activity in skeletal muscle with a modest reduction in myocardium. Storage material was present in glia and perikarya of neurons, increasing in amount in the rostrocaudal direction, involving most severely the motor neurons in the brain stem and spinal cord, dorsal root ganglia and myenteric plexi. Inclusions were also present in most organs, especially liver and skeletal muscle. Ultrastructurally, the inclusions ranged from granular aggregates of membrane-bound material concentrated in the region of Golgi apparatus to large filamentous bodies similar to polyglucosan bodies. This baby differs from other patients with infantile glycogenosis IV by the severity and onset of symptoms at birth, involvement of neuronal perikarya and widespread extraneural deposits. The combined deficiencies of branching enzyme and phosphorylase may have accounted for the unique clinical and neuropathological findings.

Angiotensin-converting enzyme in human skeletal muscle. A simple in vitro assay of activity in needle biopsy specimens.

The importance of skeletal muscle for human haemodynamics and metabolism makes it a suitable sample tissue for the measurement of angiotensin converting enzyme (ACE; EC 3.4.15.1). A study of serum and muscle angiotensin-converting enzyme was performed in 50 essential-hypertensive subjects. Muscle tissue was obtained from the vastus lateralis muscle by Bergström needle biopsies. The method used is a modification in Cushman's fluorometric assay for tissue angiotensin-converting enzyme. It utilized 5 mM hippuryl-histidyl-leucine as a synthetic substrate for a 60-min fixed-time incubation at 37 degrees C. The 0.25 ml incubation mixture consisted of 0.12 mM potassium phosphate buffer, pH 8.1, 300 mM NaCl, and 1 or 2 mg homogenized muscle tissue. The activity was found to have the properties previously reported for human ACE. The intra- and interassay coefficients of variation of the method were 13% and 21%, respectively, for the 1 mg incubation as assessed from internal controls (n = 20). the muscle ACE activity in the hypertensive subjects was 49 +/- 14 mU g-1 (mean +/- SD) with no difference between values for men and women. No correlation was found between muscle ACE activity and age, or between muscle and serum angiotensin-converting enzyme activity. We consider this assay a useful new tool for investigating the role of angiotensin-converting enzyme in the pathogenesis and treatment of hypertension, as it can be applied to patient materials.

A New Stimulation Protocol for Cardiac Assist Using the Latissimus Dorsi Muscle

When treating severe cardiac failure with dynamic cardiomyoplasty, knowledge about the optimal way of stimulating the latissimus dorsi (LD) muscle is of obvious importance. We evaluated a new stimulation protocol in four goats using in situ electrical stimulation of the left LD muscle. Stimulation was started using a burst of two pulses with an interpulse interval of 100 msec for 50 bursts/min. The number of pulses was increased every 2 weeks concomitant with a decrease in interpulse interval. This resulted after 12 weeks in 60 bursts/min using bursts of six pulses with an interpulse interval of 20 msec after 12 weeks. Force measurements, which were done every 2 weeks, showed an early decrease in contraction and relaxation speed as reflected in the ripple (= interstimulus amplitude/peak force amplitude measured at 10 Hz). Fatigue resistance increased significantly within 4 weeks of conditioning as indicated by preservation of force, positive dF/dt, and negative dF/dt. Full preservation of these variables was seen even during a 1-hour fatigue test at the end of the conditioning period. Skeletal muscle enzyme activity as an indicator of muscle damage showed a significant rise in creatine kinase enzyme activity only on the first day following the start of LD stimulation. LD muscle biopsies revealed almost complete transformation to type I muscle fibers with a significant increase in capillary/fiber ratio when compared to the nonstimulated LD muscle. However, some biopsies, in particular near the electrodes, did show some signs of skeletal muscle damage. Contraction characteristics of the fully transformed LD muscles were tested by increasing the number of bursts of six pulses from 50/min to 100/min. Interpulse intervals of 20 and 33 msec were used. These tests revealed that maximal force, positive dF/dt, and negative dF/dt was reached with 50 bursts/min using a six pulse burst with interpulse intervals of 20 msec.

Effects of Feeding and Food Deprivation on Oxygen Consumption, Muscle Protein Concentration and Activities of Energy Metabolism Enzymes in Muscle and Brain of Shallow-Living (Scorpaena Guttata) and Deep-Living (Sebastolobus Alascanus) Scorpaenid Fishes

ABSTRACT The effects of feeding and fasting were examined on the deep-living short-spine thornyhead (Sebastolobus alascanus) and the confamilial shallow-living spotted scorpionfish (Scorpaena guttata) to determine whether the low metabolic rate of the deeper-living species was in part a consequence of food deprivation in its habitat. Laboratory acclimation for periods of 90–115 days under either ad libitum feeding or complete fasting did not lead to similar rates of respiration in individuals of the two species held under identical conditions. Respiration of fish fed ad libitum was 52 % (S. guttata) or 68% (S. alascanus) higher than for fasted fish of the same species. Furthermore, the metabolic rates of freshly collected specimens of S. alascanus resembled those of laboratory-fasted fish. In white skeletal muscle, both total protein concentration and the activities of four enzymes of ATP metabolism, lactate dehydrogenase (LDH) and pyruvate kinase (PK) of glycolysis, malate dehydrogenase (MDH) and citrate synthase (CS, a citric acid cycle indicator), were lower in S. alascanus than in S. guttata. Within a species, protein concentration and activities of the four enzymes in white muscle, but not in brain, were higher in fed than in starved fish, although these differences were greater in S. alascanus than in S. guttata. During fasting, LDH and PK activity in white muscle of S. alascanus decreased much more than MDH and CS activity; decreases in enzyme activities in red muscle were smaller than those in white muscle. Activities of enzymes in white skeletal muscle of field-collected S. alascanus generally resembled those of the fasted specimens. In contrast, red muscle of field-collected S. alascanus, compared with that of either fed or starved laboratory-held specimens, had a highly glycolytic poise (high LDH and PK activities relative to MDH and CS activities), which may suggest that muscle enzyme activities in the field-collected fish reflect adaptation to the low oxygen level in its adult habitat, the oxygen minimum layer. The strong correlations found between tissue biochemical properties and respiration rate allow us to develop a predictive index for metabolic rate from simple biochemical analyses, e.g. white muscle protein content or CS activity. We conclude that the low metabolic rate of S. alascanus is due to at least four depth-related factors: reduced abundance of food, low temperature, low ambient oxygen concentration and darkness, which may select for reduced locomotory activity.

Glucocorticoid induction of epinephrine synthesizing enzyme in rat skeletal muscle and insulin resistance.

Rat skeletal muscle contains two enzymes which can make epinephrine: phenylethanolamine N-methyltransferase (PNMT) and nonspecific N-methyltransferase. We studied the time-course and mechanism by which the glucocorticoid dexamethasone increases muscle PNMT activity. We also examined the hypothesis that increased muscle E synthesis may contribute to glucocorticoid-induced insulin resistance. Dexamethasone (1 mg/kg s.c. for 12 d) increased muscle PNMT activity seven-fold but did not change NMT activity. Immunotitration with an anti-PNMT antibody indicated that the PNMT elevation was due to increased numbers of PNMT molecules. Dexamethasone rapidly increased PNMT activity and this elevation was largely maintained 6 d after glucocorticoid treatment stopped. Muscle epinephrine levels were transiently elevated by dexamethasone. Dexamethasone-treated rats had elevated insulin levels after a glucose load, and chronic administration of the PNMT inhibitor SKF 64139 reversed this increase. Chronic SKF 64139 improved glucose tolerance in normal rats. Dexamethasone induced muscle synthesis of the epinephrine-forming enzyme PNMT. A PNMT inhibitor lowered insulin levels in glucocorticoid-treated rats and glucose levels in untreated rats. These findings are compatible with antagonism of insulin-mediated glucose uptake by epinephrine synthesized in skeletal muscle.

Phosphate-dependent glutaminase of rat skeletal muscle. Some properties and possible role in glutamine metabolism

A relatively high activity (26.7 nmol/min per mg mitochondrial protein) of phosphate-dependent glutaminase (EC 3.5.1.2; L-glutamine amidohydrolase) was found in rat skeletal muscle (mixed type from hindlegs) mitochondria incubated in 200 mM potsssium phosphate (pH 8.2); the activity was lower in rat heart and diaphragm mitochondria. Phosphate-dependent glutaminase was also found in human skeletal muscle mitochondria, but the activity was about 3–5 times lower than in rat skeletal muscle. Multiplying the specific activity of mitochondrial glutaminase by the amount of mitochondrial protein present in 1 g of rat skeletal muscle the maximum glutaminase activity was found to be 0.352 μmol/min per g wet tissue. The rat skeletal muscle enzyme appears to be similar in many respects to phosphate-dependent glutaminase of the kidney (e.g., S 0.5 for glutamine, K 0.5 for phosphate, the pH activity profile, inhibition by glutamate). These properties make the skeletal muscle enzyme very similar to the ‘kidney type’ glutaminase isoenzyme of rat tissues. A significant difference between rat kidney and skeletal muscle enzymes is their adaptive response during acidosis. While the kidney enzyme increases during acidosis, the skeletal muscle glutaminase activity does not. A possible role of glutaminase in the glutamine metabolism in rat skeletal muscle is discussed.

The Skeletal Muscle Transverse Tubular Mg-ATPase: Identity with Mg-ATPases of Smooth Muscle and Brain

Purified chicken skeletal muscle transverse tubule (T-tubule, TT) membrane preparations contain a very active Ca- or Mg-ATPase (EC 3.6.1.3) previously thought to be a T-system-specific marker enzyme. The function of the Mg-ATPase has not yet been determined although its prominent activity and concentration in junctional complexes supports a possible role in the excitation-contraction cycle. An essential component of the Mg-ATPase has been identified as a M r 85,000 glycoprotein (85k-GP). Polyclonal antibodies raised against the TT 85k-GP were specific and exhibited no cross-reactivity with other skeletal muscle proteins on immunoblots. Using this anti-85k-glycoprotein IgG, we have explored other chicken tissues to determine the tissue distribution of the 85k-GP. Antibody reactive polypeptides of M r 85,000 were found in gizzard smooth muscle, brain, heart, spleen, and lung tissue. The brain and smooth muscle membrane proteins were further purified and characterized for 85k-GP-associated Mg-ATPase activity. The brain and smooth muscle enzymes exhibited properties indistinguishable from the skeletal muscle TT-specific Mg-ATPase with regard to a series of activators and inhibitors, amino terminal amino acid sequences, and the effects of deglycosylation. The enzyme in all three tissues was inhibited by the diacylglycerol kinase inhibitor R 59022. Identification of the TT Mg-ATPase in gizzard smooth muscle has allowed the investigation of the Mg-ATPase membrane topology using isolated whole smooth muscle cells. The data support an ecto-orientation for the smooth muscle cell enzyme. Although the orientations of the brain and skeletal muscle enzymes have not been conclusively determined, the nearly identical properties of all three enzymes argues for an ecto-orientation of the active sites of these enzymes as well. The responsiveness of the three enzymes to regulatory lipids suggests that the ecto-Mg-ATPase may serve as a master switch controlling extracellular ATP concentrations and ligand accessibility to P 1- and P 2-purinoceptors. It is also proposed that the ecto-Mg-ATPase may regulate ATP accessibility to ectoprotein kinases in a variety of tissues, and, in brain, the ecto-Mg-ATPase may modulate the neurotransmitter role of ATP.

714 The Effects of Aging and Training on Glutathione Status and Antioxidant Enzymes in Rat Skeletal Muscle

714 The Effects of Aging and Training on Glutathione Status and Antioxidant Enzymes in Rat Skeletal Muscle

Hierarchical Regulation by Casein Kinases I and II of the Activation of Protein Phosphatase-II by Glycogen Synthase Kinase-3 Is Ionic Strength Dependent

The roles of casein kinases I and II in the activation of protein phosphatase-1, (PP-1 i) by glycogen synthase kinase-3 (GSK-3) were studied using enzyme preparations from porcine heart. PP-1 i was activated by GSK-3 and the levels of activation achieved decreased by increasing the ionic strength (0-0.2 M KCl) in the incubation mixtures. At low ionic strength (no KCI added) casein kinase II increased the rate of activation of PP-1 i by GSK-3 and the activation proceeded to a slightly greater extent (110-120%) than that obtained by GSK-3 alone. In the presence of 0.14 M KCl only a partial activation of PP-1 i by GSK-3 was observed, but when casein kinase II was also added activation was restored to levels observed when PP-1 i was activated by GSK-3 in the absence of salt. This effect was shown to be dependent on the concentration of casein kinase II. These results would imply that at low ionic strength casein kinase II and GSK-3 synergistically activate PP-1 i as has been previously reported for the rabbit skeletal muscle enzyme (DePaoliRoach, A. A., J. Biol. Chem. 259, 12 144-12152, 1984), whereas, at physiological ionic strength, casein kinase II action may be obligatory for GSK-3 activity. Similar results were obtained when casein kinase I replaced casein kinase II.

Cadiac and skeletal muscle enzymes levels in hypertensive and ageing rats

1. 1. The activities of glycolytic, fatty acid oxidation and citric acid cycle enzymes were measured in hypertensive and ageing rat cardiac and skeletal muscles. 2. 2. Lactate dehydrogenase and β-hydroxyacyl-CoA dehydrogenase were significally decreased in hypertensive, but not senescent, cardiac muscle. 3. 3. Total phosphorylase activity was significantly increased in senescent, but not hypertensive, cardiac muscle. 4. 4. In ageing rat cardiac and skeletal muscles, calpian II titers increased significantly with age, but in normotensive and hypertensive muscles, the titers showed no significant difference.

Glutathione and antioxidant enzymes in skeletal muscle: effects of fiber type and exercise intensity.

Glutathione status and antioxidant enzymes in various types of rat skeletal muscle were studied after an acute bout of exercise (Ex) at different intensities. Glutathione (GSH) and glutathione disulfide (GSSG) concentrations were the highest in soleus (SO) muscle, followed by those in deep (DVL) and then superficial (SVL) portions of vastus lateralis. In DVL, but not in SO or SVL, muscle GSH increased proportionally with Ex intensity and reached 1.8 +/- 0.08 mumol/g wet wt compared with 1.5 +/- 0.03 (P < 0.05) in resting controls (R). GSSG in DVL was increased from 0.10 +/- 0.01 mumol/g wet wt in R to 0.14 +/- 0.01 (P < 0.05) after Ex. Total glutathione (GSH + GSSG) contents in DVL were also significantly elevated with Ex, whereas GSH/GSSG ratio was unchanged. Activities of GSH peroxidase (GPX), GSSG reductase (GR), and catalase (CAT) were significantly higher in SO than in DVL and SVL, but there was no difference in superoxide dismutase activity between the three muscle types. Furthermore, Ex at moderate intensities elicited significant increases in GPX, GR, and CAT activities in DVL muscle. None of the antioxidant enzymes was affected by exercise in SO. It is concluded that rat DVL muscle is particularly vulnerable to exercise-induced free radical damage and that a disturbance of muscle GSH status is indicative of an oxidative stress.

Electrical stimulation-induced changes in skeletal muscle enzymes of men and women

The purpose of the present study was to determine the effects of low-frequency electrical stimulation (LFES) on the skeletal muscle metabolic profile of men and women. The knee extensor muscles of sedentary men (N = 16) and women (N = 10) were submitted to 3 h.d-1 of 8-Hz neuromuscular electrical stimulation with the use of a portable stimulator (Respond II, Medtronic), 6 d.wk-1 for 6 wk. Enzyme activity levels of creatine kinase (CK), hexokinase (HK), glyceraldehydephosphate dehydrogenase (GAPDH), 3-hydroxyacyl CoA dehydrogenase (HADH), citrate synthase (CS), phosphofructokinase (PFK), and cytochrome c oxidase (COX) were determined in vastus lateralis muscle samples taken before and after the LFES protocol. The analyses of variance revealed no change in CK and in GAPDH. However, a small decrease in PFK activity, the rate-limiting enzyme of glycolysis, was observed in female (8%) and in male subjects (10%), but it reached significance in males only (P < 0.05). The activity level of HK, a regulatory enzyme of the skeletal muscle glucose phosphorylation (HK), increased significantly in female subjects only (36%; P < 0.01) in response to the stimulation protocol. Activity level of marker enzymes of the Krebs cycle (CS) and of the electron-transfert chain (COX) significantly increased in males (18% and 16%; P < 0.05) as well as in females (31% and 19%; P < 0.05). Increment in the marker enzyme activity of the fatty acid oxidation (HADH) was significant in female subjects (30%; P < 0.01) and, although significant, rather modest in male subjects (12%; P < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Pre- and Postnatal Protein Undernutrition Increases Hepatic Carnitine Palmitoyltransferase I Activity and Decreases Enzyme Sensitivity to Inhibitors in the Suckling Rat

Rats were pair-fed isocaloric diets containing either 25% (control diet) or 6% protein (low-protein diet) during the 5 weeks prior to conception and through the gestation and lactation periods; then, carnitine palmitoyltransferase I (CPT-I) activity was determined in liver and skeletal muscle mitochondria isolated from the corresponding pups. Maternal protein undernutrition increased the activity of hepatic CPT-I all along the suckling period, whereas the activity of the skeletal muscle enzyme was unaffected. Moreover, the sensitivity of hepatic CPT-I to inhibition by both malonyl-CoA and 4-hydroxyphenylglyoxylate was decreased in the low-protein group. These alterations in the properties of hepatic CPT-I may be involved in the appearance of hyperketonemia in the rat pup upon maternal administration of low-protein diets.

LDH isozymes in amazon fish—III. Distribution patterns and functional properties in Serrasalmidae (Teleostei: Ostariophysi)

1. 1. The patterns of distribution of LDH isozymes in five tissues from 12 Serrasalmidae species have been studied through two-fold serial dilution (Klebe's test) as well as the pyruvate inhibition of LDH enzyme in skeletal and heart muscles (low/high ratios). 2. 2. The species' electrophoretic patterns differ by orthologous A 4 isozyme mobilities since the orthologous B 4 isozymes present similar electrophoretic migration. These differences between Ldh-A and Ldh-B products reflect three-, four-, and five-banded patterns. Thus, different LDH isozyme numbers formed from A and B subunits should not be used as an evolutionary or phylogenetic characteristic from different taxonomic groups. 3. 3. Out of 66 pairs of species only five pairs showed significant differences in the distribution patterns in all five analyzed tissues, while no pair of species showed the same distribution in these tissues. This variation was explained as differential regulation of structural genes among tissues and/or species. 4. 4. Functional properties showed significant between the LDHs from heart and skeletal muscles, and are consistent with a preference for aerobic metabolism. We suppose that by selecting B-like subunits these fishes are able to maintain good control of aerobic/anaerobic transitions, maintaining predominantly oxidative metabolism even in hypoxic waters, with which they have to cope.

Identification of mitochondrial branched chain aminotransferase and its isoforms in rat tissues.

The tissue distribution and subcellular location of branched chain aminotransferase was analyzed using polyclonal antibodies against the enzyme purified from rat heart mitochondria (BCATm). Immunoreactive proteins were visualized by immunoblotting. The antiserum recognized a 41-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate. The 41-kDa protein was always present in mitochondria which contained branched chain aminotransferase activity, skeletal muscle, kidney, stomach, and brain, but not in cytosolic fractions. In liver mitochondria, which have very low levels of branched chain aminotransferase activity, the 41-kDa protein was not present. However, two immunoreactive proteins of slightly higher molecular masses were identified. These proteins were located in hepatocytes. The 41-kDa protein was present in fetal liver mitochondria but not in liver mitochondria from 5-day neonates. Thus disappearance of the 41-kDa protein coincided with the developmental decline in liver branched chain aminotransferase activity. Two-dimensional immunoblots of isolated BCATm immunocomplexes showed that the liver immunoreactive proteins were clearly different from the heart and kidney proteins which exhibited identical immunoblots. Investigation of BCATm in subcellular fractions prepared from different skeletal muscle fiber types revealed that branched chain aminotransferase is exclusively a mitochondrial enzyme in skeletal muscles. Although total detergent-extractable branched chain aminotransferase activity was largely independent of fiber type, branched chain aminotransferase activity and BCATm protein concentration were highest in mitochondria prepared from white gastrocnemius followed by mixed skeletal muscles with lowest activity and protein concentration found in soleus mitochondria. These quantitative differences in mitochondrial branched chain aminotransferase activity and enzyme protein content suggest there may be differential expression of BCATm in different muscle fiber types.

Metabolic Aspects of Shivering Thermogenesis in Passerines during Winter

Various passerines enhance their cold resistance in winter through a form of metabolic acclimatization. This involves an improved ability to sustain elevated rates of regulatory thermogenesis, primarily through shivering. This muscular activity depends principally on catabolism of fatty acids. Significantly, establishment and daily replenishment of substantial fat reserves are associated with winter acclimatization in many passerines. This acclimatization also appears linked with winter increases in the activities of certain catabolic enzymes in skeletal muscle, notably P-hydroxyacyl Co-A dehydrogenase and phosphofructokinase. The factors that initiate and regulate winter acclimatization in passerines are poorly understood. The metabolic character of this form of adjustment, which certainly involves processes affecting deposition, mobilization, and use of energy substrates assures that an array of hormones will be involved. We review several possible candidates, emphasizing glucagon, corticosterone, and the thyroid hormones. Recent studies indicate seasonal variation in levels of thyroid hormones, but not of corticosterone in free-living individuals of the American Goldfinch Carduelis tristis, a prominent species in studies of passerine winter acclimatization.