Homogenates Of Rat Skeletal Muscle Research Articles

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements

Preparation of Rat Skeletal Muscle Homogenates for Nitrate and Nitrite Measurements.

Nitrate ions (NO3-) were once thought to be inert end products of nitric oxide (NO) metabolism. However, previous studies demonstrated that nitrate ions can be converted back to NO in mammals through a two-step reduction mechanism: nitrate being reduced to nitrite (NO2-) mostly by oral commensal bacteria, then nitrite being reduced to NO by several mechanisms including via heme- or molybdenum-containing proteins. This reductive nitrate pathway contributes to enhancing NO-mediated signaling pathways, particularly in the cardiovascular system and during muscular exercise. The levels of nitrate in the body before such utilization are determined by two different sources: endogenous NO oxidation and dietary nitrate intake, principally from plants. To elucidate the complex NO cycle in physiological circumstances, we have examined further the dynamics of its metabolites, nitrate and nitrite ions, which are relatively stable compared to NO. In previous studies skeletal muscle was identified as a major storage organ for nitrate ions in mammals, as well as a direct source of NO during exercise. Therefore, establishing a reliable methodology to measure nitrate and nitrite levels in skeletal muscle is important and should be helpful in extending its application to other tissue samples. This paper explains in detail the preparation of skeletal muscle samples, using three different homogenization methods, for nitrate and nitrite measurements and discusses important issues related to homogenization processes, including the size of the samples. Nitrate and nitrite concentrations have also been compared across four different muscle groups.

Response of Sarcolemmal Fatty Acid Transport Proteins to Moderate Exercise in Human and Rat Muscle

Fatty acid transport proteins, including fatty acid translocase (FAT/CD36) and plasma membrane fatty acid binding protein (FABPpm) are responsible for protein-mediated uptake of long chain fatty acids (LCFA) into skeletal muscle. These proteins have been shown to exist in intracellular vesicles and translocate to the plasma membrane in response to acute electrical stimulation in rat skeletal muscle, however this has not been tested in exercising animals or humans. PURPOSE: To determine if prolonged moderate intensity exercise increases the content of fatty acid transport proteins at the sarcolemma in rat and human skeletal muscle and if this is accompanied by an increase in LCFA transport in rats. METHODS: Nine male and female subjects cycled for 120 min at ∼60 ± 2% VO2 max. Two skeletal muscle biopsies were taken at rest and again following 120 min of cycling and expired gases were sampled throughout exercise. Giant sarcolemmal vesicles were prepared from the muscle biopsies and the remaining muscle was used for whole muscle measurements. 4 male and 4 female Sprague-Dawley rats completed a 2 h treadmill run at 20 m/min while four male and four female rats acted as non-running controls. Giant sarcolemmal vesicles were prepared from the hindlimb muscles and palmitate uptake was measured. Protein content of FAT/CD36 and FABPpm were measured in both giant sarcolemmal vesicles and whole rat and human skeletal muscle homogenates. RESULTS: In human skeletal muscle, plasma membrane FAT/CD36 protein content increased by 40-300% following 120 min of exercise (p < 0.05), while whole muscle homogenate FAT/CD36 was unchanged. There was no change in FABPpm protein in human muscle. Transport increased 1.2-fold (p < 0.05) with exercise in rat muscle, which correlated with an increase in sarcolemmal FAT/CD36 (1.2-fold) and FABPpm (1.3-fold) protein. There was no change in sarcolemmal protein content in whole muscle. CONCLUSIONS: The rat and human data suggest that fat transport proteins are mobilized and translocated to the plasma membrane of skeletal muscle during prolonged endurance exercise to assist with increased LCFA uptake into the cell. The increase was restricted to FAT/CD36 in human skeletal muscle, and the increase was larger and more variable than found in rat skeletal muscle. Funded by CIHR and NSERC, Canada.

Targeted 18O-Labeling for Improved Proteomic Analysis of Carbonylated Peptides by Mass Spectrometry

Proteomic characterization of carbonylated amino acid sites currently relies on confidently matching tandem mass spectra (MS(2)) to peptides within a sequence database. Although effective to some degree, reliable proteomic characterization of carbonylated peptides using this approach remains a challenge needing new, complementary solutions. To this end, we developed a method based on partial (18)O-labeling of reactive carbonyl modifications, which produces a unique isotope signature in mass spectra of carbonylated peptides and enables their detection without reliance on matching MS(2) spectra to a peptide sequence. Key to our method were optimized measures for eliminating trypsin-catalyzed incorporation of (18)O at peptide C-termini, and for stabilizing the incorporated (18)O within the carbonyl modification to prevent its loss during liquid chromatography separation. Applying our method to a rat skeletal muscle homogenate treated with the carbonyl modification 4-hyroxynonenal (4-HNE), we demonstrated its compatibility with solid-phase hydrazide enrichment of carbonylated peptides from complex mixtures. Additionally, we demonstrated the value of (18)O isotope signatures for confirming HNE-modified peptide sequences matched via sequence database searching, and identifying modified peptides missed by MS(2) and/or sequence database searching. Combining our (18)O-labeling method with a customized automated software script, we systematically evaluated for the first time the efficiency of MS(2) and sequence database searching for identifying HNE-modified peptides. We estimated that less than half of the modified peptides selected for MS(2) were successfully identified. Collectively, our method and software should provide valuable new tools for investigators studying protein carbonylation via mass spectrometry-based proteomics.

Age-related increase in xanthine oxidase activity in human plasma and rat tissues

This study assessed the role of xanthine oxidase in vascular ageing. A positive correlation between xanthine oxidase activity and age was found in human plasma. Similar results were found in rat plasma. Xanthine oxidase expression and activity in homogenates from the aortic wall were significantly higher in samples from old rats than in their young counterparts (p<0.01). In rat skeletal muscle homogenates both xanthine oxidase expression and activity showed a similar age-related profile. Superoxide production by xanthine oxidase in aortic rings was higher in aged rats. Uric acid, the final product of xanthine oxidase has been proposed as a risk factor for coronary heart disease and an independent marker of worse prognosis in patients with moderate-to-severe chronic heart failure. These results give a possible explanation for this correlation and underscore the role of xanthine oxidase in ageing.

Fenofibrate impairs rat mitochondrial function by inhibition of respiratory complex I.

Fibrates are used for the treatment of dyslipidemia and known to affect mitochondrial function in vitro. To better understand the mechanisms underlying their mitochondrial effects, fibrate actions on complex I of the respiratory chain and cell respiration were studied in vitro. In homogenates of rat skeletal muscle, fenofibrate, and to a lesser extent clofibrate, reduced the activity of complex I (10, 30, and 100 microM fenofibrate: -41 +/- 7%, -70 +/- 2%, and -78 +/- 4%; 100 microM clofibrate: -27 +/- 7%; p < 0.005 each). Inhibition of complex I by fenofibrate (100 microM) was confirmed by reduced state 3 respiration of isolated mitochondria consuming glutamate + malate as substrates for complex I (-33 +/- 4%; p < 0.0005), but not of such consuming succinate as substrate for complex II (-8 +/- 4%; NS). In isolated rat muscle, 24-h fenofibrate exposure (25, 50, and 100 microM) decreased CO(2) production from palmitate (-15 +/- 7%, -23 +/- 8%, and -22 +/- 7%; p < 0.05 each) and increased lactate release (+15 +/- 5%, +14 +/- 5%, and + 17 +/- 6%; p < 0.02 each) indicating impaired cell respiration. Ciprofibrate and gemfibrocil (but not bezafibrate) impaired cell respiration without any inhibition of complex I. Our findings support the notion that individual fibrates induce mitochondrial dysfunction via different molecular mechanisms and show that fenofibrate predominantly acts by inhibition of complex I of the respiratory chain.

Effect of intermittent and continuous hypoxia on ryanodine receptors of rat heart

Intermittent hypoxia (IH) adaptation has been shown to exert beneficial effects on the functions of hearts that had been subjected to insult by ischemia or ischemia/reperfusion. To understand whether calcium release channels/ryanodine receptors (RyRs) were involved, the effects of IH and continuous hypoxia (CH) on [ 3H]ryanodine binding to homogenates of rat hearts were investigated. Similar studies were performed on rat skeletal muscle. The main results on cardiac muscle were as follows: 1) Ischemia for up to 45 min in normal rat hearts had no obvious effect on the equilibrium ryanodine binding constant (K d), while the maximum number of ryanodine binding sites (B max) was affected in a time-dependent manner. B max was significantly increased with 15 min ischemia, which then returned to control levels upon prolonging the ischemia to 30 min. After 45 min ischemia, a small decrease of B max was observed. 2) IH adaptation for up to 28 days did not change B max, but a significant decrease of B max was apparent after longer IH adaptation or after CH exposure. Although B max was not altered by 30 min ischemia, 30 min reperfusion following 30 min ischemia induced an evident decrease of B max. After either IH or CH adaptation, the ischemia/reperfusion- induced decrease of B max was abolished. 3) Several effects on K d of ischemia and ischemia/reperfusion, with and without IH or CH adaptation, were observed. The most distinct and consistent finding was that a clear increase of K d was induced by ischemia or ischemia/reperfusion in CH adapted rats. [ 3H]Ryanodine binding to homogenates of rat skeletal muscle was also affected by IH and CH adaptation. In contrast to that found in cardiac muscle, a decrease of B max in skeletal muscle appeared only after CH adaptation. The physiological significance of these effects is discussed.

Detoxification of cytotoxic alpha,beta-unsaturated aldehydes by carnosine: characterization of conjugated adducts by electrospray ionization tandem mass spectrometry and detection by liquid chromatography/mass spectrometry in rat skeletal muscle.

Oxidation of polyunsaturated fatty acids containing phospholipids in tissue generates lipid hydroperoxides, which are further degraded to several products, among which unsaturated aldehydes such as 4-hydroxy-trans-2-nonenal (HNE) play an important role in mediating the pathological effects of oxidative stress. While the reaction of HNE with glutathione (GSH) is a well recognized pathway of detoxification in biological systems, no data are available on HNE interactions with carnosine, a dipeptide (beta-alanyl-L-histidine) present in high concentration in skeletal muscle. The aim of this work was to study the quenching ability of carnosine towards HNE and to characterize the reaction products by electrospray ionization tandem mass spectrometry (ESI-MS/MS), using GSH as a model peptide. GSH incubation with HNE in 1 mM phosphate buffer (pH 7.4) results in the complete disappearance of HNE within 1 h owing to the formation of a Michael adduct, S-(4-hydroxynonanal-3-yl)glutathione. The reaction of HNE with carnosine was studied in different molar ratios and monitored up to 24 h by high-performance liquid chromatography (HPLC) (HNE consumption), MS/MS (infusion) and liquid chromatography mass spectrometry (LC/MS) experiments. Carnosine, although less reactive than GSH, significantly quenched HNE (48.2 +/- 0.9% HNE consumption after 1 h; carnosine:HNE molar ratio 10 : 1). Two reaction products were identified: the Michael adduct, N-(4-hydroxynonanal-3-yl)carnosine involving the imidazolic nitrogen of histidine, and the imine adduct, involving the amino group of the beta-alanine residue. Definitive structure assignment was achieved by chemical reduction with NaBH(4) and multinuclear magnetic resonance experiments. To understand whether carnosine acts as a quencher of unsaturated aldehydes in biological matrices, rat skeletal muscle homogenate was incubated with HNE and the formation of conjugated adducts was determined by LC/MS analysis. Three main products were detected and identified as Michael adducts of HNE with GSH, carnosine and anserine (the N-methylated derivative of carnosine, present in high concentrations in rat muscle). The results indicate that beside GSH, histidine-containing dipeptides could be involved in the detoxification pathway of reactive aldehydes from lipid peroxidation generated in skeletal muscle during physical endurance.

Carnosine is a quencher of 4-hydroxy-nonenal: through what mechanism of reaction?

The aim of this study was to understand the mechanism of action through which carnosine (β-alanyl- l-histidine) acts as a quencher of cytotoxic α,β-unsaturated aldehydes, using 4-hydroxy- trans-2,3-nonenal (HNE) as a model aldehyde. In phosphate buffer solution (pH 7.4), carnosine was 10 times more active as an HNE quencher than l-histidine and N-acetyl-carnosine while β-alanine was totally inactive; this indicates that the two constitutive amino acids act synergistically when incorporated as a dipeptide and that tshe β-alanyl residue catalyzes the addition reaction of the histidine moiety to HNE. Two reaction products of carnosine were identified, in a pH-dependent equilibrium: (a) the Michael adduct, stabilized as a 5-member cyclic hemi-acetal and (b) an imine macrocyclic derivative. The adduction chemistry of carnosine to HNE thus appears to start with the formation of a reversible α,β-unsaturated imine, followed by ring closure through an intra-molecular Michael addition. The biological role of carnosine as a quencher of α,β-unsaturated aldehydes was verified by detecting carnosine-HNE reaction adducts in oxidized rat skeletal muscle homogenate.

Impact of dietary conjugated linoleic acid on the oxidative stability of rat liver microsomes and skeletal muscle homogenates.

Dietary conjugated linoleic acid (CLA; 0-2.0%) increased CLA concentrations in liver microsomes and skeletal muscle homogenates from rats. Dietary CLA decreased oleic and arachadonic acid concentrations in both liver microsomes and skeletal muscle. The presence of CLA in liver microsomes had no impact on linoleic acid, arachadonic acid, and alpha-tocopherol oxidation rates. Dietary CLA (2.0%) also did not alter alpha-tocopherol oxidation rates in liver microsomes or muscle homogenates. Formation of malonaldehyde (MDA) in oxidizing liver microsomes decreased with increasing CLA concentration as determined by measurement of thiobarbituric acid-MDA complexes by HPLC. The ability of CLA to decrease MDA formation without impacting other lipid oxidation markers such as the disappearance of fatty acid and alpha-tocopherol suggests that decreased MDA concentration was the result of CLA's ability to lower polyenoic fatty acids such as arachadonic acid. While CLA does not appear to act as an antioxidant, its ability to decrease polyenoic fatty acid concentrations could decrease the formation of highly cytotoxic lipid oxidation products such as MDA.

Interaction of Neuronal Nitric-oxide Synthase with Caveolin-3 in Skeletal Muscle: IDENTIFICATION OF A NOVEL CAVEOLIN SCAFFOLDING/INHIBITORY DOMAIN

Neuronal nitric-oxide synthase (nNOS) has been shown previously to interact with alpha1-syntrophin in the dystrophin complex of skeletal muscle. In the present study, we have examined whether nNOS also interacts with caveolin-3 in skeletal muscle. nNOS and caveolin-3 are coimmunoprecipitated from rat skeletal muscle homogenates by antibodies directed against either of the two proteins. Synthetic peptides corresponding to the membrane-proximal caveolin-3 residues 65-84 and 109-130 and homologous caveolin-1 residues 82-101 and 135-156 potently inhibit the catalytic activity of purified, recombinant nNOS. Purified nNOS also binds to a glutathione S-transferase-caveolin-1 fusion protein in in vitro binding assays. In vitro binding is completely abolished by preincubation of nNOS with either of the two caveolin-3 inhibitory peptides. Interactions between nNOS and caveolin-3, therefore, appear to be direct and to involve two distinct caveolin scaffolding/inhibitory domains. Other caveolin-interacting enzymes, including endothelial nitric-oxide synthase and the c-Src tyrosine kinase, are also potently inhibited by each of the four caveolin peptides. Inhibitory interactions mediated by two different caveolin domains may thus be a general feature of enzyme docking to caveolin proteins in plasmalemmal caveolae.

Effect of dietary carnosine on plasma and tissue antioxidant concentrations and on lipid oxidation in rat skeletal muscle

The effect of dietary carnosine supplementation on plasma and tissue carnosine and alpha-tocopherol concentrations and on the formation of thiobarbituric acid reactive substances (TBARS) in rat skeletal muscle homogenates was evaluated. Plasma, heart, liver and hind leg muscle was obtained from rats fed basal semipurified diets or basal diets containing carnosine (0.0875%), alpha-tocopheryl acetate (50 ppm), or carnosine (0.0875%) plus alpha-tocopheryl acetate (50 ppm). Dietary carnosine supplementation did not increase carnosine concentrations in heart, liver and skeletal muscle. Dietary supplementation with both carnosine and alpha-tocopherol increased carnosine concentrations in liver 1.56, 1.51- and 1.51-fold as compared with diets lacking carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol, respectively. Dietary supplementation with both carnosine and alpha-tocopherol also increased alpha-tocopherol concentrations in heart and liver 1-38-fold and 1.68-fold, respectively, as compared to supplementation with alpha-tocopherol alone. Dietary supplementation with carnosine, alpha-tocopherol or both carnosine and alpha-tocopherol was effective in decreasing the formation of TBARS in rat skeletal muscle homogenate, with dietary alpha-tocopherol and alpha-tocopherol plus carnosine being more effective than dietary carnosine alone. The data suggest that dietary supplementation with carnosine and alpha-tocopherol modulates some tissue carnosine and alpha-tocopherol concentrations and the formation of TBARS in rat skeletal muscle homogenates.

Glutamine transaminase K and cysteine S-conjugate β-lyase activity stains

An activity stain to detect glutamine transaminase K subjected to nondenaturing polyacrylamide gel electrophoresis (ND-PAGE) was developed. The gel is incubated with a reaction mixture containing l-phenylalanine, α-keto-γ-methiolbutyrate (αKMB), glutamate dehydrogenase, phenazine methosulfate (PMS) and nitroblue tetrazolium (NBT). Glutamine transaminase K catalyzes a transamination reaction between phenylalanine and αKMB. The resultant methionine is a substrate of glutamate dehydrogenase. The NADH formed in the oxidative deamination of methionine reacts with PMS and NBT to form a blue band on the surface of the gel coincident with glutamine transaminase K activity. Cysteine S-conjugate β-lyase activity is detected in the gel by incubating the gel with a reaction mixture containing αKMB (to ensure maintenance of the enzyme in the pyridoxal 5′-phosphate form), S-(1,2-dichlorovinyl)- l-cysteine (DCVC), PMS, and NBT. The products of the lyase reaction interact with PMS and NBT to form a blue dye coincident with the lyase activity. In addition, a new assay procedure for measuring cysteine S-conjugate β-lyase activity was devised. This procedure couples pyruvate formation from DCVC to the alanine dehydrogenase reaction. Preparations of purified rat kidney glutamine transaminase K yield a single protein band on ND-PAGE (apparent M r ∼ 95,000). This band coincides with both the cysteine S-conjugate β-lyase and glutamine transaminase K activities. Activity staining showed that homogenates of rat kidney, liver, skeletal muscle, and heart possess a glutamine transaminase K/cysteine S-conjugate β-lyase activity with an R f value on ND-PAGE identical to that of purified rat kidney glutamine transaminase K. Activity staining of rat kidney homogenates (and to a lesser extent rat liver homogenates) for glutamine transaminase K and cysteine S-conjugate β-lyase activities revealed the presence of an additional band. The enzyme responsible for the second band of activity was estimated to have an apparent molecular weight of ∼ 330,000. The ratio of β-lyase to transaminase activity was greater for the higher molecular weight form than for the lower molecular weight form.

Properties of 5'-deiodinase of 3,3',5'-triiodothyronine in rat skeletal muscle.

To characterize rT3 5'-deiodinase (5'D) in rat skeletal muscle, the effects of altered thyroid status and PTU on rT3 5'D were studied. rT3 5'D activity was measured by incubating homogenates of rat skeletal muscle with [125]rT3, iodine labelled in the outer ring, in the presence of 20 mmol/l DL-dithiothreitol. This activity was observed to increase significantly 24 h after a single sc injection of T3 (75 micrograms/kg). The increase following the daily administration of this drug (15 or 75 micrograms/kg) for 3 and 14 days was dependent on the dose and number of previous days of injection. A significant decrease in activity was observed 2 weeks after thyroidectomy. The addition of 0.1 mmol/l 6-n-propyl-2-thiouracil (PTU) to the incubation medium in vitro caused a marked reduction in the activity in homogenates of skeletal muscle from hypothyroid, euthyroid and hyperthyroid rats. PTU, present at 0.05% in the drinking water for 2 weeks virtually abolished it. The properties of rT3 5'D in rat skeletal muscle thus appear to be essentially the same as those of type I enzyme with respect to response toward altered thyroid status and PTU.

Acyl-CoA oxidase activity and peroxisomal fatty acid oxidation in rat tissues

Acyl-CoA oxidase, the first enzyme of the peroxisomal β-oxidation, was proved to be rate-limiting for this process in homogenates of rat liver, kidney, adrenal gland, heart and skeletal muscle. Acyl-CoA oxidase activity, based on H 2O 2-dependent leuko-dichlorofluorescein oxidation in tissue extract, was compared with radiochemically assayed peroxisomal β-oxidation rates. Dichlorofluorescein production was a valid measure of peroxisomal fatty acid oxidation only in liver and kidney, but not in adrenal gland, heart or skeletal muscle. Production of 14C-labeled acid-soluble products from 1- 14C-labeled fatty acids in the presence of antimycin-rotenone appears to be a more accurate and sensitive estimate of peroxisomal β-oxidation than the acyl-CoA oxidase activity on base of H 2O 2 production. Chain-length specificity of acyl-CoA oxidase changed with the acyl-CoA concentrations used. Below 80 μM, palmitoyl-CoA showed the highest activity of the measured substrates in rat liver extract. No indications were obtained for the presence in rat liver of more forms of acyl-CoA oxidase with different chain-length specificity.

Specific inhibition of [3H] saxitoxin binding to skeletal muscle sodium channels by geographutoxin II, a polypeptide channel blocker.

Geographutoxin II (GTX II), a peptide toxin isolated from Conus geographus, inhibited [3H]saxitoxin binding to receptor sites associated with voltage-sensitive Na channels in rat skeletal muscle homogenates and rabbit T-tubular membranes with K0.5 values of 60 nM for homogenates and 35 nM for T-tubular membranes in close agreement with concentrations that block muscle contraction. Scatchard analysis of [3H]saxitoxin binding to T-tubular membranes gave values of KD = 9.3 nM and Bmax = 300 fmol/mg of protein and revealed a primarily competitive mode of inhibition of saxitoxin binding by GTX II. The calculated KD values for GTX II were 24 nM for T-tubules and 35 nM for homogenates, respectively. In rat brain synaptosomes, GTX II caused a similar inhibitory effect on [3H]saxitoxin binding at substantially higher concentrations (K0.5 = 2 microM). In contrast, binding of [3H]batrachotoxin A 20-alpha-benzoate and 125I-labeled scorpion toxin to receptor sites associated with Na channels in synaptosomes was not affected by GTX II at concentrations up to 10 microM. Furthermore, [3H]saxitoxin binding to membranes of rat superior cervical ganglion was only blocked 10% by GTX II at 10 microM. These results indicate that GTX II interacts competitively with saxitoxin in binding at neurotoxin receptor site 1 on the sodium channel in a highly tissue-specific manner. GTX II is the first polypeptide ligand for this receptor site and the first to discriminate between this site on nerve and adult muscle sodium channels.

The metabolic fate of branched-chain amino acids and 2-oxo acids in rat muscle homogenates and diaphragms

1. 1. After incubation of muscle preparations with [U- 14C]branched-chain amino acids or 2-oxo acids, radioactive metabolites were separated, identified and quantified. 2. 2. Homogenates of rat heart and skeletal muscle incubated with 4-methyl-2-oxopentanoate accumulated isovalerate, 3-hydroxyisovalerate and the corresponding carnitine esters. 3. 3. Incubation with 3-methyl-2-oxobutanoate resulted in the production of isobutyrate, 3-hydroxyisobutyrate and their carnitine esters. Addition of L-carnitine increased the production of the esters. 4. 4. The enzymes 3-methylcrotonyl-CoA carboxylase and 3-hydroxyisobutyric acid dehydrogenase apparently are inactive during incubation of muscle homogenates. 5. 5. With liver homogenates the degradation of both 2-oxo acids was more complete. 6. 6. Rat hemidiaphragms incubated with leucine, valine and isoleucine accumulated the corresponding branched-chain 2-oxo acids, fatty acids and hydroxylated fatty acids. 7. 7. The degradation of valine was markedly limited by the release of these metabolites. 8. 8.Considerable amounts (relatively smaller for valine) of radioactivity were also recovered in CO 2 and glutamine and glutamate. 9. 9. Incubations with branched-chain 2-oxo acids gave the same radioactive products, except for glutamine and glutamate. 10. 10. Radioactivity was never found in lactate, pyruvate or alanine. 11. 11. These data indicate that the carbon-chains of amino acids entering the citric acid cycle in muscle, are not used for oxidation or for alanine synthesis, but are converted exclusively to glutamine.

Characterization of triglyceride lipase activities in rat skeletal muscle

Triglyceride lipase activity was determined in particulate and soluble fractions from rat skeletal muscle homogenates. The fractions exhibited an acid (pH 5,0) optimum with an impressive enhancement in the combined P17 100 fraction. Methylamine inhibited this acid lipase activity. A further lipase was observed with maximal activity at pH 7,0 and only a small enhancement in the combined P17 100 fraction and inhibition by diethyl p-nitrophenyl-phosphate but not by protamine sulfate. Lipoprotein lipase activity was identified by the following in vitro criteria: Stimulation of activity by serum, maximal activity at alkaline pH (pH 8,5 – 9,0) and inhibition of activity by NaCl and protamine sulfate. There was a definite enhancement of lipoprotein lipase activity in the bombined P17 100 fraction after the lipase activity has been washed out from the capillary bed with heparin.

( [formula omitted] activity of crude homogenates of rat skeletal muscle as estimated from their K +-dependent [formula omitted] activity

A highly sensitive fluorimetric assay using 3-O- methylfluorescein phosphate as substrate was used in the determination of K +-dependent phosphatase activity in preparations of rat skeletal muscle. The gastrocnemius muscle was chosen because of mixed fibre composition. Crude, detergent treated homogenate was used so as to avoid loss of activity during purification. K +-dependent phosphatase activities in the range 0.19–0.37 μmol · (g wet weight) −1 · min −1 were obtained, the value decreasing with age and K +-deficiency. Complete inhibition of the K +-dependent phosphatase was obtained with 10 −3 M ouabain. Using a KSCN-extracted muscle enzyme the intimate relation between K +-dependent phosphatase activity and ( Na + + K +)- activated ATP hydrolysis could be demonstrated. A molecular activity of 620 min −1 was estimated from simultaneous determination of K +-dependent phosphatase activity and [ 3H]ouabain binding capacity using the partially purified enzyme preparation. The corresponding enzyme concentration in the crude homogenates was calculated and corresponded well with the number of [ 3H]ouabain binding sites measured in intact muscles or biopsies hereof.

Drug distribution and binding to muscle in rat.

Drug binding to rat skeletal muscle homogenate was studied using quinidine and quinine as basic drugs, and furosemide and phenylbutazone as anionic drugs. Characteristically different binding fashions were observed among basic and anionic drugs. Quinidine and quinine which are stereoisomers bound to muscle not depending on drug concentration and showed similar binding ratios, while furosemide and phenylbutazone bound to muscle depending on drug concentration. Quinidine and quinine bound mainly to 1000 x g precipitates of muscle homogenate while furosemide and phenylbutazone bound exclusively to cytosol fraction. Binding to 1000 x g precipitates was not explained by binding to actin and myosin alone, while the second protein fraction eluted from cytosol by Sephadex G-75 gel filtration was found to have binding properties for furosemide.