Membrane fatty acid composition of tissues is related to body mass of mammals

Phospholipids were extracted from tissues (heart, skeletal muscle, kidney cortex, liver and brain) of mammals representing a 9,000-fold range in body mass (mouse, rat, rabbit, sheep and cattle) and their fatty acid composition was determined. In heart, skeletal muscle and kidney cortex, there were significant allometric decreases in the Unsaturation Index (UI; average number of double bonds per 100 fatty acid molecules) with increasing body mass. There were significant inverse allometric relationships between body mass and the proportion of docosahexaenoic acid (2263) in heart and skeletal muscle. In heart, skeletal muscle and kidney cortex, larger mammals also had shorter fatty acid chains in their phospholipids and a higher proportion of monounsaturates. In liver, smaller mammals had a higher UI than larger mammals (except the rabbit, which had the lowest UI and very low proportions of 3 fatty acids). The brain of all mammals maintained a high UI with similar levels of polyunsaturated fatty acids, especially 2263. Our results suggest that in heart, skeletal muscle and kidney cortex the activity of the elongases and desaturases are reduced in large mammals compared to small mammals. The allometric trends in membrane composition may be involved in modifying membrane permeability. It is proposed that the elevated degree of polyunsaturation in the membranes of several tissues from small mammals is related to their higher metabolic activity.This work was supported by an Australian Commonwealth Postgraduate Research Scholarship from the University of Wollongong to P. Couture and by a grant from the Australian Research Council to A.J. Hulbert. We wish to thank Voytek Mantaj for technical assistance.     

Oxidative phosphorylation in myocardial mitochondria 'in situ': a calorimetric study on permeabilized cardiac muscle preparations

A novel flow calorimetric technique was developed to study the energy turnover of myocardial mitochondria. Cylindrical strands of cardiac muscle (trabeculae) weighing 100–500 µg were isolated from guinea-pig heart and mounted in a tubular recording chamber which was continuously perfused with physiological salt solution at 37°C. The temperature difference between the upstream and the downstream side of the chamber, which is proportional to the rate of heat production of the trabecula, was measured at high resolution. In this way the rate of energy expenditure of isolated cardiac muscle could be recorded continuously for several hours. When the preparations were superfused with an 'intracellular' solution containing 5 mM pyruvate and 2 mM malate as substrates, permeabilization of the sarcolemma with 25 µM digitonin induced a marked increase in the measured heat rate in the presence of 2 mM ADP. The major fraction of the ADP sensitive heat production (83%) could be blocked with 400 µM at ractyloside, an inhibitor of the adeninenucleotide translocase, and by 600 µM -cyano-4-hydroxycinnamate, an inhibitor of monocarboxylate/H⁺ co-transport. The atractyloside sensitive heat production was abolished in anoxic solution. These results suggest that the atractyloside-sensitive heat production (21.8 ± 3.5 mW cm^-3 of tissue) was attributable to oxidative phosphorylation. The mitochondria apparently remained intact after treatment with digitonin, since application of the uncoupler 2,4-dinitrophenol (DNP) produced a very large increase in heat rate. A minor fraction of the heat rate induced by ADP in permeabilized cardiac muscle preparations (17%) was not sensitive to atractyloside. This component was also seen before application of digitonin and was probably related to ectonucleotidases. In conclusion, our calorimetric technique allows investigation of the energy metabolism of myocardial mitochondria 'in situ', i.e. without destroying the microarchitecture of cardiac muscle cells. (Mol Cell Biochem 174: 101–113, 1997)     

Utilization of amino acids by Frankia sp. strain CpI1

The utilization of some amino acids, added at 1 mM and 10 mM concentrations, as the sole combined nitrogen sources by Frankia sp. strain CpI1, has been investigated. Glutamine, like NH ₄ ⁺ , provided rapid growth without N₂ fixation. Histidine at 1 mM yielded poor N₂-fixing activity but better cell growth than N₂. Aspartate, glutamate, alanine, proline, each at 1 mM concentration, supported similar levels of N₂ fixation and growth. Growth on 10 mM glutamate, proline, or histidine resulted in poor N₂-fixing activity and poor cell growth. Cells grown on 10 mM alanine had about half the N₂-fixing activity of cells grown on N₂ but growth was good. Aspartate at 10 mM concentration, however, stimulated N₂-fixing activity dramatically and promoted faster growth. Enzyme analysis suggested that asparate is catabolized by glutamate-oxaloacetate transaminase (GOT), since GOT specific activity was induced, and aspartase activity was not detected, in cells grown on aspartate as the sole combined nitrogen source. Thinlayer chromatography (TLC) of metabolites extracted from N₂-grown cells fed with [¹⁴C]-aspartate showed that label was rapidly accumulated mainly on aspartate and/or glutamate, depending on the cells' physiological state, without detectable labeling on fumarate or oxaloacetate (OAA). These findings provide evidence that aspartate is catabolized by GOT to OAA which, in turn, is rapidly converted to -ketoglutarate through the TCA cycle and then to glutamate by GOT or by glutamate synthase (GOGAT). The stimulation of N₂ fixation and growth by aspartate is probably caused by an increased intracellular glutamate pool.     

Species differences in localization of cardiac cAMP-phosphodiesterase activity: A cytochemical study

The localization of the membrane-bound cyclic 3,5-AMP phosphodiesterasein cardiac tissues of both, rat and dog was studied by cytochemical method.40 µm thick slices from glutaraldehyde fixed heart tissue wereincubated in the medium with cAMP as a substrate and Pb ions as a capturemetal of the reaction product. The cAMP-PDE activity in the rat ventriclewas only shown positive on the sarcolemma. Whereas, in canine ventriculartissue the cAMP-PDE activity in cardiomyocytes was shown on the sarcolemma,on the junctional sarcoplasmic reticulum and on subsarcolemmal cisternae.The results confirm differences in the localization of cAMP-PDE in dog andrat heart.     

Resistance of Surface Phosphohydrolases of Barley Root Cells to Nickel Salts Is One of Factors of Plant Adaptation to Excessive Nickel in the Environment

We studied the effect of nickel ions on the activity of ecto-phosphohydrolases (acid phosphatase and Ca-stimulated nucleotidase) from root surface of etiolated barley seedlings as well as from root microsomal fraction. The presence of nickel nitrate (25 M) proved to stop root growth and insignificantly (on average by 20%) decreased specific hydrolytic activity of both enzymes determined on root surface as well as in the root microsomal fraction. At the same time, direct addition of nickel to the incubation mixture when measuring the substrate hydrolysis demonstrated high resistance of the microsomal fraction enzymes to the salts. A significant decrease in Ca-stimulated nucleotidase activity was observed only for nickel nitrate concentrations above 100 M, reaching 50–60% for 3 mM Ni(NO₃)₂. The presence of an activator ion as well as extended duration of the microsomal fraction pretreatment with nickel nitrate (2.5 h) did not increase its effect on the enzyme activity. The pattern of nickel effect on acid phosphatase activity depended on the presence of magnesium ions in the mixture but did not change after extended duration of the microsomal fraction pretreatment (3 h). Inhibition of acid phosphatase activity in the presence of magnesium was observed only for nickel nitrate concentrations above 500 M being no more than 20% for 3 mM Ni(NO₃)₂. Hence, the hydrolytic enzymes of the apoplast of plant root cells have different tolerance to nickel salts. We propose that an insignificant decrease in specific activity of surface hydrolases of plant roots grown on a medium containing nickel salts in concentrations inhibiting growth processes (25 M) is not related to direct effect of Ni on the apoplastic enzymes. The significance of hydrolytic enzyme resistance in plant adaptation to high nickel content in the soil is discussed.     

Molecular and kinetic alterations of muscle AMP deaminase during chronic creatine depletion

We examined apossible mechanism to account for the maintenance of peak AMPdeamination rate in fast-twitch muscle of rats fed the creatine analog-guanidinopropionic acid (-GPA), in spite of reduced abundance ofthe enzyme AMP deaminase (AMPD). AMPD enzymatic capacity (determined atsaturating AMP concentration) and AMPD protein abundance (Western blot)were coordinately reduced ~80% in fast-twitch white gastrocnemiusmuscle by -GPA feeding over 7 wk. Kinetic analysis of AMPD in thesoluble cell fraction demonstrated a single Michaelis-Menten constant(K_m; ~1.5 mM) in control muscle extracts. An additional high-affinityK_m (~0.03 mM)was revealed at low AMP concentrations in extracts of -GPA-treated muscle. The kinetic alteration in AMPD reflects increased molecular activity at low AMP concentrations; this could account for high ratesof deamination in -GPA-treated muscle in situ, despite the loss ofAMPD enzyme protein. The elimination of this kinetic effect bytreatment of -GPA-treated muscle extracts with acid phosphatase invitro suggests that phosphorylation is involved in the kinetic controlof skeletal muscle AMPD in vivo.

     

Ethylmalonic Acid Inhibits Mitochondrial Creatine Kinase Activity from Cerebral Cortex of Young Rats in Vitro

Short-chain acyl-CoA dehydrogenase (SCAD) deficiency is an inherited metabolic disorder biochemically characterized by tissue accumulation of predominantly ethylmalonic acid (EMA) and clinically by neurological dysfunction. In the present study we investigated the in vitro effects of EMA on the activity of the mitochondrial (Mi-CK) and cytosolic (Cy-CK) creatine kinase isoforms from cerebral cortex, skeletal muscle, and cardiac muscle of young rats. CK activities were measured in the mitochondrial and cytosolic fractions prepared from whole-tissue homogenates of 30-day-old Wistar rats. The acid was added to the incubation medium at concentrations ranging from 0.5 to 2.5 mM. EMA had no effect on Cy-CK activity, but significantly inhibited the activity of Mi-CK at 1.0 mM and higher concentrations in the brain. In contrast, both Mi-CK and Cy-CK from skeletal muscle and cardiac muscle were not affected by the metabolite. We also evaluated the effect of the antioxidants glutathione (GSH), ascorbic acid, and a-tocopherol and the nitric oxide synthase inhibitor L-NAME on the inhibitory action of EMA on cerebral cortex Mi-CK activity. We observed that the drugs did not modify Mi-CK activity per se, but GSH and ascorbic acid prevented the inhibitory effect of EMA when co-incubated with the acid. In contrast, L-NAME and -tocopherol could not revert the inhibition provoked by EMA on Mi-CK activity. Considering the importance of CK for brain energy homeostasis, it is proposed that the inhibition of Mi-CK activity may be associated to the neurological symptoms characteristic of SCAD deficiency.     

Glycogen synthase in diabetic rat skeletal muscle: activation by insulin

Summary Glycogen synthase in skeletal muscle of 3-day alloxan-diabetic rats was found to be in a less active state than in normal muscle. Both the activity ratio (activity without G6P divided by activity with 7.2 mM G6P at 4.4 mM UDPG, pH 7.8) and fractional velocity (activity with 0.25 mM G6P divided by activity with 10 mM G6P at 0.03 mM UDPG, pH 6.9) were significantly lower in the diabetic tissue. Correspondingly, the S_0.5 for UDPG and A_0.5 for G6P were significantly higher in diabetic tissue, suggesting decreased affinity for substrate and activator, respectively. The kinetic changes in the diabetic synthase were identical whether the alloxan-treated animals were maintained on insulin for 7 days prior to withdrawal for 3 days, or studied 3 days immediately after alloxan treatment. The diabetes-induced changes in synthase could be reversed by injecting the diabetic rat with insulin 10 min prior to sacrifice. After insulin treatment, the S_0.5 for UDPG and A_0.5 for G6P decreased to control levels or lower and the activity ratios and fractional velocities increased to control levels or higher.The activity of glycogen synthase phosphatase was not decreased in diabetic skeletal muscle. This observation, coupled with the rapid response of the diabetic synthase to in vivo insulin treatment, suggests that, unlike the phosphatase in cardiac muscle and liver, the glycogen synthase phosphatase in skeletal muscle is not altered by the diabetic state.Abbreviations UDPG uridine diphosphoglucose - G6P glucose 6-phosphate - EDTA ethylene diamine tetraacetic acid - IP intraperitoneally - MOPS morpholinopropane sulfonic acid - -ME -mercaptoethanol - V_G6P calculated velocity of the enzyme in the presence of infinite G6P concentration - V_UDPG calculated velocity of the enzyme in the presence of infinite UDPG concentration     

Caffeic acid ethanolamide prevents cardiac dysfunction through sirtuin dependent cardiac bioenergetics preservation

Background

Cardiac oxidative stress, bioenergetics and catecholamine play major roles in heart failure progression. However, the relationships between these three dominant heart failure factors are not fully elucidated. Caffeic acid ethanolamide (CAEA), a synthesized derivative from caffeic acid that exerted antioxidative properties, was thus applied in this study to explore its effects on the pathogenesis of heart failure.

Results

In vitro studies in HL-1 cells exposed to isoproterenol showed an increase in cellular and mitochondria oxidative stress. Two-week isoproterenol injections into mice resulted in ventricular hypertrophy, myocardial fibrosis, elevated lipid peroxidation, cardiac adenosine triphosphate and left ventricular ejection fraction decline, suggesting oxidative stress and bioenergetics changes in catecholamine-induced heart failure. CAEA restored oxygen consumption rates and adenosine triphosphate contents. In addition, CAEA alleviated isoproterenol-induced cardiac remodeling, cardiac oxidative stress, cardiac bioenergetics and function insufficiency in mice. CAEA treatment recovered sirtuin 1 and sirtuin 3 activity, and attenuated the changes of proteins, including manganese superoxide dismutase and hypoxia-inducible factor 1-α, which are the most likely mechanisms responsible for the alleviation of isoproterenol-caused cardiac injury

Conclusion

CAEA prevents catecholamine-induced cardiac damage and is therefore a possible new therapeutic approach for preventing heart failure progression.     

Biochemical conditions for the production of polysialic acid by Pasteurella haemolytica A2

The capsular polysaccharide of Pasteurella haemolytica A2 consists of a linear polymer of N-acetylneuraminic acid (Neu5Ac) with (2–8) linkages. When the bacterium was grown at 37°C for 90 h in 250 ml shake flasks at 200 rpm in Brain heart infusion broth (BHIB), it accumulated, attaining a level of 60 g/ml. Release of this polymer was strictly regulated by the growth temperature, and above 40° no production was detected. The pathway for the biosynthesis of this sialic acid capsular polymer was also examined in P. haemolytica A2 and was seen to involve the sequential presence of three enzymatic activities: Neu5Ac lyase activity, which synthesizes Neu5Ac by condensation of N-acetyl-D-mannosamine and pyruvate with apparent Km values of 91 mM and 73 mM, respectively; a CMP-Neu5Ac synthetase, which catalyzes the production of CMP-Neu5Ac from Neu5Ac and CTP with apparent Km values of 2 mM and 0.5 mM, respectively, and finally a membrane-associated polysialyltransferase, which catalyzes the incorporation of sialic acid from CMP-Neu5Ac into polymeric products with an apparent CMP-Neu5Ac Km of 250 M.     

A novel callose synthase from pollen tubes of Nicotiana

Pollen-tube cell walls are unusual in that they are composed almost entirely of callose, a (1,3)--linked glucan with a few 6-linked branches. Regulation of callose synthesis in pollen tubes is under developmental control, and this contrasts with the deposition of callose in the walls of somatic plant cells which generally occurs only in response to wounding or stress. The callose synthase (uridine-diphosphate glucose: 1,3--d-glucan 3--d-glucosyl transferase, EC 2.4.1.34) activities of membrane preparations from cultured pollen tubes and suspension-cultured cells of Nicotiana alata Link et Otto (ornamental tobacco) exhibited different kinetic and regulatory properties. Callose synthesis by membrane preparations from pollen tubes was not stimulated by Ca²⁺ or other divalent cations, and exhibited Michaelis-Menten kinetics only between 0.25 mM and 6 mM uridine-diphosphate glucose (K _m 1.5–2.5 mM); it was activated by -glucosides and compatible detergents. In contrast, callose synthesis by membrane preparations from suspension-cultured cells was dependent on Ca²⁺, and in the presence of 2 mM Ca²⁺ exhibited Michaelis-Menten kinetics above 0.1 mM uridine-diphosphate glucose (K _m 0.45 mM); it also required a -glucoside and low levels of compatible detergent for full activity, but was rapidly inactivated at higher levels of detergent. Callose synthase activity in pollen-tube membranes increased ten fold after treatment of the membranes with trypsin in the presence of detergent, with no changes in cofactor requirements. No increase in callose synthase activity, however, was observed when membranes from suspension-cultured cells were treated with trypsin. The insoluble polymeric product of the pollen-tube enzyme was characterised as a linear (1,3)--d-glucan with no 6-linked glucosyl branches, and the same product was synthesised irrespective of the assay conditions employed.Abbreviations Ara l-arabinose - CHAPS 3-[(3-cholamidopropyl)dimethylammonia]-1-propane sulphonic acid - DAP diphenylamine-aniline-phosphoric acid stain - Gal d-galactose - Glc d-glucose - Man d-mannose - Mes 2-(N-morpholino)ethane sulphonic acid - Rha d-rhamnose - Rib d-ribose - TFA trifluoroacetic acid - UDPGlc uridine-diphosphate glucose - Xyl d-xylose This research was supported by funds from a Special Research Centre of the Australian Research Council. H.S. was funded by a Melbourne University Postgraduate Scholarship and an Overseas Postgraduate Research Studentship; S.M.R. was supported by a Queen Elizabeth II Research Fellowship. We thank Bruce McGinness and Susan Mau for greenhouse assistance, and Deborah Delmer and Adrienne Clarke for advice and encouragement throughout this project.     

Peroxisomes of the rat cardiac and soleus muscles increase after starvation

Summary We have investigated the change of catalase activity in the homogenates of rat cardiac and skeletal muscles. After 7 days' starvation, the catalase activity of heart increased about 3-fold and that of soleus muscle enhanced 2-fold higher than that of control rats. Immunoblot analysis of catalase showed a single band in the homogenates of cardiac and soleus muscles and increase of catalase antigen after starvation. Light microscopic immunoenzyme staining showed that after starvation catalase positive granules markedly increased in both the cardiac and soleus muscle. Quantitative analysis of the staining showed that number of the granules per 100 m² of tissue section was about 1.4-fold in the soleus muscle and 1.7-fold in the cardiac muscle after starvation. By electron microscopy of alkaline DAB staining, we confirmed that the granules were peroxisomes, which increased in both number and size. Furthermore, we stained the peroxisomes for catalase by a protein A-gold technique. Labeling density (gold particles/m²) of the cardiac and soleus muscles from the starved rat increased approximately 1.4 times as much as that of normal animal. When the numerical density is multiplied by the labeling density, the values are largely consistent with the enhancement of catalase activity. These results show that increase in the catalase activity of the muscle tissue after starvation is caused by increase in number and size of peroxisomes.     

Resistance adaptation of the freshwater crayfish and thermal inactivation of membrane-bound enzymes

Summary Heat death and resistance adaptation of freshwater crayfish are thought to be properties of its muscle membranes. The inactivation at high temperatures of a membrane-bound enzyme, the Ca⁺⁺-stimulated ATPase of crayfish abdominal muscle sarcoplasmic reticulum, and the effect of thermal acclimation of crayfish upon the inactivation kinetics have been investigated. In the absence of KCl, the Ca⁺⁺-stimulated ATPase is irreversibly inactivated with pseudo-first order kinetics at temperatures that cause heat death in the whole animal. 0.1–10.0 mM KCl resulted in slower inactivation, while 100 mM KCl activated the enzyme to 120–180% of its original activity. Enzyme activation by KCl and heat involved a shift in the enzyme concentration/activity curve. Thermal acclimation of crayfish had no significant effect upon the kinetics or Arrhenius activation energy for enzyme inactivation (100.6±10.5 and 92.3±14.6 kcal/mole for preparations from 4°C and 25°C acclimated crayfish).Ca⁺⁺-stimulated ATPase isolated from heat dead crayfish exhibited normal in vitro activity due presumably to the high intracellular K⁺ concentration. Nevertheless, the close correspondence between heat death temperatures and inactivation temperatures for several membrane-bound enzymes of muscle is thought to reflect some perturbation of muscle structure that occurs during heat death.Abbreviations ATP Ademosine 5-Triphosphate - EGTA Ethyleneglycol-bis [-amino-ethyl ether] - N N-tetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - FSR Fragmented sarcoplasmic reticulum - Tris Tris (hydroxymethyl)aminomethane     

The ultrahistochemical picture of the so-called reversed ATPase in the gastrocnemius muscle of the rat

Summary The ultrahistochemical localization of the reversed ATPase activity was investigated. Red muscle fibres showed permanent sarcomere contraction, enzymatic activity in the inner membrane and matrix of mitochondria, and large, osmiophilic, probably calcium-containing structures within mitochondria and on their outside. White muscle fibre sarcomeres were relaxed, and activity within their sarcoplasmic reticulum was marked, but slight in the mitochondria. The relaxed state of the sarcomere in the white muscle fibres is supposed to be connected with inactivation of myofibrillar ATPase by acid preincubation, whereas red muscle contraction indicates that acid preincubation does not inactivate their myofibrillar ATPase. That the product of its activity failed to become visible in the sarcomeres is probably due to imperfection of the method.Two sub-types of red muscle fibres were distinguished: those showing only enzymatic activity in mitochondria, and those containing large intra-and extramitochondrial osmiophilic structures. The origin and composition of these structures is difficult to explain. A relation seems to exist between their presence within mitochondria and outside.This work was supported by a Fellowship from the Muscular Dystrophy Association of America and under Research Project No. 05-002-1 of the National Institutes of Health, IND, Bethesda, USA. Preliminary experiments were made at the Medical Neurology Branch, INDS, NIH, Bethesda, USA     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

Phosphoenol-pyruvate-carboxylase activity in cotton and Sorghum seeds and its relation to seedling development

Cotton (Gossypium hirsutum) seeds and Sorghum vulgare caryopses are able to incorporate CO₂ through a PEP-carboxylating enzyme (EC 4.1.1.38). The enzyme activity is optimal at pH 8.2 and is unaffected by ATP, GDP or acetyl CoA. The partially purified cotton enzyme is stimulated by inorganic phosphate with an apparent Km of 0.3 mM. The enzymes from both cultivars are inhibited by pyrophosphate, malate, and aspartate but not by succinate. Kinetic studies for Sorghum and cotton seed enzymes show apparent Km values for carbonate of 5 mM and 1.2 mM and for PEP of 36 M and 5 mM, respectively. The V_max values are 90 and 3.3 nmol min^-1 mg protein^-1, respectively.A two-fold increase in the enzyme activity from cotton seeds occurs after 2 h under laboratory germination conditions after which the activity drops sharply to 1/3 of the original activity after 5 h imbibition. No such change was observed in Sorghum caryopses enzyme. A correlation between PEP-carboxylase activity and seed vigor in both cultivars was demonstrated.Abbreviations GOT glutamicoxaloacetic-transaminase - MDH malic dehydrogenase-NADH₂ - RH relative humidity     

Oxygen requirements,morphology, cell coat and membrane permeability of calcium-tolerant myocytes from hearts of adult rats

Summary The morphological, functional, and biochemical properties of freshly isolated heart muscle cells were examined. A reproducible method for the separation and purification of such cells isolated from adult rat heart was developed. It yields an average of 5×10⁶ striated rectangular cells which retain normal morphology (range) 2.5 to 11×10⁶ and 4×10⁶ calcium-tolerant cells (range) 2.5 to 5.5×10⁶ per heart. After purification, 85 to 95% of the cells retain normal morphology in solutions of calcium ion activity equal to 10M, and 65 to 79% of the cells are rectangular in solutions of calcium ion activity equal to 1 mM.Under the light microscope we were able to identify functionally intact individual cells that are calcium-tolerant and contract only in response to electrical stimulation, as well as dying myocytes that beat spontaneously. The examination of such cells under the electron microscope permitted us to address the question: What is the sequence of structural changes in a dying cell? The sarcomere lengths measured both in the living state and after preparation for electron microscopy are in the physiological range. In steady states of oxygen tension, respiration of the intact cells is undiminished from 50 torr to 2 torr. The oxygen tension for half maximal respiration is 0.15 torr. Therefore, the limitation of oxygen diffusion to the mitochondria of isolated heart muscle cells must be remarkably small.This work was supported in part by NIH HL 19299 (BAW), and NIH HL 24336 and N.Y. Heart Grant in Aid (TFR.)Dr. Thomas F. Robinson is the recipient of NIH, Research Career Development Award HL 00568     

Inorganic phosphate speeds loaded shortening in rat skinned cardiac myocytes

Force generation in striated muscle is coupled with inorganic phosphate (P_i) release from myosin, because force falls with increasing P_i concentration ([P_i]). However, it is unclear which steps in the cross-bridge cycle limit loaded shortening and power output. We examined the role of P_i in determining force, unloaded and loaded shortening, power output, and rate of force development in rat skinned cardiac myocytes to discern which step in the cross-bridge cycle limits loaded shortening. Myocytes (n = 6) were attached between a force transducer and position motor, and contractile properties were measured over a range of loads during maximal Ca²⁺ activation. Addition of 5 mM P_i had no effect on maximal unloaded shortening velocity (V_o) (control 1.83 ± 0.75, 5 mM added P_i 1.75 ± 0.58 muscle lengths/s; n = 6). Conversely, addition of 2.5, 5, and 10 mM P_i progressively decreased force but resulted in faster loaded shortening and greater power output (when normalized for the decrease in force) at all loads greater than 10% isometric force. Peak normalized power output increased 16% with 2.5 mM added P_i and further increased to a plateau of 35% with 5 and 10 mM added P_i. Interestingly, the rate constant of force redevelopment (k_tr) progressively increased from 0 to 10 mM added P_i, with k_tr 360% greater at 10 mM than at 0 mM added P_i. Overall, these results suggest that the P_i release step in the cross-bridge cycle is rate limiting for determining shortening velocity and power output at intermediate and high relative loads in cardiac myocytes. muscle mechanics; force-velocity relationship; cross-bridge cycle     

Simultaneous purification of L-malate dehydrogenase and L-lactate dehydrogenase from bovine heart by biomimetic-dye affinity chromatography

Two commercially important enzymes, L-lactate dehydrogenase (LDH) and L-malate dehydrogenase (MDH) were purified simultaneously from bovine heart, on an agarose affinity adsorbent. This adsorbent bears a dye-ligand composed of an anthraquinone chlorotriazine chromophore linked to a biomimetic terminal 4-aminophenyloxanylic acid moiety. The purification protocol exploited the biomimetic affinity adsorbent, in combination with a cross-linked agarose DEAE anion-exchanger. The procedure comprised a preliminary anion-exchange first step, for the separation of the three enzyme activities, mMDH, cMDH and LDH. In the second step, that of affinity chromatography, the unbound mMDH obtained from the first step, was purified by specific elution with NAD⁺/sulphite (22.5-fold purification, 55% step-yield). The procedure afforded mMDH preparation of specific activity approx. 1,300?u/mg (25?°C) at 45% overall yield, free of cytoplasmic MDH, glutamic-oxaloacetic transaminase (GOT) and fumarase. The LDH activity, which, bound to the anion-exchanger during the first step, was recovered from the adsorbent in 200?mM KCl, and finally purified by biomimetic-dye affinity chromatography (NAD⁺/sulphite elution) and a second ion-exchange chromatography step (elution with 200?mM KCl). The LDH preparation exhibited specific activity approx. 500?u/mg at 25?°C (content of impurities: pyruvate kinase and GOT were not detected; MDH, 0.01%).     

The phosphorylation of troponin B by phosphorylase b kinase in skeletal muscle of mice carrying the phosphorylase b kinase deficiency gene

In skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene there is < 1% of the normal activity to convert phosphorylase $\text{b}$ to $\text{a}$ in the presence of Ca⁺⁺, Mg⁺⁺, and ATP (1). Correspondingly, there is < 1% of the normal activity to phosphorylate phosphorylase $\text{b}$. Nevertheless, under the same conditions, these extracts catalyze the phosphorylation of troponin at a rate 57% of normal. Phosphorylase $\text{b}$ converting activity can be sedimented from skeletal muscle of control mice by centrifugation. This fraction isolated from I strain skeletal muscle extracts phosphorylates troponin at a rate 29–39% of the control. EGTA¹ (15 mM) inhibits troponin phosphorylation by 50–60% in this fraction from both strains. The EGTA inhibition is reversed by 15 mM Ca⁺⁺. Thus the phosphorylase $\text{b}$ kinase in skeletal muscle of animals with the phosphorylase $\text{b}$ kinase deficiency gene can phosphorylate troponin B, although it shows little or no activity with phosphorylase as a substrate. This observation is consistent with the normal muscle contractility of I strain animals.