Reprint of: Comparative Studies of Rabbit Cardiac and Skeletal Myosins.

Rabbit cardiac myosin contains fewer cysteine residues than the skeletal myosin (7 and 8.8 moles/105 gm. of myosin, respectively). A similar difference is found between the cysteine content of rabbit cardiac and skeletal heavy meromyosins; the cardiac heavy meromyosin contains 8.9 moles/105 gm. of protein as compared to 11 moles in the skeletal heavy meromyosin. In skeletal myosin, actomyosin, and myofibrils the Ca++-ATPase, Ca++-ITPase, and EDTA-ATPase activities are about three times higher than those of cardiac myosin, actomyosin, and myofibrils; whereas the skeletal to cardiac actomyosin-ATPase activity ratio is higher. The ATPase activities of both cardiac and skeletal myosins, actomyosins, and myofibrils, however, are close to each other when determined in the presence of Mg++ at high ionic strength. The abilities of cardiac and skeletal myosins to combine with actin at either high or low ionic strength are also essentially the same. The Ca++-ATPase, Ca++-ITPase, EDTA-ATPase, and actomyosin-ATPase activities of cardiac myosin, heavy meromyosin, and myofibrils, unlike those of skeletal myosin, heavy meromyosin, and myofibrils, do not increase over pH 8.0. The ATPase activities of cardiac and skeletal myosins in the presence of Mg++ at high ionic strength, on the other hand, are affected similarly by changes of pH. In cardiac myosin, heavy meromyosin, and myofibrils, the Ca++activated ATPase is less sensitive to high KC1 concentrations than is that of skeletal myosin, heavy meromyosin, and myofibrils.

Analysis of living tissue by phosphorus-31 magnetic resonance.

Nuclear magnetic resonance is a new method for assaying the content of phosphate metabolites in intact tissues. Its nondestructive nature allows simultaneous and repeated determinations of these compounds with a minimum perturbation of tissue. Changes in the concentrations of the phosphates as a function of time characterize the metabolic machinery of the tissue and reveal alterations in enzymic activity that result from drug treatment or disease. The entire phosphate profile shows differences between normal and diseased muscle which should be of diagnostic value. Further, by examining phosphate profiles we detected a family of chemical compounds that were not previously known to exist as major constituents in muscle. Of these, two have been isolated and one has been identified as glycerol 3-phosphorylcholine. Finally, shifts in the positions of resonances monitor the internal environment of the living system, its hydrogen ion concentration, the complexing of alkaline earth metals with ATP, and compartmentalization within the cell.

Calponin phosphorylation does not accompany contraction of various smooth muscles.

Calponin phosphorylation was quantitated in 32P-labeled porcine arterial, uterine, tracheal, stomach and bladder smooth muscles. The negligible amount of 0.003-0.008 mol [32P]phosphate/mol calponin in resting muscles did not increase upon contraction induced by various agents, under conditions when myosin light-chain phosphorylation increased several-fold. The results indicate no involvement of calponin phosphorylation is smooth-muscle contraction.

Application of high-field 1H-NMR spectroscopy for the study of perifused amphibian and excised mammalian muscles.

Frog sartorius and gastrocnemius muscles were perifused at 20 degrees C, the intracellular pH (pHi) and the concentration of phosphocreatine were determined in the resting muscle by 1H-NMR spectroscopy at 470 MHz; values of pHi = 7.31 +/- 0.05 (n = 7) and concentration of phosphocreatine = 20.4 +/- 1.1 mumol/g wet wt. (n = 6) were found. The hydrolysis of phosphocreatine and the simultaneous increase in lactate upon perifusion with 10 mM caffeine (in Ringer's solution) was followed with a time resolution of 1 min. Lactate increased at a rate of 1.0 mumol/g per min, but no pHi change was recorded during the time monitored. The lower limit for the buffering capacity of the muscle cytosol was estimated to be 16.7 mumol/g muscle per pH unit from the uncertainty in pHi determination (+/- 0.03 pH units) and from the amount of lactate produced and phosphocreatine hydrolyzed. Changes in pHi, lactate concentration and fatty acyl chain intensity were monitored by 1H-NMR spectroscopy at 361 MHz in ischemic rat skeletal muscle, excised and stored at 20 degrees C. The resonances in the 1H-NMR spectrum of a human skeletal muscle perchloric acid extract are reported and tentatively assigned.

Isoforms of the phosphorylatable myosin light chain in arterial smooth muscle.

Two isoforms of the phosphorylatable myosin light chain of arterial smooth muscle have been identified, at proportions of 15 and 85%. The isoforms have similar tryptic peptide maps and can be mono-, di- and triphosphorylated. In intact or homogenized muscle, monophosphorylation and, to a small degree, diphosphorylation occur, whereas in isolated actomyosin diphosphorylation and triphosphorylation are manifested. Serine and threonine residues are phosphorylated in all three systems, but the ratio of phosphothreonine to phosphoserine is much higher in actomyosin than in muscle.

Estimation of tissue phospholipids by natural abundance 13C-NMR.

The total phospholipid content of excised rat muscle, liver, brain and kidney and of human muscle biopsies was estimated by natural abundance 13C-NMR after complete solubilization of the tissue membranes with excess halothane. An external dioxane capillary, calibrated against pure palmitic acid and phospholipid vesicles with known phosphate concentration, was inserted into the tissues, and the repeating methylene carbon peak area in the spectra of the halothane-treated tissues was integrated versus the dioxane reference peak area. The amount of tissue used for NMR analysis was quantitated by dry weight determination after 13C spectroscopy was completed. The phospholipid content estimated by the indirect NMR method was in good agreement with that measured by direct phosphate analysis and with literature data. For human muscle biopsies, the NMR method can also estimate the fraction of the total phospholipids which are mobile without treating the muscles with halothane. In this respect human muscles could be separated into three different groups: normal and nonspecific muscle diseases, myotonia and myopathy, Duchenne dystrophy; with increasing fraction of the mobile phospholipids in this order.

Protein phosphorylation in arterial muscle contracted by high concentration of phorbol dibutyrate in the presence and absence of Ca2+.

Porcine carotid arterial muscles were 32P-labeled then contracted with 8 microM phorbol dibutyrate (PDBu) in normal physiological salt solution (PSS) and in Ca(2+)-free PSS containing 0.5 mM ethylene glycol-bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetate. Significant incorporation of [32P]phosphate into the 20-kDa myosin light chain, a 28-kDa protein, desmin and caldesmon was measured with no apparent difference between normal and Ca(2+)-depleted muscles. Ca-determination showed that the Ca(2+)-depleted muscle contained 15% of the total Ca of the normal muscle. However, determination of the actin-bound Ca revealed that all the Ca in the Ca(2+)-depleted muscle could be accounted for by its actin-bound Ca. Accordingly, protein phosphorylation during the slow PDBu-induced contraction may proceed in the virtual absence of free Ca2+. Phosphopeptide mapping of the myosin light chain isolated from muscles contracted with PDBu either in the presence or absence of Ca2+ showed that two-thirds of the incorporated [32P]phosphate was attributable to myosin light chain kinase catalyzed phosphorylation and one-third was due to phosphorylation by protein kinase C. PDBu increased the phosphorylation of the 28-kDa protein, desmin and caldesmon two- to threefold, as compared with that in muscles contracted by KCl depolarization or by the receptor mediated agonists norepinephrine and histamine. Muscles contracted by high concentration of PDBu in the presence or absence of Ca2+ could be fully relaxed and recontracted.

Analysis of myosin light chain phosphopeptides in phorbol dibutyrate-contracted artery.

The incorporation of [32P]phosphate into the 20 kDa myosin light chain of phorbol dibutyrate-contracted artery was slightly increased as compared to that of resting muscle. Addition of K+ to the 1-h phorbol dibutyrate-contracted artery immediately doubled the force and greatly increased the light chain phosphorylation. Two-dimensional phosphopeptide mapping of light chain from phorbol dibutyrate-contracted muscle showed distinct peptides phosphorylated on serine residues by myosin light chain kinase and protein kinase C. In addition, the peptide phosphorylated on threonine residue by protein kinase C was revealed for the first time in intact muscle. Upon addition of K+, the distribution of phosphopeptides shifted toward the myosin light chain kinase catalyzed pattern.

Modification of myosin light chain phosphorylation in sustained arterial muscle contraction by phorbol dibutyrate.

The decrease in phosphorylation of the 20 kDa myosin light chain during prolonged K(+)-stimulation of arterial smooth muscle was counteracted by treating this muscle with phorbol dibutyrate. Quantitative phosphopeptide analysis revealed that phorbol dibutyrate induced phosphorylation of serine and threonine residues in the light chain by protein kinase C and phosphorylation of a threonine residue by myosin light chain kinase. The same residues of light chain were also phosphorylated when phorbol dibutyrate was added to muscles pretreated either with the Ca2(+)-channel-blocking agents nifedipine and verapamil, or with the Ca2(+)-chelating agent ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. The results indicate an interrelationship between protein kinase C and myosin light chain kinase phosphorylated sites of light chain in intact arterial smooth muscle.

Dephosphorylation of distinct sites in myosin light chain by two types of phosphatase in aortic smooth muscle.

Two types of myosin light chain phosphatase from aortic smooth muscle extract were separated by chromatography on heparin-agarose. The phosphatase which appeared in the flow-through fractions had low activity on actomyosin, its apparent molecular mass was 260 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 36-39 kDa as determined by gel filtration. This phosphatase preferentially dephosphorylated the alpha-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. The phosphatase retained by heparin-agarose had high activity on actomyosin, its apparent molecular mass was 150 kDa and upon ethanol treatment it generated a catalytic subunit with an apparent molecular mass of 39-42 kDa. It preferentially dephosphorylated the beta-subunit of phosphorylase kinase and its phosphorylase phosphatase activity was not inhibited by heparin, inhibitor-1 or inhibitor-2. Myosin light chain was phosphorylated by myosin light chain kinase in peptides AB (Ser-P) and CD (Thr-P), and/or by protein kinase C in peptides E (Ser-P) and F (Thr-P) as determined by one-dimensional phosphopeptide mapping. The catalytic subunit of heparin-agarose flow-through phosphatase preferentially dephosphorylated peptide F over peptides AB, CD and E in both isolated light chain and actomyosin. The catalytic subunit of heparin-agarose bound phosphatase could effectively dephosphorylate all sites in isolated light chain, whereas it was less effective on dephosphorylation of peptide E in actomyosin.

Isolation of phosphorylated acid chloroform/methanol-soluble proteins from live frog muscle.

About 6-7% of the total proteins from trichloroacetic acid-washed and freeze-dried frog muscle could be extracted with acid chloroform/methanol. Three of these proteins were found to be phosphorylated in the live frog. They were purified to apparent homogeneity by gel chromatography and preparative gel electrophoresis. The apparent molecular weights, determined by sodium dodecyl sulfate gel electrophoresis, were 34 000, 19 000 and 10 000. Each phosphorylated protein contained 3 mol of a covalently bound neutral sugar but they did not contain any tightly bound lipids. All three proteins incorporated 32P into serine phosphate. The 10 000 dalton protein, which had the highest specific radioactivity contained an unusually high proportion of serine, 14% of the total amino acids. It also did not stain with Coomassie Blue.

The separation of phosphocreatine from creatine, and pH determination in frog muscle by natural abundance 13C-NMR.

Phosphocreatine can be separated from creatine in superfused frog muscle by natural abundance 13C-NMR, based on the difference in resonance frequency of their guanidino carbons. After taking into account the longitudinal relaxation times and nuclear Overhauser enhancement factors, the integrated peak areas of the guanidino carbons could be used for determination of the phosphocreatine-to-creatine ratio in the muscle. The pH dependence of the chemical shift of the C-2 carbon in the histidine ring of carnosine was used for estimation of the intracellular pH in the intact muscle.

Characterization of the phosphorylatable myosin light chain in rat uterus.

The 20 kDa myosin light chain of 32P-labeled rat uterus exhibited four spots on two-dimensional gel electrophoretograms; the corresponding autoradiograms revealed that three spots were radioactive. Completely dephosphorylated light chain exhibited three spots on electrophoretograms. Serine and threonine residues of the light chain were found to be phosphorylated in the uterus at a ratio of 6 to 1. During contraction, the amount of each phosphoamino acid increased proportionally to the increase in the total phosphate content of the light chain.

Evidence for isoforms of the phosphorylatable myosin light chain in rat uterus.

The phosphorylatable myosin light chain of rat uterus was resolved into four spots by two-dimensional gel electrophoresis. Antibody against the 20 kDa light chain from smooth muscle myosin recognized these four spots. Purified light chain exhibited four spots on a diagonal upon isoelectrofocusing in both first and second dimensions, proving that these spots are not due to artifactual charge modification. Accordingly, the four spots contain light chain isoforms derived from myosin.

Effects of ATP reduction on the pattern of force development and myosin light chain phosphorylation in intact arterial smooth muscle.

The effect of reduction of ATP content on phosphorylation of the 20 kDa light chain of myosin (MLC) and force development in intact carotid arterial smooth muscle was investigated. With reduction of ATP to 23% of control by treatment with 2-deoxyglucose there was reduction in basal, in peak and 30 min MLC phosphorylation during contraction (P less than 0.001). The rate of force development was reduced, but maximal force was the same as control. By treatment with 50 microM iodoacetate, the resting ATP content was unchanged but fell to 22% after 30 min contraction. Basal MLC phosphorylation was the same as control, but peak (P less than 0.001) and 30 min phosphorylation were lower (P less than 0.005), even though the rate and magnitude of force development were greater. The results indicate that neither rate nor magnitude of force development correlate with MLC phosphorylation. Basal and initial MLC phosphorylation may play a cooperative role in contractile function.

Oxidation of acetate and octanoate and its relation to glucose metabolism in contracting porcine carotid artery.

The oxidation of octanoate and acetate was measured in segments of porcine carotid arteries to ascertain whether the oxidation of exogenous fatty acid substrates (acetate and octanoate) is augmented during contraction induced by K(+)-depolarization. The oxidation of acetate increased from 7 +/- 1 to 14 +/- 2 nmol/min/g (P < 0.01) during sustained isometric contraction. Octanoate oxidation increased from 11 +/- 1 to 14 +/- 1 nmol/min/g (P < 0.05). The rate of oxidation of neither acetate nor octanoate was affected by the presence or absence of glucose either in resting or contracting arteries Acetate or octanoate oxidation could account for the majority of O2 consumption during contraction. Octanoate but not acetate inhibited glucose uptake and glycolysis in resting muscles. In contrast to augmented acetate and octanoate metabolism during contraction, there was a "down-regulation" of glucose metabolism in contracting muscles as evidenced by a decrease in the rate of glucose uptake, glycolysis and lactic acid production during sustained isometric contraction. Thus, contractile activation of vascular smooth muscle is associated with a shifting pattern of substrate utilization. Exogenous acetate or octanoate can serve as the primary oxidative substrate during sustained isometric contraction.

ATPase activity of myosin correlated with speed of muscle shortening.

Myosin was isolated from 14 different muscles (mammals, lower vertebrates, and invertebrates) of known maximal speed of shortening. These myosin preparations were homogeneous in the analytical ultracentrifuge or, in a few cases, showed, in addition to the main myosin peak, part of the myosin in aggregated form. Actin- and Ca(++)-activated ATPase activities of the myosins were generally proportional to the speed of shortening of their respective muscles; i.e. the greater the intrinsic speed, the higher the ATPase activity. This relation was found when the speed of shortening ranged from 0.1 to 24 muscle lengths/sec. The temperature coefficient of the Ca(++)-activated myosin ATPase was the same as that of the speed of shortening, Q(10) about 2. Higher Q(10) values were found for the actin-activated myosin ATPase, especially below 10 degrees C. By using myofibrils instead of reconstituted actomyosin, Q(10) values close to 2 could be obtained for the Mg(++)-activated myofibrillar ATPase at ionic strength of 0.014. In another series of experiments, myosin was isolated from 11 different muscles of known isometric twitch contraction time. The ATPase activity of these myosins was inversely proportional to the contraction time of the muscles. These results suggest a role for the ATPase activity of myosin in determining the speed of muscle contraction. In contrast to the ATPase activity of myosin, which varied according to the speed of contraction, the F-actin-binding ability of myosin from various muscles was rather constant.

Absence of calponin phosphorylation in contracting or resting arterial smooth muscle.

We have tested the hypothesis of Winder and Walsh [(1990) J. Biol. Chem. 265, 10148] that the contractile state of smooth muscle is regulated by calponin phosphorylation. Porcine carotid arterial muscles were highly labeled with 32P, then contracted with four different agents for various times. No radioactivity was detected in calponin isolated by 2D or 1D gel electrophoresis from the muscles. Similarly, resting muscles showed no [32P]phosphate in calponin. Apparently the sites of calponin available for phosphorylation in vitro are rendered unavailable in the intact muscle.

1H NMR of intact muscle at 11 T.

1H NMR spectra of intact frog, and chicken skeletal muscles, were recorded at 470 MHz with the Plateau and Guéron pulse sequence for the suppression of water [(1982) J. Am. Chem. Soc. 104, 7310]. Only a few transients were required to resolve the resonances from the protons of muscle metabolites. The previously unobserved exchangeable protons of muscles were also recorded and thereby phosphocreatine and creatine could be measured simultaneously. During aging of dissected frog muscle, changes in levels of phosphocreatine, creatine and lactic acid, and the decrease in the intracellular pH were followed by 1H NMR.

Proton magnetic resonance spectroscopy of the brain in schizophrenic and affective patients.

Water-suppressed 1H magnetic resonance spectra were recorded from two brain regions of psychiatric patients and normal volunteers. The two regions studied were (a) the basal ganglia structures surrounding the anterior horn of the lateral ventricle and (b) the occipital cortex. N-Acetylaspartate (NAA), phosphocreatine-creatine (PCr-Cr), choline and inositol resonances were seen in both regions. Ratios of metabolite peak integrals to PCr-Cr peak integral were calculated for each spectrum. To control for partial volume effects, comparisons between patients and controls were made only from identical regions i.e. basal ganglia vs basal ganglia, and likewise for occipital cortex. Metabolite ratios from the occipital region of patients were similar to those from the occipital region of normal subjects. Bipolar patients being treated with lithium had elevated NAA/PCr-Cr in the basal ganglia region when compared to normals. These patients also demonstrated elevated choline/PCr-Cr and inositol/PCr-Cr ratios in the basal ganglia region.