Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1.

Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K+- and Ca2+-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.     

The effect of physostigmine on (Na+ + K+)-ATPase activity in different rat brain regions

The activity of (Na+ + K+)-ATPase and acetylcholine esterase were followed in rat brain cerebral cortex, caudate, thalamus, hippocampus and medulla after i.v. administration of physostigmine. Both enzymes were found to be inhibited in a dose-dependent manner. The most pronounced inhibition of (Na+ + K+)-ATPase was found in caudate, where the highest activity of acetylcholine esterase is found.     

Inhibition of hepatic Na +, K + -adenosinetriphosphatase in taurolithocholate-induced cholestasis in the rat.

Na +, K + -adenosinetriphosphatase (Na +, K + -ATPase) activity was decreased in liver plasma membranes from rats in which cholestasis had been induced by i.v. administration of sodium taurolithocholate (5 mumoles/100 g b. wt). Incubation of liver plasma membranes with taurolithocholate (10--1300 muM) caused significant and dose dependent reductions of Na +, K + -ATPase activity at taurolithocholate concentrations above 100 muM. These findings lend support to the hypothesis that cholestasis induced by monohydroxy bile acids is at least partially the result of an inhibition of hepatic Na +, K + -ATPase activity.     

Possible role of intestinal alkaline phosphatase activity in thiamine transport.

Thiamine deficiency caused a marked decrease of intestinal alkaline phosphatase (al-Pase) activity, but had no effect on the Ca++-ATPase activity and Ca++-absorption in rats. The al-Pase activity was significantly decreased 1 h after oral administration of ethanol at 0.5 and 2.5 g/kg. In contrast, Mg++-, Ca++-and (Na+ + K+)-ATPase activities did not change after the administration of ethanol. These findings show that the al-Pase activity, unlike the Ca++-ATPase activity, is not related to Ca++-absorption. A possible role of al-Pase activity in the active transport of thiamine in the intestine was discussed.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Transients in myoplasmic [Ca2+] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K+ -stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca2+ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an improved estimate of activation rates.     

Affinity chromatographic preparation of arterial heavy meromyosin subfragment-1

Summary Heavy meromyosin subfragment-1 (HMM S-1) was prepared by papain digestion of arterial myosin or actomyosin and was purified by agarose-ATP affinity chromatography. Proteolysis of crude arterial myosin suspensions was preceded by solubilization. HMM-S-1 thus obtained consisted mainly of a 90,000 dalton polypeptide and fully retained the K⁺- and Ca²⁺-ATPase of the parent myosin. Its affinity to agarose-ATP was comparable to that of skeletal muscle HMM S-1.The support of this work by an EMBO short time fellowship (to R. L.) and by the Deutsche Forschungsgemeinschaft (No. SFB 90, B 7) and the excellent technical assistance byM. Troschka andC. Köhler are gratefully acknowledged.     

Myosin heavy chains in fast skeletal muscle of chick embryo

Summary The peptide map obtained by electrophoresis after digestion of purified myosin heavy chains from pectoralis muscle of embryonic chicken with theStaphylococcus aureus V8 protease, produces a peptide pattern very similar but not identical to that of adult fast myosin. In fact, some components that are present in a small amount in the map of slow adult myosin are visible in the embryonic pattern.Acknowledgments. I wish to acknowledge the enthusiastic encouragement of Dr Ugo Carraro and the help of Prof. Stefano Schiaffino's group and of Dr Marcello Cantini for embryonic tissue isolation. The skillful technical assistance of Mr Silvio Belluco is also gratefully acknowledged. This work was supported in part by a grant of Muscular Dystrophy Association of America to Prof. Massimiliano Aloisi.     

The (Na+ + K+)-ATPase activity in brain of Quaking mice.

The (Na+ 4 K+)- and Mg2+-dependent ATPase distribution in several brain areas has been investigated in Quaking mutant mice characterized by myelin deficiency. A marked decrease of (Na+ + K+)-ATPase activity has been found in limbic structures, hypothalamus and cerebellum. The Mg2+-dependent activity did not change. A possible involvement of the impairment of the (Na+ + K+)-ATPase activity in the seizure susceptibility of this mice is discussed.     

Increase in Na+, K+ -ATPase enzyme units in liver and kidneys from essential fatty acid deficient rats.

(3H)-Ouabain binding to liver and kidney preparations was utilized to estimate the number of Na+, K+-ATPase enzyme units in livers and kidneys from rats fed 2% corn oil supplemented or fat-free diets. The specific (3H)-ouabain binding in liver and kidney preparations from fatty acid deficient rats was increased approximately 40%, but the affinity of the binding sites for ouabain (Kd-value) remained unchanged. The increased concentration of Na+, K+-ATPase enzyme units observed in the essential fatty acid deficient rats may contribute to the reduced body fat accumulation and elevated heat production observed in these animals.     

A histochemical demonstration of the Na+ + K+-ATPase activity in the thyroid and the effect of cyclic adenosine monophosphate (c-AMP).

With a suitable modification of the Farquhar and Palade technique the Na+ + K+-ATPase activity in guinea-pig thyroid is demonstrated. The addition of c-AMP (5 X 10(-6) M or 1.5 X 10(-5) M) to the incubation media produced an apparent intensification of the Na+ + K+ -ATPase activity in the thyroid.     

The role of myosin phosphorylation in the contraction-relaxation cycle of smooth muscle

Summary Considerable evidence from a variety of experimental procedures indicates that the phosphorylation of myosin is involved in the regulation of contractile activity in smooth muscle. Phosphorylation of the 20,000-dalton myosin light chains is required to initiate crossbridge cycling and this is consistent with the observation that the actin-activated Mg²⁺-ATPase activity of myosin is phosphorylation-dependent. In the simplest interpretation of this process it may be proposed that phosphorylation acts as an on-off switch. Clearly this cannot explain the observed complexity of smooth muscle contractile behavior and such may imply either that additional mechanisms are involved or that the role of myosin phosphorylation is not fully appreciated. Recently it has been shown that monomeric smooth muscle myosin can exist in a folded and an extended conformation and that each form is characterized by distinct enzymatic properties. Under appropriate solvent conditions phosphorylation of myosin favors the extended conformation. It is tentatively suggest that this, or an analogous, transition might be involved in the regulation of the smooth muscle contractile apparatus, and this possibility is discussed.The authors are supported by grants HL 23615 and HL 20984 from the National Institutes of Health.     

Insulin effect in vitro on human erythrocyte plasma membrane

The effect of porcine insulin has been tested in vitro on human erythrocyte plasma membrane (Na+-K+) and Mg2+-ATPase activities as well as on membrane fluidity. The results indicate that the hormonal treatment significantly inhibits (Na+-K+)-ATPase activity, and at the same time decreases membrane fluidity.     

Velocity and myosin phosphorylation transients in arterial smooth muscle: effects of agonist diffusion

Summary Transients in myoplasmic [Ca²⁺] and in phosphorylation of the 20,000 dalton light chain of myosin have been reported following stimulation of vascular smooth muscle by various agonists. Since these transients are rapid compared with the time required to attain a steady-state stress, agonist diffusion rates may be a significant limitation in activation. The purpose of this study was to estimate the effect of agonist diffusion rates on the time course of activation as assessed by mechanical measurements of stress development and isotonic shortening velocities and by determinations of the time course of myosin phosphorylation. The approach was to measure these parameters in K⁺-stimulated preparations of the swine carotid media of varying thicknesses and to estimate the theoretical contributions imposed by diffusion rates and the presence of a diffusion boundary layer surrounding the tissue. The results show that the time course of parameters which are tissue averages such as stiffness, active stress, and myosin phosphorylation is dominated by agonist diffusion rates. The sequence of events involved in excitation-contraction coupling including agonist actions on the cell membrane, Ca²⁺ release, activation of myosin light chain kinase, and cross-bridge phosphorylation appear to be very rapid events compared with stress development. Estimates of unloaded or lightly loaded shortening velocities which are not simple tissue averages appear to provide an imporoved estimate of activation rates.Supported by a grant from the National Institutes of Health 5-PO1-HL19242. K. E. Kamm was supported by a National Heart, Lung, and Blood Institute Research Service Award HL-05957.     

Effect of mycotoxin (T-2 toxin) on catecholamine and Na+, K+-ATPase levels in rat epididymis

The effect of mycotoxin (T-2 toxin) on catecholamines and Na+, K+-ATPase activities in rat epididymis has been evaluated. Dopamine and norepinephrine levels were significantly elevated in the caput and corpus regions whereas their levels remained unchanged in the caudal part of the epididymis. Na+, K+-ATPase activity was significantly increased in all the three regions of rat epididymis as a result of the toxin treatment. These changes may suggest an adverse effect on epididymal functions in rats.     

Differences in cardiac myosin light chain LC1 among human, monkey and sheep

The atrial and ventricular myosin light chains of human, monkey and sheep hearts were compared by dodecylsulfate polyacrylamide gel electrophoresis. The atrial light chain 2 and ventricular light chain 2 are similar among these mammals. However, the atrial light chain 1 of monkey has different electrophoretic mobility from those of human and sheep. The monkey ventricular light chain 1 has same mobility as that of sheep but different from that of human.     

The effect of aging on rat liver regeneration

Summary The effect of age on hepatocyte mensuration and mitotic activity 48 h after partial hepatectomy was investigated in rats. Both age and partial hepatectomy had significant effects upon hepatocyte counts per microscopic field. The number of hepatocytes per microscopic field declined with age in the control groups of different advancing ages and in the experimental groups of advancing ages. There was essentially no mitotic activity in the livers of the control groups. However, mitotic counts were greatly increased in livers from those animals that were partially hepatectomized; the increase in mitotic activity in the 13-month-old animals was double over that observed in both the very young and the very old.Acknowledgment. This research was supported in part by an Eastern Virginia Medical School Biomedical Research Development Fund. The investigators acknowledge the Gerontology Research Center, NIA, Baltimore, Maryland for their support.     

Increase in superoxide dismutase activity induced by thyroid hormones in the brains of neonate and adult rats

Summary The activity of cytoplasmic superoxide dismutase (SOD) in the liver was about twice as high in adult rats as it was in neonates. In the brain and in the interscapular brown adipose tissue (IBAT), SOD activity was not changed during postnatal development, although it was slightly higher in the brain than in the IBAT (p>0.1). Thyroid hormones produced an increase in SOD activity in the brain of newborn rats, as well as in those animals 30 and 60 days old. The same quantity of hormones did not produce any significant changes in the liver or in the IBAT.     

Cytochemical localization of the K+ regulation interface between blood and brain.

Phosphatase activity identified with Na+-K+-ATPase was localized at the basal surface of cerebral cortical capillary endothelium by perfusion with a p-nitrophenyl phosphate-strontium medium. The relationship of this to the blood-brain barrier to K+ is discussed.     

Differences in cardiac myosin light chain LC1 among human,monkey and sheep

Summary The atrial and ventricular myosin light chains of human, monkey and sheep hearts were compared by dodecylsulfate polyacrylamide gel electrophoresis. The atrial light chain 2 and ventricular light chain 2 are similar among these mammals. However, the atrial light chain 1 of monkey has different electrophoretic mobility from those of human and sheep. The monkey ventricular light chain 1 has same mobility as that of sheep but different from that of human.Acknowledgments. This work was supported by MRC of Canada No. MA-8559. Dr G. Jackowski is a scholar of the Medical Research Council of Canada, and is the author to whom all correspondence should be addressed.