Jan Teisinger

Enzyme activities in quadriceps femoris muscle of obese diabetic male patients.

SummaryIn biopsy samples of the lateral part of the quadriceps femoris muscle of 6 obese diabetic male patients and of 11 obese males with a normal glucose tolerance, the activities of 7 enzymes of energy metabolism were estimated: hexokinase, cytoplasmic glycerol-3-phosphate: NAD dehydrogenase, triosephosphate dehydrogenase, lactate dehydrogenase, citrate synthase, malate dehydrogenase and 3-hydroxyacyl-CoA dehydrogenase. The obese diabetic male patients exhibited decreased activities of enzymes of carbohydrate breakdown and cytoplasmic NAD regeneration. Enzymes connected functionally with aerobic metabolism were less affected. The unchanged activity of 3-hydroxyacyl-CoA dehydrogenase points to an increased role of fatty acid catabolism in the muscle.

Effects of sleep deprivation on the activity of selected metabolic enzymes in skeletal muscle

SummaryThe effect of 120-h sleep deprivation on the activity of selected enzymes of energy metabolism in skeletal muscle was studied in seven healthy volunteers. The results showed a significant decrease in the activity of malate dehydrogenase, citrate synthase, glycerol-3-phosphate dehydrogenase and lactate dehydrogenase. Triosephosphate dehydrogenase, hexokinase, and hydroxyacyl-CoA-dehydrogenase activities showed an insignificant decrease. The findings are indicative of (1) decreased aerobic oxidation capacity; (2) reduced function of reducing-equivalent carriers from cytosol across the mitochondrial membrane; (3) relative accentuation of the non-aerobic glycolytic pathway; (4) a prediabetic type of muscle metabolism.

Activation of membrane Na+/K+-ATPase of mouse skeletal muscle by acetylcholine and its inhibition by α-bungarotoxin, curare and atropine

The effect of acetylcholine and of three cholinolytic compounds (α-bungarotoxin, curare and atropine) on electrogenic Na+/K+ pump and activity of the membrane Na+/K+-ATPase of mouse skeletal muscles was studied. It was found that acetylcholine potentiated both the muscle electrogenic ionic pump and the Na+/K+-ATPase activity of crude membrane fractions. The cholinolytic drugs had inhibitory effects on both parameters, with the exception of curare which was ineffective in blocking the electrogenic ionic pump.

Different sensitivity of ATP + Mg + Na (I) and Pi + Mg (II) dependent types of ouabain binding to phospholipase A2

SummaryThe effect of phospholipase A2 and of related agents on ouabain binding and Na,K-ATPase activity were studied in intact and detergent-treated membrane preparations of rat brain cortex and pig kidney medulla. It was found that phospholipase A2 (PLA2) may distinguish or dissociate ouabain binding complexes I (ATP+Mg+Na) and II (Pi+Mg), stimulating the former and inhibiting the latter. Procedures which break the permeability barriers of vesicular membrane preparations, such as repeated freezing-thawing, sonication or hypoosmotic shock failed to mimic the effect of PLA2, indicating that it was not acting primarily by opening the inside-out oriented vesicles. The detergent digitonin exhibited similar effects on ouabain binding in both ATP+Mg+Na and Pi+Mg media. Other detergents were ineffective.The ability of PLA2 to distinguish between ouabain binding type I and II can be manifested even in SDS-treated, purified preparations of Na,K-ATPase. The number of ATP+Mg+Na-dependent sites is unchanged, while the Pi+Mg-dependent sites are decreased in number in a manner similar to that seen in original membranes. This inhibition is completely lost in the reconstituted Na,K-ATPase system, where the ATP- as well as Pi-oriented ouabain sites are inhibited by PLA2.

Na, K-ATPase and the development of Na+ transport in rat distal colon

SummaryNa, K-ATPase function was studied in order to evaluate the mechanism of increased colonic Na+ transport during early postnatal development. The maximum Na+-pumping activity that was represented by the equivalent short-circuit current after addition of nystatin (IscN) did not change during postnatal life or after adrenalectomy performed in 16-day-old rats.IscN was entirely inhibited by ouabain; the inhibitory constant was 0.1mm in 10-day-old (young) and 0.4mm in 90-day-old (adult) rats. The affinity of the Na, K pump for Na+ was higher in young (11mm) than in adult animals (19mm). The Na, K-ATPase activity (measured after unmasking of latent activity by treatment with sodium dodecylsulfate) increased during development and was also not influenced by adrenalectomy of 16-day-old rats. The inhibitory constant for ouabain (KI) was not changed during development (0.1–0.3mm). Specific [3H]ouabain binding to isolated colonocytes increased during development (19 and 82 pmol/mg protein), the dissociation constant (KD) was 8 and 21 μm in young and adult rats, respectively. The Na+ turnover rate per single Na, K pump, which was calculated fromIscN and estimated density of binding sites per cm2 of tissue was 500 in adult and 6400 Na+/min·site in young rats. These data indicate that the very high Na+ transport during early postnatal life reflects an elevated turnover rate and increased affinity for Na+ of a single isoform of the Na, K pump. The development of Na+ extrusion across the basolateral membrane is not directly regulated by corticosteroids.

Vanadyl (VO2+) and vanadate (VO3−) ions inhibit the brain microsomal Na,K-ATPase with similar affinities. Protection by transferrin and noradrenaline

The activity of Na,K-ATPase was measured in brain microsomes as the function of increasing concentrations of vanadyl (VOSO4, V4+) and the vanadate (NaVO3, V5+) ions. Both forms of vanadium inhibited the Na,K-ATPase activity with high affinity -Ki (vanadate) = 3 X 10(-7)M and Ki (vanadyl = 1 X 10(-6)M. The stability of V4+ in ATPase reaction media (Tris buffers) was measured by electron spin resonance spectroscopy. Without any reducing agent, V4+ was quickly oxidised by atmospheric oxygen. When a reducing agent such as dithiothreitol was added, the V4+ was stable for at least 30 min and the inhibition pattern of Na,K-ATPase by V4+ was not changed. The blocking effect of V4+ in the presence of dithiothreitol was counteracted by pre-incubation with equimolar concentrations of transferrin or 100 times excess of noradrenaline. The regulation of brain Na,K-ATPase by vanadate may be represented by competition between low-capacity inhibitory binding sites localized on the enzyme molecule and high-capacity sites of intracellular proteins. Preferential binding of vanadyl to the latter type of sites will decrease the intracellular concentration of the free metal and thus eliminate the enzyme inhibition.

The disparity between effects of vanadate (V) and vanadyl (IV) ions on (Na+K+)-ATPase and K+-phosphatase in skeletal muscle

Abstract On crude membrane fractions of skeletal musccle, vanadyl (IV) and vanadate (V) compounds inhibited the membrane (Na + K + )-ATPase and neutral (K + -)p-nitrophenylphosphatase equally with K i 4×10 −8 mol.1 −1 . Only vanadate (V) inhibited significantly the muscle (Na + K + )ATPase with K i 1×10 −6 mol.1 −1 , whereas vanadyl (IV) ions were almost without effect. Extracellular application of both forms of vanadium failed to inhibit the electrogenic (Na + K + ) pump in intact mouse diaphragm fibres.

Enzyme activity patterns of energy supplying metabolism in the quadriceps femoris muscle(Vastus lateralis)

Summary1.In 3 groups of men, differing as to the amount and intensity of physical training loads, increasing in the order “sedentary”:“sporting”:“athletic”, enzyme activities were estimated in biopsy samples of m. quadriceps femoris (vastus lateralis). The enzymes were: Hexokinase (HK), NAD: glycerol-3-phosphate dehydrogenase (GPDH), triosephosphate dehydrogenase (TPDH), lactate dehydrogenase (LDH), citrate synthase (CS), NAD: malate dehydrogenase (MDH), and 3-hydroxyacyl-CoA dehydrogenase (HOADH). Indicators of laboratory performance and whole-body metabolic capacities (maximal oxygen consumption etc.) were estimated in the “sporting” and “athletic” groups.2.In the 2 latter groups, distinguished by greater physical activity, the atypical enzyme activity pattern, remarkable by a low activity of LDH and high relative activities of GPDH and HK, as reported earlier in a sedentary group (Basset al., 1975a), disappeared. The possibility of the atypical low LDH enzyme activity pattern as resulting from lack of bodily exertion is discussed.3.The moderately trained “sporting” group distinguishes itself from the “sedentary” one mainly by a higher activity of LDH and by lower activities of GPDH and MDH. In the intensively trained “athletic” group, enzymes connected to aerobic oxidation (MDH, CS, HOADH) and GPDH also show higher activities than in the “sporting” group. The difference between the two more active groups is further borne out by a higher maximum oxygen uptake and carbon dioxide release of the well-trained “athletic” group. This difference of enzyme activity pattern may not be confined to the quadriceps femoris muscle.

Effect of catecholamines and metal chelating agents on the brain and brown adipose tissue Na,K-ATPase

Catecholamines stimulate Na,K-ATPase activity in the microsomal membranes of the brain and brown adipose tissue. This stimulation is apparent in the absence of soluble, cytosolic inhibitors and exhibits the same characteristics in both tissues: it occurs at high concentrations (10(-6)-10(-4) M) only; there is no difference in potency between isoprenaline, norepinephrine and epinephrine (EC50 = 1-2 X 10(-5) M); the D-stereoisomer of isoprenaline is equally as effective as the L-form; stimulation of Na,K-ATPase may also be achieved by the metal chelators EDTA, EGTA and desferal; the hydrophobic beta-blockers, propranolol and alprenolol, inhibit both the norepinephrine-stimulated and basal levels of enzyme activity at concentrations of 10(-5)-10(-3) M; phenoxybenzamine, an irreversible alpha-adrenergic blocker, inhibits basal Na,K-ATPase as well as norepinephrine-stimulated enzyme activity (EC50 = 2.5 X 10(-5) M). Because none of these observations can be related to the properties of the stereospecific adrenergic receptor (alpha or beta), it may be concluded that the catecholamine-Na,K-ATPase interaction is not mediated by the receptor. More probably, catecholamines may antagonize the Na,K-ATPase inhibition caused by some tightly membrane-bound metals (but not vanadium) via the ortho-catechol moiety of the catecholamine molecule. The stimulation of brown fat Na,K-ATPase by catecholamines does not have much relevance to the norepinephrine-stimulated thermogenesis in this tissue.

Bleomycin stimulates both membrane (Na+-K+) ATPase and electrogenic (Na+-K+) pump and partially removes the inhibition by vanadium ions.

Bleomycin 2 X 10(-6) and 6 X 10(-6) mol.1(-1) increased the activity of specific (Na+-K+) ATPase of the rat brain microsomes. It also stimulated the electrogenic (Na+-K+) pump in intact skeletal muscle cells. The blocking effect of vanadyl (+4V) on membrane (Na+-K+) ATPase was eliminated completely by the drug, but the action of vanadate (+5V) was counteracted only partially. Electron paramagnetic resonance spectra revealed the formation of a +4V - bleomycin complex which is still able to activate the (Na+-K+) ATPase.