Enzymatic characteristics of the guanylate cyclase of KB cells: their change as a function of the development of the cultures]

We have localized 71% of the guanylate cyclase activity in the (G X 105,000) supernatent fraction of broken KB cells. The reaction follows Michaelis-Menten kinetics, the apparent Km for GTP is 0,5 mM, as long as GTP is lower than a limited concentration, then activity is inhibited. The ion Mn++ is an absolutely required activator, it does not change enzyme-substrate affinity. The enzyme shows several types of binding sites of Mn++. Guanylate cyclase, studied over a period of development of culture, shows, in KB cells without cell contact, an activity higher than that observed in confluent cells. This is not due to the fact of a change in enzyme-substrate affinity but to a modification of Mn++ influence.     

Normal adenylate-cyclase activity in platelets of patients with Huntington's disease

Summary Adenylate-cyclase activity and cyclic-AMP concentration in platelets of 7 Huntington's disease patients were found to be similar to control values.     

Influence of ethanol on calcium, inositol phospholipids and intracellular signalling mechanisms

Studies have implicated Ca++ in the actions of ethanol at many biochemical levels. Calcium as a major intracellular messenger in the central nervous system is involved in many processes, including protein phosphorylation enzyme activation and secretion of hormones and neurotransmitters. The control of intracellular calcium, therefore, represents a major step by which neuronal cells regulate their activities. The present review focuses on three primary areas which influence intracellular calcium levels; voltage-dependent Ca++ channels, receptor-mediated inositol phospholipid hydrolysis, and Ca++/Mg++-ATPase, the high affinity membrane Ca++ pump. Current research suggests that a subtype of the voltage-dependent Ca++ channel, the dihydropyridine-sensitive Ca++ channel, is uniquely sensitive to acute and chronic ethanol treatment. Acute exposure inhibits, while chronic ethanol exposure increases 45Ca++-influx and [3H]dihydropyridine receptor binding sites. In addition, acute and chronic exposure to ethanol inhibits, then increases Ca++/Mg++-ATPase activity in neuronal membranes. Changes in Ca++ channel and Ca++/Mg++-ATPase activity following chronic ethanol may occur as an adaptation process to increase Ca++ availability for intracellular processes. Since receptor-dependent inositol phospholipid hydrolysis is enhanced after chronic ethanol treatment, subsequent activation of protein kinase-C may also be involved in the adaptation process and may indicate increased coupling for receptor-dependent changes in Ca++/Mg++-ATPase activity. The increased sensitivity of three Ca++-dependent processes suggest that adaptation to chronic ethanol exposure may involve coupling of one or more of these processes to receptor-mediated events.     

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.     

Specific phosphorylation of a calf serum protein by a kinase from the cell surface]

A protein of molecular weight approximately 200,000, which exists in trace amounts in calf serum, is specifically phosphorylated by an enzyme located at the surface of cultured cells. The emzymatic reaction utilizes ATP, is enhanced by Mg++ and Zn++ ions, and is not dependent on cyclic AMP. This kinase activity is associated with normal growing fibroblasts but disappears when they are contact-inhibited. It remains high, however, in transformed and malignant cells, whatever their growth state.     

A technical improvement in the radioimmunoassay of cyclic-AMP.

An improvement in the technique for the radioimmunoassay of cyclic-AMP, wherein ammonium sulfate precipitation is replaced with zirconyl phosphate gel, is presented. This substitution produces a more stable pellet than that obtained with ammonium sulfate. This greatly reduces a potential source of error due to pellet instability.     

Extracellular calcium ions modify the effects of Anemonia sulcata toxin (ATX II) in guinea-pig papillary muscles

In isolated guinea-pig papillary muscle ATX II prolonged the action potential duration to a lesser extent at high extracellular Ca++-concentrations. This is interpreted as an interference of Ca++ with ATX II-binding sites.     

Effects of chronic treatment with ACTH on the intracellular levels of cyclic-AMP and cyclic-GMP in the rat adrenal cortex.

The effects of chronic ACTH treatment on the increase in the intracellular concentration of cyclic-AMP and cyclic-GMP acutely elicited by ACTH in the rat adrenal cortex were investigated. The results are consistent with the hypothesis that chronic ACTH treatment stimulates a) the de novo synthesis of adenylate- and guanylate-cyclase or b) the synthesis of new specific membrane receptors for ACTH.     

Role of tropomyosin in the sensitivity of (Na+ + K+) ATPase to ouabain]

EDTA treatment of isolated plasma membranes from MF2S cells increased 1,000 fold the sensitivity of (Na+ + K+) ATPase activity to ouabain. The original sensitivity of the enzyme to the drug is recovered after addition of tropomyosin together with Ca++ ions to the treated membranes.     

Purification of rat brain guanylate cyclase]

The purification of guanylate cyclase has been achieved. After electrophoresis on polyacrylamide gel only one protein band was observed. The low factor of purification must therefore be ascribed to the loss of an activator of guanylate cyclase. Stimulation of the activity by nitroprusside is also lost during purification. The purified enzyme follows Michaelis--Menten kinetics, it is activated by Mn++ ions and inhibited by triphospho nucleotides.     

Evolution in time of transmitter release at frog neuromuscular junction]

It has previously been shown that the unitary quantal end-plate potentials observed at synapses blocked by low Ca++ high Mg++ ringer, belong to distinct clusters according to their amplitude, time to peak and latency characteristics. These clusters correspond probably to distinct releasing units dispersed along the presynaptic terminal branches. The distribution versus time of the occurence of unitary potentials belonging to one latency cluster has been studied over long lasting evoked nerve activity (stimulation frequencies: 1 to 10 Hz). It was observed that transmitter release at one releasing site is statistically periodic with emitting periods separated by rest periods. At a given end-plate the period of emitting activity seems to be independent from one emitting site to another.     

Characterization of the (Na+-K+)-ATPase from endometrial plasma membranes of immature mammals]

The (Na+-K+)-ATPase in plasma membrane from Mammiferous endometrium is characterized by the Mg/ATP ratio equal to one, and by a distinct affinity for Na+ (1.3 mM) and K+ (2 mM). The activity is maximum for pH 7.4-7.5 in presence of Mg++ 2mM and ATP 2 mM, Na+ 140 mM and K+ 10 mM.     

Effects of chronic treatment with ACTH on the intracellular levels of cyclic-AMP and cyclic-GMP in the rat adrenal cortex

Summary The effects of chronic ACTH treatment on the increase in the intracellular concentration of cyclic-AMP and cyclic-GMP acutely elicited by ACTH in the rat adrenal cortex were investigated. The results are consistent with the hypothesis that chronic ACTH treatment stimulates a) the de novo synthesis of adenylate- and guanylate-cyclase or b) the synthesis of new specific membrane receptors for ACTH.Part of this work was presented at the 3rd International Conference on Cyclic Nucleotides, July 1977, New Orleans, Abstr. Book, p. 79.     

Some properties of external NADH oxidation by human placental mitochondria.

Isolated human term placenta mitochondria catalyse oxidation of external NADH in the presence of cytochrome c. This reaction is insensitive to the respiratory chain inhibitors such as rotenone and antimycin A, and is not coupled to phosphorylation. Comparison of the effect of Mg++ ion on NADH plus cytochrome c oxidation by human term placental, human skeletal muscle and rat skeletal mitochondria showed that Mg++ ion exerts an inhibitory effect in the case of human mitochondria and a stimulatory effect in the case of rat skeletal muscle mitochondria.     

Specific inhibition of a calcium dependent activation of brain cyclic AMP phosphodiesterase activity by vinblastine.

Vinblastine selectively inhibits the activation of brain cyclic AMP phosphodiesterase activity by Ca++-protein activator (50% inhibition by 2 x 10(-5) M). This inhibitory effect was reversed by excessive amounts of the activator, whereas large quantities of Ca++ caused only a slight suppression of the vinblastine effect. This result of vinblastine suggests a new site of its action and also suggests the possible role of protein activator, phosphodiesterase proteins or cyclic nucleotides in the previously known effects of vinblastine in vivo and in vitro.     

Permeability of the isolated human amniotic membrane to divalent cations]

Membrane potential and resistance recordings, in vitro, show that Mg++ does not pass through the amnion from the inside of the amniotic compartment to the outside of the amniotic membrane. Mg++ may become fixed on the surface or in the midst of the amniotic membrane. However, Mg++ diffuses in the opposite direction. Ca++, Ba++, Sr++ diffuse in both directions across the amniotic membrane.     

Presence of a specific uridine 5'-monophosphate pyrophosphorylase in baker's yeast.

Uridine 5'-monophosphate pyrophosphorylase was found to be present in baker's yeast. The enzyme preparation, purified about 30-fold, shows a strict specificity toward uracil and requires Mg++ for its activity.     

Effects of cadmium on the immune system of mice.

Chronic oral exposure of mice to Cd++ inhibits cell-mediated immunity of delayed type hypersensitivity induced by sheep red blood cells (SRBC). No effect was detected on humoral immune response to SRBC. Spleen cells derived from mice exposed to Cd++ showed in vitro enhanced response to T and B cell mitogens. These results demonstrate that Cd++ exposure alters the immune system of mice.     

Identification of cAMP dependant protein kinases in human lymphocytes]

Chromatographic purification by "DEAE" cellulose resolves the cAMP binding proteins in human lymphocytes into three parts. In presence of Mg++ each one possesses cAMP dependent protein-kinase activity, one of them showing allosteric characteristics.     

Kinetic study of membrane 5'-nucleotidase activity in cultured diploid cells]

5'-nucleotidase activity of cultured diplo?d cells was measured using intact suspended cells. This depended on the substrate amount, but did not vary with Mg ++ concentration-Kinetic data were determined using Con. A,a specific inhibitor.