Some characteristics of Ca2+ uptake by yeast cells

Summary Experiments were performed to obtain information on: (i) the specific properties of Ca²⁺ binding and transport in yeast (ii) the relationship between both parameters; (iii) similarities to or differences from other biological systems as measured by the effects of inhibitors; and (iv) the effects of mono and divalent cations, in order to get some insight on the specificity and some characteristics of the mechanism of the transport system for divalent cations in yeast.The results obtained gave some kinetic parameters for a high affinity system involved in the transport of Ca²⁺ in yeast. These were obtained mainly by considering actual concentrations of Ca²⁺ in the medium after substracting the amounts bound to the cell. Ak _m of 1.9 m and aV _max of 1.2 nmol (100 mg·3 min)^–1 were calculated.The effects of some inhibitors and other cations on Ca²⁺ uptake allow one to postulate some independence between binding and transport for this divalent cation.Of the inhibitors tested, only lanthanum seems to be a potent inhibitor of Ca²⁺ uptake in yeast.The effects of Mg²⁺ on the uptake of Ca²⁺ agree with the existence of a single transport system for both divalent cations.The actions of Na⁺ and K⁺ on the transport of Ca²⁺ offer interesting possibilities to study further some of the mechanistic properties of this transport system for divalent cations.     

Inhibitor of renal gluconeogenesis (IGN): Additional physiological modulator?

• 1.1. A procedure for the partial purification of IGN has been developed: it is insoluble in 80% ethanol but soluble in 6% TCA. It moves in void volume on a column of Sephadex G 25, passes through a membrane with cut-off at 25,000 dallons but not through a membrane with cut-off at 10,000 daltons.
• 2.2. IGN inhibits gluconeogenesis both in kidney cortex and liver slices. The inhibition is small during the first 30 min of incubation but it increases during the following periods of time. Liver slices are much less sensitive to the inhibitory action of IGN than kidney cortex slices.
• 3.3. IGN exerts its effect even in the absence of either K⁺, Ca²⁺ or Mg²⁺ from Krebs-Ringer bicarbonate.

     

Influence of calcium and magnesium on ethylene production by apple tissue slices

The decline in ethylene production in apple (Pyrus malus L. cv. Golden Delicious) tissue slices during 24 hours incubation in 600 millimolar sorbitol and 10 millimolar 2-(N-morpholino)ethanesulfonic acid buffer (pH 6.0) is recognized as a senescent phenomenon. The inclusion of very high concentrations (100 millimolar) of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ severely inhibited ethylene production during the first 6 hours of incubation. However, after 6 hours and up to 24 hours the ethylene-forming system was stablized. These high concentrations of Ca²⁺, Mg²⁺, or Ca²⁺ plus Mg²⁺ virtually eliminated lipid peroxidation and protein leakage from these slices. Also conversion of 1-aminocyclopropane-1-carboxylic-1-acid to ethylene and the influence of indoleacetic acid on ethylene production was stabilized after 24 hours of incubation by these high concentrations of Ca²⁺, Mg²⁺, and Ca²⁺ plus Mg²⁺. Addition of divalent ionophores severely inhibited ethylene production, but this inhibition was prevented by Ca²⁺ in concentrations greater than the ionophore. These data suggest that the loss of ethylene production by aging tissue slices results from degradation of membranes. They support previous work that indicates that the ethylene-forming system, perhaps the segment of the pathway from 1-aminocyclo-propane-1-carboxylic-1-acid to ethylene, resides in the plasma membrane.     

Release of endogenous dopamine, 3,4-dihydroxyphenylacetic acid,and amino acid transmitters from rat striatal slices

The release of endogenous dopamine (DA) and 3,4-dihydroxyphenylacetic acid (DOPAC) was measured in superfused striatal slices of the rat and the results compared with data obtained for the release of endogenous (a) DA and DOPAC in the cerebral cortex, nucleus accumbens and thalamus; (b) 5-hydroxytryptamine (5-HT), 5-hydroxyindoleacetic acid (5-HIAA), GABA, and glutamate in the striatum; and (c) GABA, glutamate and 5-HT in the cerebral cortex. In superfused slices of all four CNS regions, there appeared to be a Ca²⁺-dependent, K⁺-stimulated release of endogenous DA. In addition, in slices of the striatum and nucleus accumbens there also appeared to be a Ca²⁺-dependent, 60 mM K⁺ stimulated release of endogenous DOPAC. In the striatum, 16 mM Mg²⁺ was as effective as 2.5 mM Ca²⁺ in promoting the 60 mM K⁺-stimulated release of DOPAC. In addition, 16 mM Mg²⁺ appeared to function as a weak Ca²⁺ agonist since it also promoted the release of DA to approximately 40% of the level attained with Ca²⁺ in the presence of 60 mM K⁺. On the other hand, in the striatum, 16 mM Mg²⁺ inhibited the Ca²⁺-dependent, 60 mM K⁺-stimulated release of GABA and glutamate. Similar Mg²⁺-inhibition was observed in the cerebral cortex not only for GABA and glutamate but also for DA and 5-HT. With the use of -methyl -tyrosine (tyrosine hydroxylase inhibitor), cocaine (uptake inhibitor) and pargyline (monoamine oxidase inhibitor), it was determined that (a) most of the released DA and DOPAC was synthesized in the slices during the superfusion; (b) DOPAC was not formed from DA which had been released and taken up; and (c) DA and DOPAC were released from DA nerve terminals. In addition, the data indicate a difference in the release process between the amino acids and the monoamines from striatal slices since Mg²⁺ inhibited the Ca²⁺-dependent, K⁺-stimulated release of GABA and glutamate and appeared to promote the release of DA and 5-HT.     

Modulation of nitrate reductase in vivo and in vitro: Effects of phosphoprotein phosphatase inhibitors,free Mg2+ and 5′-AMP

Nitrate reductase in spinach (Spinacia oleracea L.) leaves was rapidly inactivated in the dark and reactivated by light, whereas in pea (Pisum sativum L.), roots, hyperoxic conditions caused inactivation, and anoxia caused reactivation. Reactivation in vivo, both in leaves and roots, was prohibited by high concentrations (10–30 M) of the serine/threonine-protein phosphatase inhibitors okadaic acid or calyculin, consistent with the notion that protein dephosphorylation catalyzed by type-1 or type-2A phosphatases was the mechanism for the reactivation of NADH-nitrate reductase (NR). Following inactivation of leaf NR in vivo, spontaneous reactivation in vitro (in desalted extracts) was slow, but was drastically accelerated by removal of Mg²⁺ with excess ethylenediaminetetraacetic acid (EDTA), or by desalting in a buffer devoid of Mg²⁺. Subsequent addition of either Mg²⁺, Mn²⁺ or Ca²⁺ inhibited the activation of NR in vitro. Reactivation of NR (at pH 7.5) in vitro in the presence of Mg²⁺ was also accelerated by millimolar concentrations of AMP or other nucleoside monophosphates. The EDTA-mediated reactivation in desalted crude extracts was completely prevented by protein-phosphatase inhibitors whereas the AMP-mediated reaction was largely unaffected by these toxins. The Mg²⁺-response profile of the AMP-accelerated reactivation suggested that okadaic acid, calyculin and microcystin-LR were rather ineffective inhibitors in the presence of divalent cations. However, with partially purified enzyme preparations (5–15% polyethyleneglycol fraction) the AMPmediated reactivation was also inhibited (65–80%) by microcystin-LR. Thus, the dephosphorylation (activation) of NR in vitro is inhibited by divalent cations, and protein phosphatases of the PP1 or PP2A type are involved in both the EDTA and AMP-stimulated reactions. Evidence was also obtained that divalent cations may regulate NR-protein phosphatase activity in vivo. When spinach leaf slices were incubated in Mg²⁺ -and Ca²⁺-free buffer solutions in the dark, extracted NR was inactive. After addition of the Ca²⁺ /Mg²⁺-ionophore A 23187 plus EDTA to the leaf slices, NR was activated in the dark. It was again inactivated upon addition of divalent cations (Mg²⁺ or Ca²⁺). It is tentatively suggested that Mg²⁺ fulfills several roles in the regulatory system of NR: it is required for active NR-protein kinase, it inactivates the protein phosphatase and is, at the same time, necessary to keep phospho-NR in the inactive state. The EDTA- and AMP-mediated reactivation of NR in vitro had different pH optima, suggesting that two different protein phosphatases may be involved. At pH 6.5, the activation of NR was relatively slow and the addition or removal of Mg²⁺ had no effect. However, 5-AMP was a potent activator of the reaction with an apparent K _m of 0.5 mM. There was also considerable specificity for 5AMP relative to 3- or 2-AMP or other nucleoside monophoposphates. We conclude that, depending upon conditions, the signals triggering NR modulation in vivo could be either metabolic (e.g. 5-AMP) or physical (e.g. cytosolic [Mg²⁺]) in nature.Abbreviations DTT dithiothreitol - Mops 3-(N-morpholino)propanesulfonic acid - NR NADH-nitrate reductase - NRA nitrate-reductase activity - PP protein phosphatase This paper is dedicated to Prof. O.K. Volk on the occasion of his 90th birthdayThe skilled technical assistance of Elke Brendle-Behnisch is gratefully acknowledged. The investigations were cooperatively supported by the Deutsche Forschungsgemeinschaft (SFB 251), the U.S. Department of Agriculture, Agricultural Research Services, Raleigh, NC. This work was also supported in part by a grant from the U.S. Department of Energy (Grant DE-A I05-91 ER 20031 to S.C.H.).     

Ca2+ - and Mg2+-dependent ATPase activities in the deoxycholate-treated rat heart sarcolemma

• 1.1. Isolated rat heart sarcolemma was treated with different concentrations of an ionic detergent, deoxycholate (DOC) and ATP hydrolysis in the presence of Ca²⁺ or Mg²⁺ was determined.
• 2.2. Both Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were decreased in the DOC-treated membranes; however, the depression of Mg²⁺-dependent ATPase activity was greater than that of Ca²⁺-dependent ATPase.
• 3.3. The differential changes in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were apparent when incubations with DOC were carried out for different time intervals and at different temperatures.
• 4.4. In DOC-treated preparations, the K_m value for Ca²⁺-dependent ATPase was decreased whereas that for Mg²⁺-dependent ATPase was increased. The half maximal velocities of the Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase enzyme reactions in the treated preparations were obtained at a DOC: membrane protein ratio of 3.0 and 0.6, respectively.
• 5.5. In the DOC-treated membranes exhibiting the half maximal velocities of enzyme reactions, the K_i value for Ca²⁺-dependent ATPase was drastically reduced but remained unchanged for Mg²⁺-dependent ATPase.
• 6.6. The DOC treatment was associated with a loss of protein as well as phospholipids and resulted in changes in the ultrastructural integrity of the membrane.
• 7.7. Varying degrees of decreases in the activities of sarcolemmal adenylate cyclase. (Na⁻-K⁺)-ATPase. 5'-nucleotidase and calcium binding were seen upon DOC treatment.
• 8.8. The extent of reduction in Ca²⁺-dependent ATPase and Mg²⁺-dependent ATPase activities were also different when the membrane was treated with a non-ionic detergent, Lubrol PX.
• 9.9. These data suggest that Ca²⁺-dependent ATPase in heart sarcolemma is more resistant than Mg²⁺-dependent ATPase to detergent treatments and further indicate some differences in the properties of these enzymes.

     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

High-affinity Ca2+-Mg2+-ATPase in kidney of euryhaline Gillichthys mirabilis: kinetics,subcellular distribution and effects of salinity

• 1.1. Two components of Ca²⁺-Mg²⁺-ATPase are observed in kidneys of G. mirabilis. The high-affinity component has a K_0.5Ca of 0.23μM; the low-affinity activity K_0.5Ca is 90–110μM. The high-affinity activity requires Mg²⁺, displays Michaelis-Menten kinetics, has peak activity at 1.2 μM Ca²⁺, and is insensitive to ouabain and Na⁺ azide.
• 2.2. In subcellular fractions, the high-affinity component segregates with Na⁺-K⁺-ATPase and is localized predominantly in BLM. The low-affinity component is broadly distributed among membranous organelles, including brush border, and may be equivalent to alkaline phosphatase.
• 3.3. Specific activity of the high-affinity Ca²⁺-Mg²⁺-ATPase is modestly increased following adaptation of fish to FW, but total renal high-affinity activity is greatest in the hypertrophied kidneys of FW-adapted fish and is least in kidneys of fish adapted to 200% SW.
• 4.4. High-affinity Ca²⁺-Mg²⁺-ATPase may be associated with active Ca²⁺ transport or with regulation of intracellular Ca²⁺ concentration of tubular cells.

     

Effect of jasmonic acid on Ca<Superscript>+2</Superscript> transport through the plasmalemma of potato tuber cells

The rate of Ca²⁺ accumulation in plasmalemma vesicles isolated from quiescent and sprouting potato (Solanum tuberosum L.) tubers and the effect of 10^?5–10^?10 M jasmonic acid on the accumulation of Ca⁺² in plasmalemma vesicles and its efflux were studied. It was found that potato tuber plasmalemma contains a Ca⁺²,Mg⁺²-ATPase whose activity decreases upon the transition from forced quiescence to growth. The direction of the effect of jasmonic acid on Ca⁺²,Mg⁺²-ATPase (stimulation or suppression) depends on the physiological state of tubers and the phytohormone concentration.