Action Of Mg2 Research Articles

Identification of a Mg(2+)- and guanyl nucleotide-dependent glucagon receptor cycle by use of permeabilized canine hepatocytes.

We have investigated (by use of intact and saponinpermeabilized canine hepatocytes) the roles of Mg2+ and guanyl nucleotides in regulating glucagon-receptor interactions. In contrast to intact cells, saponinpermeabilized hepatocytes bind [[125I]iodo-Tyr10]glucagon according to a single first-order process and exhibit a single apparent dissociation constant for glucagon binding during steady-state incubations. Further analysis of the permeabilized cell system demonstrated (a) the temperature-sensitive action of Mg2+ to enhance the extent and affinity of glucagon-receptor interactions at steady-state, (b) the conversion of Mg(2+)-independent hormone-receptor complexes to Mg(2+)-dependent complexes, (c) the effect of guanyl nucleotides to inhibit specifically the Mg(2+)-dependent component of glucagon-receptor interactions, (d) the more rapid association of glucagon with receptor during cell incubations occurring in the presence of guanyl nucleotides or in the absence of Mg2+, and (e) the ability of guanyl nucleotides to induce both high and low affinity states of glucagon-receptor interactions. Additional experiments identified an effect of cell incubations in the presence of glucagon to limit the subsequent binding of hormone, the ability of GDP, GTP, or guanosine-5'-3-O-(thio)triphosphate (GTP gamma S) to dissociate previously bound glucagon, and a specific requirement for GDP to re-activate the glucagon receptor for additional cycles of hormone binding. A model is presented in which (a) glucagon binds to receptor in a Mg(2+)-independent fashion, (b) glucagon-receptor complexes are converted to a Mg(2+)-dependent state, (c) guanyl nucleotide exchange initiates both an alteration in glucagon-receptor affinity and the subsequent dissociation of hormone, and (d) in the context of the intact cell, G protein-mediated hydrolysis of GTP to GDP is required to reinitialize the system.

The condensing action of Mg 2+, hexamminecobalt 3+ and spermidine 3+ on chromatin with gene regions activated by 17-β estradiol

1. 1. Hepatic vitellogenin synthesis was induced by injecting 17-β estradiol into fish. Liver nuclei were incubated with the endonuclease EcoRI at an increasing concentration of Mg 2+ (0.15–1.50mM), hexamminecobalt 3+ or spermidine 3+ (0.10–1.00 mM). Chromatin was separated into a 5000 g supernatant, S-fraction, and pellet fraction. 2. 2. The release of chromatin into the S-fraction was higher for the induced than the control chromatin. Hybridization of the vitellogenin gene retained in the pellet fraction of the controls was unaffected by the individual cations. After activation of the vitellogenin gene, Mg 2+ at its lowest concentration retained a high amount of the vitellogenin gene in the pellet fraction. The level of hybridization decreased by increasing the Mg 2+ concentration. Retention of the gene rose by adding hexamminecobalt 3+ and more so by adding spermidine 3+. 3. 3. The condensing action of spermidine 3+ was extended to the activated vitellogenin gene regions of chromatin.

Autophosphorylation of protein kinase C may require a high order of protein-phospholipid aggregates

The activation of protein kinase C (PKC) usually displays cofactor requirements that include phosphatidylserine (PS), diacylglycerol, and calcium. A complicating factor is that good exogenous substrates of PKC are polycationic proteins or peptides that form aggregates with PS in the assay. This study examined the autophosphorylation of PKC using assays with phospholipid provided in the form of vesicles or phospholipid-Triton mixed micelles. The results showed a close correlation between PKC autophosphorylation and the formation of aggregated assay components. Aggregation occurred primarily by the action of Mg2+ on phospholipids and appeared to underlie a number of major features of PKC autophosphorylation. For example, autophosphorylation required higher concentrations of PS than phosphorylation of exogenous substrates. This appeared to be the result of the different PS requirements of aggregation by divalent metal ions and cationic substrates. An unanticipated result was that aggregation of mixed micelles showed specificity for PS, high cooperativity with respect to several agents, and a requirement for calcium. These parameters were remarkably similar to those describing PKC autophosphorylation. Several major implications are evident in this study. Since the autophosphorylation assay is not a well defined system of monodisperse materials, autophosphorylation of PKC may proceed by intra- or interpeptide mechanism. The uniform correlation between aggregation and production of PKC activity suggested that kinetic parameters may represent interactions of assay components other than the enzyme. Aggregation, which appeared necessary for in vitro activation of PKC, may represent the expression of important but undefined in vivo requirements for this enzyme's function.

Evidence that the loss of the voltage-dependent Mg2+ block at the N-methyl-D-aspartate receptor underlies receptor activation during inhibition of neuronal metabolism.

In this study, the importance of the Mg2+ blockade of the N-methyl-D-aspartate (NMDA) receptor during metabolic stress was examined in embryonic day 13 chick retina. Retina exposed to mild conditions of metabolic stress (i.e., blockade of glycolysis with 1 mM iodoacetate for 30 min) underwent acute histological somal and neuritic swelling and an increase in gamma-aminobutyric acid (GABA) release into the medium. These acute signs of metabolic stress were eliminated by NMDA antagonists present during pharmacological blockade of glycolysis, occurred in the absence of a net increase in extracellular glutamate or aspartate, and were not affected by the presence or absence of Ca2+ in the incubation medium. One possible explanation for the activation of NMDA receptors in the absence of an increase in extracellular ligand is that NMDA sensitivity during metabolic stress may be governed at the receptor level. Depolarization of membrane potential during metabolic stress may result in the loss of the Mg2+ blockade from the NMDA receptor channel, resulting in an increased potency for glutamate. To test this, the dose-response characteristics for NMDA, glutamate, and kainate in the presence or absence of extracellular Mg2+ and the effects of Mg2+ on metabolic inhibition were examined. The potency for NMDA- or glutamate-mediated acute toxicity was enhanced two- to fivefold in the absence of Mg2+. Omission of Mg2+ greatly decreased the minimal concentration of agonist needed to produce acute excitotoxicity; 25 versus 5 microM for NMDA and 300 versus 10 microM for glutamate in 1.2 or zero Mg2+, respectively. Elevating external Mg2+ to 20 mM completely protected against NMDA-mediated acute toxic effects. In contrast, varying external Mg2+ had no effect on kainate-induced toxicity. Acute toxicity caused by inhibition of metabolism was not potentiated in the absence of Mg2+ but was attenuated by elevating extracellular Mg2+. The protective effect of Mg2+ during metabolic inhibition was not additive with NMDA antagonists, suggesting that the action of Mg2+ was at the level of the NMDA receptor. These findings are consistent with the hypothesis that the Mg2+ block is lifted during metabolic inhibition and may be the primary event resulting in NMDA receptor activation.

NMDA receptors in invertebrates

The concept of an NMDA receptor has evolved over some thirty years. The initial impetus was the need to better distinguish “glutamate preferring” from “aspartate preferring” receptors among neurons of the mammalian spinal cord (Curtis and Watkins, 1963; McCulloch et al., 1974). This search gave rise to substantial evidence for a diversity of ionotropic excitatory amino acid (EAA) receptors with quantitatively different sensitivities for various EAA analogues. The properties of these receptors are reviewed in Mayer and Westbrook (1987), Shinozaki (1988), Cotman et al. (1988), Sansom and Usher-wood (1990) and Watkins (1989). The vertebrate receptors are named after the preferred selective agonist. The most important distinction is between the NMDA and non-NMDA (quisqualate and kinate) receptors. Although both receptor types are highly sensitive to glutamate and mediate nonspecific cation conductances, the NMDA receptor is particularly significant because the ion channel is voltage dependent and highly permeable to Ca*+ (Mayer and Westbrook, 1987). To our knowledge there has never been a major effort to determine whether any invertebrate species possesses an NMDA receptor. Rather, the modest data available have been produced in the context of structure-activity studies to better characterize particular glutamate and/or aspartate receptors. Although there are some gross similarities between some vertebrate and invertebrate EAA receptors, significant differences are invariably found in the details. Altogether we have identified 21 studies in which NMDA action was tested on an invertebrate glutamate/aspartate receptor. Although the overwhelming majority of results are negative, a few positive indications emerge. These are summarized in Table 1. In the foregoing these results are considered relative to the vertebrate NMDA receptor and the EAA receptors of the invertebrate species tested. Operationally, the NMDA receptor is characterized by the effects of selective agonists and antagonists, the action of modulators (Mg’+, glycine), voltage dependence in the presence of Mg*+, a large single channel conductance and ionic permeability. When agonist affinities are measured by the capacity to displace selective antagonists from the NMDA receptor, L-glutamate is the most potent (Watkins, 1989; Oliverman and Watkins, 1989). NMDA, ibotenate and L-homocysteate are strong competitors for the NMDA binding site while L-quisqualate, L-kainate and AMPA are not. D-a-aminoadipate (Davies and Watkins, 1979) was the first competitive antagonist discovered, to exhibit a preference for the NMDA receptor. Subsequently, more potent blockers were developed in which a phosphonate group is substituted for the w-carboxyl group and additional methylene groups extend the molecule length (Evans et al., 1982). The most effective of these are 2-amino-5phosphonopentanoate (APV or D-AP5) and 2-amino-7-phosphonoheptanoate (D-AP7). Recently these have been superseded by higher affinity antagonists derived from piperdine (Olverman and Watkins, 1989). Activation of the NMDA receptor requires glytine (K, N 200 nM), a requirement normally satisfied in vivo by the interstitial fluid (Johnson and Ascher, 1987; Thomson et al., 1989). The receptor’s glycine binding site is insensitive to strychnine but is competitively blocked by kynurenate, 7-Cl-kynurenate and HA-966 (Ascher and Johnson, 1989). M$+(Kd = 72 nM) blocks current flow through the open NMDA receptor channel and imparts a voltage-dependence to channel activation (Davies and Watkins, 1977; Ault et al., 1980; Ascher and Nowak, 1988). At 1 .O mM Mg*+, membrane depolarizations to above 30 mV are sufficient to overcome the Mg*+ block. Co*+ and Mn*+ can mimic the action of Mg*+ but Ba*+, Ca*+ and Cd*+ do not. In physiological salines the single channel conductance is about 50 pS which is substantially larger than that of the non-NMDA receptor channels. Elevated external Ca*+ reduces the single channel conductance at negative membrane potentials from 56 pS (in 0 Caz+) to 15 pS at 100 mM Ca*+ (Ascher and Nowak, 1988). The NMDA channel is permeable to Na + , K + and Ca*+ and the Ca*+ permeability is unusually high; Pc,Z+/PN,+ * 1.0 (Ascher and Johnson, 1989). In normal physiological salines the reversal potential of the NMDA-elicited current is -0 mV. In many neurons, NMDA receptors are interspersed among non-NMDA receptors (Jahr and Stevens, 1987; Cull-Candy and Usowicz, 1987) and the voltage-sensitivity and slower kinetics of the NMDA activated channels are thought to have a

The effect of Mg 2+ on the Ca 2+ binding to troponin C in rabbit fast skeletal myofibrils

The effect of Mg 2+ on the Ca 2+ binding to rabbit fast skeletal troponin C and the Ca 2+ dependence of myofibrillar ATPase activity was studied in the physiological state where troponin C was incorporated into myofibrils. The Ca 2+ binding to troponin C in myofibrils was measured directly by 45Ca using the CDTA-treated myofibrils as previously reported (Morimoto, S. and Ohtsuki, I. (1989) J. Biochem. 105, 435–439). It was found that the Ca 2+ binding to the low and high affinity sites of troponin C in myofibrils was affected by Mg 2+ competitively and the Ca 2+- and Mg 2+-binding constants were 6.20 · 10 6 and 1.94 · 10 2 M −1, respectively, for the low affinity sites, and 1.58 · 10 8 and 1.33 · 10 3 M −1, respectively, for the high affinity sites. The Ca 2+ dependence of myofibrillar ATPase was also affected by Mg 2+, with the apparent Ca 2+- and Mg 2+-binding constants of 1.46 · 10 6 and 2.76 · 10 2 M −1, respectively, suggesting that the myofibrillar ATPase was modulated through a competitive action of Mg 2+ on Ca 2+ binding to the low affinity sites, though the Ca 2+ binding to the low affinity sites was not simply related to the myofibrillar ATPase.

Divalent cation-sensitive antagonist binding to heart muscarinic receptors

1. 1. The binding of (−)[ 3H]quinuclidinylbenzilate (QNB), a potent muscarinic antagonist, to cardiac muscarinic receptors was examined in washed particles and microsomes isolated from rat heart atria. 2. 2. Addition of divalent cations (Mg 2+, Mn 2+ and Ca 2+) to the binding assays increased (6 to 8-fold) the antagonist binding to microsomes but exerted little or only weak (up to 35%) stimulation of the antagonist binding to washed particles. 3. 3. Mg 2+ ions present only in the dilution and washing buffer prior to rapid filtration of the binding reaction were capable of markedly increasing the antagonist binding to microsomes but not to washed particles. 4. 4. Such differential action of Mg 2+ on antagonist binding to subcellular fractions was seen when fractions were either isolated and/or incubated in media containing either 10 or 50 mM imidazole-Cl, Tris-phosphate or Tris-Cl buffer, each at pH 7.5. While the modulatory effect of Mg 2+ was not seen in Na-K-phosphate buffer, pH 7.5, the antagonist binding per se was much higher in Na-K-phosphate buffer compared to other buffers.

Fructose-1,6-bisphosphatase in mantle of the sea mussel Mytilus galloprovincialis Lmk—I. Purification and ion requirements

1. 1. The enzyme fructose-1,6-bisphosphatase was purified from the mantle of the sea mussel Mytilus galloprovincialis Lmk. The purified enzyme showed a single band in SDS-polyacrylamide gel electrophoresis. The mol. wt and subunit mol. wt of the enzyme were 105,000 and 27,000, respectively. 2. 2. Divalent cations are essential for the enzyme activity. In the absence of chelating agents, FBPase 1 exhibits hyperbolic kinetics with respect to Mn 2+, Zn 2+ and Mg 2+. The K m for Mg 2+ is lower than the physiological concentration of cation in the tissue, whereas its K m for Mn 2+ and Zn 2+ is greater than the respective in vivo concentrations. 3. 3. The joint action of Mg 2+ and Zn 2+ increases the affinity of the enzyme for the substrate Fru-1,6-P 2, though V max is reduced. 4. 4. Na + strongly inhibits the enzyme even at very low concentrations. K + has no effect whatsoever.

Study on normal and otosclerotic bone cell cultures: An advance in understanding the pathogenesis of otosclerosis

The authors first reviewed the main theories concerning the pathogenesis of otosclerosis and studied the morphologic and functional characteristics of cell cultures derived from normal and otosclerotic bones. Light transmission and scanning electron microscopy did not permit definite identification of the cultured cells as predominantly osteoblasts, nor did these techniques show significant differences between cultured cells derived from normal and pathologic bone. Functional tests of the cell cultures proved more interesting. First, the bony nature of the cultured cells was demonstrated by studying the intracellular 45Ca++ uptake after stimulation with calcitonin and dybutryl-cAMP. Second, cell cultures derived from otosclerotic bone behaved differently from those derived from normal bone. Their peak uptake of calcium appeared later, and post-stimulatory values were higher, suggesting that cells derived from otosclerotic bone store a greater quantity of 45Ca++. Furthermore, after stimulation with calcitonin and propranolol, we observed an inhibition of the calcium uptake and decreased intracellular cAMP levels in normal bone cell cultures. In contrast, the cell cultures derived from otosclerotic bone exhibited an initial inhibition of calcium absorption followed by massive calcium penetration. The response of adenylate cyclase to the action of Mg++, Ca++, and F- ions was evaluated in cultures derived from normal bone, otosclerotic bone, and normal skin fibroblasts. The resulting data show that activation due to Mg++ is much lower in cultured cells derived from otosclerotic bone than in those from either normal bone or skin fibroblasts. No significant differences were found after Ca++ inhibition in any of the cell cultures. Moreover, in cell cultures derived from normal bone, F- ions induced a strong activation that was lower than the levels observed in cultures of otosclerotic bone or in normal fibroblasts. We hypothesize that an alteration at the calcitonin receptor site is responsible for the difference in calcium uptake and cAMP levels observed in the cells derived from otosclerotic bone as compared to those cultured from normal cells.

Acetaldehyde oxidation in rat liver mitochondria, action of Mg2+, ATP and rotenone on acetaldehyde oxidation in intact mitochondria, and on some purified aldehyde dehydrogenase isozymes.

In intact rat liver mitochondria acetaldehyde is oxidized by three functionally distinct dehydrogenase systems. Two of these reduce intramitochondrial nicotinamide adenine dinucleotide (NAD): one is operative with micromolar acetaldehyde concentrations and is stimulated by Mg2+, the other is operative with millimolar acetaldehyde concentrations and is stimulated by adenosine 5'-triphosphate (ATP). The third system reduces added NAD and is stimulated by rotenone. Connected to these systems, three aldehyde dehydrogenase isozymes (ALDH) have been purified: a low-Km ALDH activated by Mg2+, a high-Km ALDH activated by ATP and Mg2+, a high-Km ALDH activated by rotenone. The properties of some isozymes are affected by detergents. Thus, deoxycholate augments the stimulation of low-Km isozyme by Mg2+ and confers sensitivity to Mg2+ and ATP on one of the high-Km isozymes. A fourth isozyme has been purified. Its affinity for acetaldehyde is so low that it is very unlikely that acetaldehyde is the physiological substrate.

Electrophysiologic action of Mg2++ and its clinical application

Electrophysiologic action of Mg<SUP>2+</SUP>+ and its clinical application

Rapid release of [3H]dopamine from median eminence and striatal synaptosomes.

Release of preaccumulated, tritium-labeled dopamine ([3H]DA) from preparations of isolated nerve terminals (synaptosomes) of rat median eminence (ME) and corpus striatum (CS) was examined over short time intervals (1-20 s). In both preparations, basal efflux of [3H]DA was linear with time. Depolarization with high K+ resulted in an initial rapid release of [3H]DA which stabilized by 20 s, whereas veratridine elicited an increased rate of release over basal levels that was linear over the first 20 s. The calculated rate constants of release for both the initial phase of K+- and the veratridine-stimulated release were approximately threefold greater in CS than in ME synaptosomes. The major component of the high K+-induced release of [3H]DA from both synaptosome preparations increased as a graded function of [Ca2+]o. However, a smaller component, independent of external Ca2+, existed in both ME and CS synaptosomes. Increasing the [Mg2+] in the external solution resulted in a right shift of both the [K+]o and the [Ca2+]o dose-response curves, consistent with actions of Mg2+ on screening surface membrane charges and blocking voltage-dependent Ca2+ channels. In all studies, steady-state uptake of the [3H]DA was about twofold greater into CS than into ME synaptosomes. Moreover, the fraction of incorporated [3H]DA released by stimulation from the CS was much greater than that released from ME synaptosomes. These data are consistent with differences between these two types of dopaminergic terminals with respect to packaging and/or distribution of the accumulated neurotransmitter in intraneuronal pools, as well as marked differences in the apparent kinetics of DA release.

Characterization of reacted ohmic contacts to GaAs

In the calculation of the true value of specific resistivity of reacted ohmic contacts, a modification in the conductivity of the semiconductor that may occur below the contact must be considered. Contacts to GaAs are discussed. A substantial decrease in the sheet resistance of p-type GaAs is measured for Pt-reacted contacts in which a single thin layer of Mg is interposed. It is pointed out that this lowering, which is attributed to the doping action of Mg, if not taken into account, can lead to serious errors in the estimation of specific contact resistivity.

The combined action of Mg 2+ and Cl − ions in cement pastes

In order to study the phenomenon of seawater attack on hydrated cement components, we focused our interest on the combined action of Mg 2+ and Cl − ions on hydrated cement pastes. Thus, cement pastes were prepared from portland cement and its mixture with 30% pozzolan (Santorin Earth). These pastes were cured in baths of varied concentrations of Mg 2+ and Cl − ions and stored in 18 ± 2° C. The hydration phenomena were studied in these cement pastes, by XRD and SEM.

Quantitative studies on some antagonists of N-methyl D-aspartate in slices of rat cerebral cortex.

Coronal sections of rat brain (500 micron thick) were trimmed to form 'wedges' of tissue consisting of cerebral cortex and corpus callosum. When these slices were placed in a two-compartment bath, the cortical tissue could be depolarized, relative to the corpus callosum, by superfusions of high K+, or by amino acids such as L-glutamate, L-aspartate, quisqualate, kainate and N-methyl D-aspartate (NMDA). Responses to NMDA were reduced by magnesium ions, by the organic antagonists (-)-2-amino 5-phosphonovalerate (APV) and 2-amino 7-phosphonoheptanoate (APH), and by the dissociative anaesthetic ketamine. In this preparation, all these antagonists shifted the NMDA dose-response curve to the right in a parallel manner. A Schild plot for Mg2+ had a slope significantly less than unity, indicative of a non-competitive action, whilst Schild plots for (-)-APV, APH and ketamine appeared linear and had slopes of approximately 1. Analysis of the results of combination experiments suggested that the presumed competitive antagonists, (-)-APV and APH, share a common site of action as NMDA antagonists, and that this site is distinct from that at which ketamine exerts its action. The action of Mg2+ is clearly different from that of either (-)-APV or ketamine. It is concluded that ketamine is a non-competitive antagonist of NMDA and may act at an allosteric site on the NMDA receptor complex to influence its function.

Aluminium—thiourea inhibitors of mild steel corrosion in high specific salinity water. Compatibility with calcium, magnesium and ferrous ions at various temperatures

Aluminium—thiourea (Althi) inhibitors were found to provide good inhibition of mild steel in water of a high specific salinity (1.0% Na 2SO 4) considered characteristic for recirculating water of zero-discharge cooling systems in steam plants. As in a real cooling system this water would contain calcium, magnesium and ferrous ions, a successful application of Althi inhibitors would depend on their compatibility with these ions. In order to check this compatibility the inhibitive efficiencies of two representative Althi inhibitor formulations were measured in the presence of Ca 2+ (as CaSO 4), Mg 2+ (as MgSO 4) and Fe 2+ (as FeSO 4 and FeCl 2) using the weight loss method according to NACE Standard TM 01-69 at 20–40°C. It was found that the inhibitive efficiencies of the tested formulations sharply decreased in the presence of Ca 2+ while the action of Mg 2+ and Fe 2+ does not appear to have a critical influence on the inhibitive properties of the same formulations. This finding showed that the application of Althi inhibitors in zero-discharge cooling systems can be successful if a complete removal of Ca 2+ from both makeup and recirculating water would be foreseen. Due to the high cost of this removal, in some cases the application of mild steel in conjunction with Althi inhibitors cannot be justified in zero-discharge cooling systems.

Regulation of hormone-responsive Sertoli cell adenylyl cyclase in a cell-free system

Homologous desensitization of either FSH- or isoproterenol-responsive adenylyl cyclases in Sertoli cell membranes can be achieved in a cell-free system. Incubation of membrane particles from cultured immature Sertoli cells with either FSH or isoproterenol resulted in a time- and concentration-dependent loss of subsequent adenylyl cyclase response to the homologous hormone. Half-maximal refractoriness was achieved within 20–30 min of incubation. The concentration of FSH required to obtain half-maximal loss of adenylyl cyclase response (400 ng/ml) was in the same order of magnitude as the apparent K m for FSH-stimulated adenylyl cyclase activity (300 ng/ml). Hormone-induced homologous desensitization was dependent on the presence of both ATP and Mg 2+. Increasing concentrations of ATP in the presence of FSH caused a concentration-dependent loss of subsequent response to the homologous hormone. Halfmaximal desensitization was achieved at an ATP concentration of 0.2 mM. Increasing concentrations of Mg 2+ in the presence of either FSH or isoproterenol caused desensitization for the homologous hormone. The concentration of Mg 2+ giving half-maximal effect was approximately 5 mM in excess of ATP and EDTA. However, higher concentrations of free Mg 2+, in the absence of hormone, caused desensitization of both FSH- and isoproterenol-sensitive adenylyl cyclase with half-maximal effect at approximately 30 mM. Hormone-specific desensitization was obtained in the presence of GTP. However, when GTP was substituted with the non-hydrolysable analogue GMPP(NH)P the hormonal activation remained constant throughout 90 min of incubation. The hormone-induced refractoriness was probably not due to cAMP, since all incubations were performed in the presence of an excess of cAMP (1 mM) to block phosphodiesterase (PDE) activity. However, the finding that both ATP and Mg 2+ were required for hormone-induced desensitization suggests that the process may be mediated via a cAMP-independent phosphorylation reaction. Furthermore, the hormone appears to accelerate desensitization of the adenylyl cyclase system by increasing its sensitivity to the actions of Mg 2+.

Modulation of myosin filament conformation by physiological levels of divalent cation

The effect of divalent cation, in particular Mg 2+, on the properties of synthetic myosin filaments has been studied; and substantial changes in sedimentation and light scattering demonstrated to occur in the physiological range of free Mg 2+. A pre-requisite for these studies has been the definition of a modified method for the preparation of myosin in highly monodisperse filament form, rigorously free from thin filament proteins. The sedimentation coefficient at infinite dilution shows a large increase (169 S to 193 S) in the range 0.2 m m to 3 m m in Mg 2+. The anomalous frictional increment found for these filaments is thus substantially reduced. The concentration dependence ( k s), however, shows a substantial decrease (470 ml/g to 334 ml/g) in the same range of Mg 2+, and the calculated filament molecular weight is virtually unchanged. A change in the filament conformation is thus indicated. This is confirmed by an analysis of the turbidity of the filaments in the centrifuge cell, which shows a similar increase in response to the addition of Mg 2+. These effects have been found to be independent of ionic strength (0.07 to 0.11), pH (7.0 to 7.6), the presence of MgATP or the presence of low levels of Ca 2+ (~100 μ m). These effects studied indicate the action of Mg 2+ through a low-affinity binding site ( K d ~ 1.5 × 10 −3m). We consider that a significant change in crossbridge conformation can adequately explain these changes in physical and enzymic properties. A provisional model is proposed, in which the effect of Mg 2+ is to bring the crossbridges into closer proximity to the filament shaft.

Effects of external calcium reduction on biphasic potassium contractures and action of divalent cations on the calcium reduction in frog single twitch muscle fibers.

The effects of external Ca2+ reduction on the biphasic potassium (K) contractures and the action of divalent cations under conditions of the Ca2+ reduction were examined in detail, using frog single twitch muscle fibers. The peak tension of the initial component of 80 mM K+ contractures was greatly potentiated by exposing the fiber to low Ca2+ solution for 30 sec, and the degree of this potentiation was decreased with increasing the exposing time. In contrast, the peak tension of the secondary component was rapidly inhibited by Ca2+ reduction. The potentiation of the initial component was removed by 3 mM Mg2+ or 0.5 mM Ni2+. The inhibition of the secondary component, especially the shortening of its time course, was reversed partially by 3 mM Mg2+ and almost completely by 0.5 mM Ni2+. The difference between the inhibitory effect of 10 mM Mg2+ or 1-3 mM Ni2+ on the initial component and that on the secondary component was also demonstrated. The tension development of the secondary component was completely inhibited by external Ca2+ reduction for 20 min, but it was observed in the presence of 3 mM Mg2+, although its time course was shorter. These results indicate that the actions of Mg2+ and Ni2+ on the initial component differ from those on the secondary component and suggest the possible mechanisms of the actions of these divalent cations on the biphasic K contractures.

Dogfish pressor response to potassium blocked by magnesium and phentolamine.

In vivo infusion of MgCl2 blocks the dogfish pressor response to K+. This action of Mg2+ was contrasted to phentolamine in in vivo and in vitro experiments. Mg2+ blocks the spontaneous release of catecholamines from dogfish chromaffin tissue but does not alter the norepinephrine-induced contraction of the isolated dogfish artery. In vivo infusion of Mg2+ causes a significant decrease in resting catecholamine levels and diminishes the catecholamine release caused by K+ challenge. Both Mg2+ and phentolamine block the pressor action of K+, Mg2+ by preventing the K+-induced release of catecholamines and phentolamine by preventing the circulating catecholamines from interacting with alpha-adrenergic receptor sites.