Micromolar Concentrations Of Ca2 Research Articles

Mechanism of activation of protein kinase C: roles of diolein and phosphatidylserine.

We studied the roles of lipid concentration, phosphatidylserine (PS), and diolein (DG) contents, as well as Ca2+ concentration, on the partitioning of protein kinase C (PKC) between aqueous and membrane environments as well as the relationship of this partitioning to the activation of the enzyme. Physiological concentrations of 1 mol % DG increased the apparent binding constant of PKC to the 3:1 PC/PS membrane 500 times. This increase was proportional to the mol % DG. Over 50% PKC was bound to that membrane at micromolar concentrations of Ca2+ and physiologically relevant total concentration of lipid only when 1 mol % DG was included. PKC bound either to PS alone or to PS and DG was enzymatically competent; however, the rate of phosphorylation was doubled in the presence of 1 mol % diolein. The dependence of PKC binding on the mol % PS was highly sigmoidal. The Hill coefficient was in the range of 4-6, with the higher values found at the lower lipid concentrations. These results suggest that the observed apparent cooperativity is due, at least in part, to the change in dimensionality when PKC binds to the membrane.

Phosphoinositide kinases in rat heart sarcolemma: biochemical properties and regulation by calcium.

Phosphatidylinositol (PtdIns) kinase and phosphatidylinositol 4-phosphate (PtdIns4P) kinase have been studied in a purified sarcolemmal fraction isolated from rat heart. Both enzymes were Mg(2+)-dependent and their activities were maximal at 2.5 mM Mg2+ and pH 7.5. Kinetic analysis of endogenous substrate phosphorylation by ATP showed that the apparent Km and Vmax values for PtdIns kinase were 292 +/- 17 microM and 1390 +/- 80 pmol.mg-1.min-1, respectively, while the apparent Km and Vmax values for PtdIns4P kinase were 398 +/- 25 microM and 382 +/- 24 pmol.mg-1.min-1. Under normal conditions, the activity of PtdIns4P kinase was lower than that of PtdIns kinase; however, the former activity increased several fold in the presence of PtdIns4P as an exogenous substrate. The enzymatic synthesis of intramembranal PtdIns4P and phosphatidylinositol 4,5-bisphosphate (PtdIns (4,5)P2) was maximally enhanced by 0.1% Triton X-100 and inhibited by micromolar concentrations of Ca2+. Inhibition of PtdIns and PtdIns4P kinase showed IC50 values for Ca2+ of 20 and 6 microM, respectively, and was independent of either Ca(2+)-induced activation of phospholipase C and polyphosphoinositide monophosphoesterases or low ATP concentrations. The results indicate that purified rat heart sarcolemmal membranes contain a very active phosphoinositide phosphorylation system which is regulated by micromolar levels of Ca2+. The Ca2+ effect may contribute to the feedback inhibition of the receptor-activated formation of inositol 1,4,5-trisphosphate.

Characterization and identification of an epidermal-growth-factor-activated phospholipase A2.

The production of arachidonic acid (AA), which is involved in mitogenic signalling by epidermal growth factor (EGF), is most directly accomplished by the action of phospholipase A2 (PLA2). We demonstrate that EGF treatment of intact NEF cells rapidly activates a cytosolic PLA2, as measured in cell-free extracts by the release of radiolabelled AA from exogenously added 1-stearoyl-2-[1-14C]arachidonoyl phosphatidylcholine. Activation of PLA2 by EGF resulted in an enhanced Vmax. and no change in Km. The PLA2 activity was eluted in a single peak at 0.4 M-NaCl from a Mono Q anion-exchange column, and migrated with an approximate molecular mass of 70 kDa on a Superose 12 gel-filtration column. The EGF-activated PLA2 activity co-migrated with the basal PLA2 activity upon gel filtration, and persisted after partial purification, which indicates that the activation is due to a stable modification of the enzyme. The EGF-stimulated PLA2 is Ca(2+)-dependent, with maximal activity at micromolar concentrations of Ca2+, has a pH optimum at 9, associates with the particulate cell fraction in a Ca(2+)-dependent fashion, and is selective for arachidonoyl at the sn-2 position. These data demonstrate the EGF-induced activation of a PLA2, which is similar to a recently cloned high-molecular-mass AA-selective cytosolic PLA2, thus providing a link between EGF-receptor tyrosine kinase activation and AA metabolism.

Mutational analysis of the role of Glu 309 in the sarcoplasmic reticulum Ca 2+-ATPase of frog skeletal muscle

Site-specific mutagenesis was used to analyse the role of the residue, Glu 309, in the function of the Ca 2+-ATPase of frog skeletal muscle sarcoplasmic reticulum by substitution with Ala or Lys. At pH 6.0, 100 μM Ca 2+ was unable to prevent phosphorylation from P i, consistent with previous observations on the Ca 2+-ATPase of rabbit fast twitch muscle [Clarke, D.M., Loo, T.W, Inesi, G. and MacLennan, D.H. (1989) Nature 339, 476–478]. At neutral pH, however, micromolar concentrations of Ca 2+ were sufficient to inhibit phosphorylation of the Glu 309→Lys mutant from inorganic phosphate, suggesting that at least one high-affinity Ca 2+ site was relatively intact in this mutant. The Glu 309→Lys mutant was unable to form a phosphoenzyme from ATP at all Ca 2+ concentrations studied (up to 12.5 mM), whereas phosphorylation of the Glu 309→Ala mutant occurred at 12.5 mM Ca 2+, but not at Ca 2+ concentrations in the submillimolar range. Kinetic studies demonstrated a reduced rate of dephosphorylation of the E 2P intermediate in the Glu 309→Lys mutant. A less pronounced stabilization of E 2P was observed with the Glu 309→Ala mutant, suggesting a possible role of the charge at the position of Glu 309 in phosphoenzyme hydrolysis.

Calcium-sensitive, actin-related gelation of cell extracts of thermosensitive Chinese hamster lung cells capable of anchorage-independent growth.

The extent of actin-related gelation of extracts of thermosensitive Chinese hamster lung (CHL) cells capable of anchorage-independent growth was studied quantitatively by monitoring the total protein in the gel obtained by low-speed centrifugation. The gelation depended on the presence of ATP, KCl, MgCl2, and a reducing agent. Micromolar concentrations of Ca2+ and low doses of cytochalasin B inhibited the actin-related gelation of these extracts. The gelation was more sensitive to inhibition by Ca2+ and cytochalasin B when the extracts were prepared from cells cultured at the permissive rather than the nonpermissive temperature. When various ts mutants were examined, the half-maximal inhibitory dose (HMID) of Ca2+ and cytochalasin B for gelation of extracts of cells cultured at the nonpermissive temperature was between 1.25 and 2.19 times higher than that for extracts of the same cells cultured at the permissive temperature. The values of the HMID for Ca2+ and cytochalasin B changed shortly after the shift in temperature of cell cultures from the nonpermissive to permissive temperature. When cell extracts were incubated at the permissive temperature in vitro for only 15 minutes, these changes in values of HMID were also observed. Analysis of polypeptides of cell extracts and gel pellets on polyacrylamide gel electrophoresis suggested that the decrease in amount of a high-molecular-weight actin-binding protein (250 kDa) may play an essential role in the increased sensitivity to inhibition by Ca2+ and cytochalasin B of actin-related gelation in extracts of these ts mutants.

Polyphosphoinositide phospholipase C in wheat root plasma membranes. Partial purification and characterization

The effect of various detergents on polyphosphoinositide-specific phospholipase C activity in highly purified wheat root plasma membrane vesicles was examined. The plasma membrane-bound enzyme was solubilized in octylglucoside and purified 25-fold by hydroxylapatite and ion-exchange chromatography. The purified enzyme catalyzed the hydrolysis of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP 2) with specific activities of 5 and 10 μmol/min per mg protein, respectively. Phosphatidylinositol (PI) was not a substrate. Optimum activity was between pH 6–7 (PIP) and pH 6–6.5 (PIP 2). The enzyme was dependent on micromolar concentrations of Ca 2+ for activity, and millimolar Mg 2+ further increased the activity. Other divalent cations (4 mM Ca 2+, Mn 2+ and Co 2+) inhibited (PIP 2 as substrate) or enhanced (PIP as substrate) phospholipase C activity.

Caffeine- and ryanodine-sensitive Ca2+ stores of canine cerebrum and cerebellum neurons

[3H]ryanodine binding to and Ca2+ release from microsomal fractions derived from canine cerebrum (CBR) and cerebellum (CBL) were investigated. High-affinity ryanodine binding sites were detected in both cerebrum and cerebellum microsomes [CBR: maximal binding capacity (Bmax) = 446 fmol/mg protein, dissociation constant (Kd) = 9 nM, Hill coefficient (n) = 0.95; CBL: Bmax = 650, Kd = 12, n = 1.8]. Ryanodine binding in both fractions was increased by millimolar concentrations of ATP [or its nonhydrolyzable analogue beta, gamma-methyleneadenosine 5'-triphosphate (AMP-PCP)] and micromolar concentrations of Ca2+ but was decreased by micromolar concentrations of ruthenium red, similar to that found in sarcoplasmic reticulum (SR) of striated muscle. The addition of caffeine or the sudden elevation of extravesicular Ca2+ induced a rapid La(3+)-sensitive Ca2+ release from both CBR and CBL microsomal fractions with rate constants of approximately 100 s-1, as determined by stopped-flow photometry of the Ca2+ indicator arsenazo III. The release of Ca2+ was activated by either millimolar ATP or AMP-PCP, blocked by micromolar concentrations of La3+, and significantly inhibited by 50 microM ryanodine. Mg2+ and ruthenium red in millimolar and micromolar concentrations, respectively, caused only a slight inhibition of Ca2+ release. These results indicate that rapid Ca2+ release occurs from caffeine-, Ca2+- and ryanodine-sensitive Ca2+ stores in both CBR and CBL microsomal fractions.

Effect of calcium ions and inhibitors on internal NAD(P)H dehydrogenases in plant mitochondria

Both the external oxidation of NADH and NADPH in intact potato (Solanum tuberosum L. cv. Bintje) tuber mitochondria and the rotenone-insensitive internal oxidation of NADPH by inside-out submitochondrial particles were dependent on Ca2+. The stimulation was not due to increased permeability of the inner mitochondrial membrane. Neither the membrane potential nor the latencies of NAD(+)-dependent and NADP(+)-dependent malate dehydrogenases were affected by the addition of Ca2+. The pH dependence and kinetics of Ca(2+)-dependent NADPH oxidation by inside-out submitochondrial particles were studied using three different electron acceptors: O2, duroquinone and ferricyanide. Ca2+ increased the activity with all acceptors with a maximum at neutral pH and an additional minor peak at pH 5.8 with O2 and duroquinone. Without Ca2+, the activity was maximal around pH 6. The Km for NADPH was decreased fourfold with ferricyanide and duroquinone, and twofold with O2 as acceptor, upon addition of Ca2+. The Vmax was not changed with ferricyanide as acceptor, but increased twofold with both duroquinone and O2. Half-maximal stimulation of the NADPH oxidation was found at 3 microM free Ca2+ with both O2 and duroquinone as acceptors. This is the first report of a membrane-bound enzyme inside the inner mitochondrial membrane which is directly dependent on micromolar concentrations of Ca2+. Mersalyl and dicumarol, two potent inhibitors of the external NADH dehydrogenase in plant mitochondria, were found to inhibit internal rotenone-insensitive NAD(P)H oxidation, at the same concentrations and in manners very similar to their effects on the external NAD(P)H oxidation.

Effects of Inorganic Cations on Na+Adsorption to the Gill and Body Surface of Rainbow Trout,Oncorhynchus mykiss, in Dilute Solutions

Using the shortest practical exposure time (15 s) to 0.2 mmol∙L−1NaCl solutions containing22Na, over 80% of the Na+taken up by rainbow trout, Oncorhynchus mykiss, was located on the gill or body surface (adsorption). Influx to the blood (absorption) was less than 20% of the total taken up, even though Na+entry into the blood was detected in less than 30 s. Sodium adsorption to the fish surface was strongly reduced by micromolar concentrations of Ca2+or by acid in the range pH 7–5. At higher NaCl concentrations (2 mmol∙L−1in the medium, Ca2+effects on adsorption were greatly attenuated. Aluminum had a negligible effect on adsorption at pH 4. It is concluded that adsorption has an important role in presenting Na+to the apical surface of gill ion transporting cells, since conditions which inhibit the branchial influx of Na+also reduce Na+adsorption. The results suggest that adsorption is controlled by ion exchange events at gill surface ligands, and by the effects of other cations on the mobility of Na+in the bulk water. Adsorption phenomena are likely to influence ionic regulation during normal fluctuations in environmental conditions.

Effect of mucosal halides on Ca(2+)-blockable currents through the skin of Rana ridibunda

The present study deals with the interaction of mucosal anions with apical Ca(2+)-blockable cation channels of the skin of Rana ridibunda. The intracellular potential was depolarized by exposing the basolateral membranes to K2SO4 Ringer solution. The apical bathing medium consisted of nominal Ca(2+)-free K+ or Na+ solutions with SO4(2-), Cl-, Br-, or I- as the major anion. The effects of mucosal anion substitutions were studied by analyzing 1) the fluctuation in K+ current across the apical membrane driven by imposed transepithelial clamping potentials and 2) alterations of the transepithelial current (It) and conductance (Gt) as well as the Lorentzian parameters in response to anion substitution in the mucosal bathing solution. It and current noise spectra were recorded at different transepithelial potentials (Vt). A Lorentzian component was present in the power density spectrum when Vt was clamped at mucosa-positive voltages. Such noise components were never observed with mucosa-negative potentials. These findings suggest a rectifying behavior of the transepithelial cation currents. The Lorentzian noise component and the inward-oriented cation currents were depressed by the addition of micromolar concentrations of Ca2+ to the apical solutions as well as by replacing mucosal K+ or Na+ by N-methyl-D-glucamine. The Ca(2+)-blockable current and Lorentzian noise plateau (So) were gradually increased by raising Vt. Both parameters, as well as the corner frequency (fc), depended strongly on the major anion species in the apical solution; replacing mucosal SO4(2-) by one of the halides tested reduced fc and elevated So, It, and Gt considerably.

Cleavage of phosphorylase kinase and calcium-free calmodulin by HIV-1 protease

Phosphorylase kinase and calcium-free calmodulin are digested by human immunodeficiency virus-1 protease. In phosphorylase kinase, the α subunit is preferentially hydrolyzed at arg 748-val 749. The β subunit is cleaved only slowly at leu 678-pro 679, and calmodulin, the integral δ subunit of phosphorylase kinase, is not cleaved at all. However, free calmodulin in the calcium-depleted form showed to be a good substrate for the protease. Here the cleavage occurs at phe 65-pro 66 and met 71-met 72. This fast hydrolysis of free calmodulin can be blocked by micromolar concentrations of Ca 2+ or millimolar concentrations of Mg 2+.

シグナル伝達と細胞機能調節 カルシウムイオンが阻害的に作用することによってなされる生体制御機構について

The intracellular concentration of Ca2+ ([Ca2+]i) in eukaryotic cells is low under resting conditions. Membrane stimulation can produce a transient increase in [Ca2+]i from submicromolar to micromolar levels. Classically, the effects of elevated [Ca2+]i on eukaryotic cell responses are excitatory: they induce muscle contraction or activate metabolic systems. However, micromolar concentration of Ca2+ has been found to inhibit the actin-myosin-ATP interaction of the lower eukaryote Physarum polycephalum, which is the first example of an effect transiently abolished by elevation of [Ca2+]i in response to extracellular stimulation (Kohama, K.: Trends Pharmacol. Sci. 22, 433-435, 1991). The inhibitory effects on the interaction are closely related to myosin phosphorylation. The activities of kinases to phosphorylate myosin was also inhibited by Ca2+. This article describes the inhibitory mode for Ca2+ regulation of the actomyosin system in Physarum and reviews this inhibitory mode discovered subsequently in many different cell types in both plants and animals.

Marcroscopic and single-channel studies of two Ca2+ channel types in oocytes of the ascidianCiona intestinalis

Whole-cell and single-channel patch-clamp experiments were performed on unfertilized oocytes of the ascidian Ciona intestinalis to investigate the properties of two voltage-dependent Ca2+ currents found in this cell. The peak of the low threshold current (channel I) occurred at -20 mV, the peak of the high-threshold current (channel II) at +20 mV. The two currents could be distinguished by voltage dependence, kinetics of inactivation and ion selectivity. During large depolarizing voltage pulses, a transient outward current was recorded which appeared to be due to potassium efflux through channel II. When the external concentrations of Ca2+ and Mg2+ were reduced sufficiently, large inward Na currents flowed through both channels I and II. Using divalent-free solutions in cell-attached patch recordings, single-channel currents representing Na influx through channels I and II were recorded. The two types of unitary events could be distinguished on the basis of open time (channel I longer) and conductance (channel I smaller). Blocking events during channel I openings were recorded when micromolar concentrations of Ca2+ or Mg2+ were added to the patch pipette solutions. Slopes of the blocking rate constant vs. concentration gave binding constants of 6.4 X 10(6) M-1 sec-1 for Mg2+ and 4.5 X 10(8) M-1 sec-1 for Ca2+. The Ca2+ block was somewhat relieved at negative potentials, whereas the Mg2+ block was not, suggesting that Ca2+, but not Mg2+, can exit from the binding site toward the cell interior.

Porcine diacylglycerol kinase sequence has zinc finger and E–F hand motifs

Cell stimulation causes diacylglycerol kinase (DGK) to convert the second messenger diacylglycerol into phosphatidate, thus initiating the resynthesis of phosphatidylinositols and attenuating protein kinase C activity. Of the DGK isoforms so far reported, only porcine DGK from lymphocytes has been characterized in detail. Here we report the isolation and sequencing of complementary DNA clones that together cover the entire region encoding porcine DGK (relative molecular mass 80,000 (80K)). The deduced primary structure of this DGK contains the putative ATP-binding sites, two cysteine-rich zinc finger-like sequences similar to those found in protein kinase C, and two E-F hand motifs, typical of Ca2(+)-binding proteins like calmodulin. Indeed, we find that the activity of this DGK isoform is enhanced by micromolar concentrations of Ca2+ in the presence of deoxycholate or sphingosine. These properties of 80K DGK indicate that its action is probably linked with both of the second messengers diacylglycerol and inositol 1,4,5-trisphosphate.

Localization of phosphoserine residues in the a subunit of rabbit skeletal muscle phosphorylase kinase

The alpha subunit of skeletal muscle phosphorylase kinase, as isolated, carries phosphate at the serine residues 1018, 1020 and 1023. Employing the S-ethyl-cysteine method, these residues are found to be phosphorylated partially, i.e. differently phosphorylated species exist in muscle. Serine 1018 is a site which can be phosphorylated by the cyclic-AMP-dependent protein kinase. The serine residues 972, 985 and 1007 are phosphorylated by phosphorylase kinase itself when its activity is stimulated by micromolar concentrations of Ca2+. These phosphorylation sites are not identical to those found to be phosphorylated already in the enzyme as prepared from freshly excised muscle. A 'multiphosphorylation loop' uniquely present in this but not in the homologous beta subunit contains all the phosphoserine residues so far identified in the alpha subunit.

Degradation of skeletal muscle plasma membrane proteins by calpain

Observations described here provide the first demonstration that calpain (Ca2+-dependent cysteine protease) can degrade proteins of skeletal muscle plasma membranes. Frog muscle plasma membrane vesicles were incubated with calpain preparations and alterations of protein composition were revealed by sodium dodecyl sulfate polyacrylamide gel electrophoresis. Calpain II (activated by millimolar concentrations of Ca2+) was isolated from frog skeletal muscle, but the activity of calpain I (activated by micromolar concentrations of Ca2+) was lost during attempts at fractionation. Calpain I obtained from skeletal muscle and erythrocytes of rats was tested instead, and exerted effects similar to those of frog muscle calpain on the membrane proteins. All of the calpain preparations caused striking losses of a major membrane protein of molecular mass of approximately 97 kDa, designated band c, and diminution of a thinner band of approximately 200 kDa. There were concomitant increases in 83- and 77-kDa polypeptides. These effects were absolutely dependent on the presence of free Ca2+, and were completely blocked by calpastatin, a specific inhibitor of calpain action. Frog muscle calpain differed only in being relatively more active at 0 degree C than were the calpains from rat tissues. Experimental observations suggest that calpain acts at the cytoplasmic surface of the plasma membrane.

The control of glycogen metabolism in yeast

Two interconvertible forms of glycogen synthase and glycogen phosphorylase, one active (a) or the other less active (b), were predominantly present in a thermosensitive adenylate-cyclase-deficient mutant that had been preincubated at the restrictive temperature of 35 degrees C, either in the presence or in the absence of glucose. Glycogen phosphorylase was at least 20-fold less active after incubation of the cells in the presence of glucose, but this residual activity had kinetic properties identical to those of the active form of enzyme, obtained after incubation in the absence of glucose; this suggests that the b form might be completely inactive and that the low activity measured after glucose treatment must be attributed to a residual amount of phosphorylase a. By contrast, the kinetic properties of the two forms of glycogen synthase were very different. When measured in the absence of glucose 6-phosphate, the two forms of enzyme had a similar affinity for UDP-Glc but differed essentially by their Vmax. Glucose 6-phosphate had no effect on synthase a, but increased both Vmax and Km of synthase b; these effects, however, were in great part counteracted by sulfate and by inorganic phosphate, the latter also having the property of increasing the Km of the a form, without affecting Vmax. It was estimated that at physiological concentrations of substrates and effectors, synthase a was about 20-fold more active than synthase b. When an extract of cells that had been preincubated in the absence of glucose was gel-filtered and then incubated at 30 degrees C, phosphorylase was progressively fully inactivated and synthase was partially activated; these reactions were severalfold faster and, in the case of glycogen synthase, more complete in the presence of 10 mM glucose 6-phosphate. When a gel-filtered extract of cells that had been preincubated in the presence of glucose was incubated at 30 degrees C in the presence of ATP-Mg and EGTA, phosphorylase became activated and synthase was inactivated; the first of these two reactions was severalfold stimulated by micromolar concentrations of Ca2+, whereas both reactions were completely inhibited by 10 mM glucose 6-phosphate and only slightly and irregularly stimulated by cyclic AMP.

The structure of the macrophage actin skeleton.

The actin skeleton of the macrophage consists of a three-dimensional network of actin filaments and associated proteins. The organization of this multiprotein structure is regulated at several levels in cells. Receptor stimulation induces a massive actin polymerization at the cell cortex, changes in cell shape and active cellular movements. Gelsolin may have a pivotal role in restructuring the actin skeleton in response to agonist stimulation, as the activity of this potent actin-modulating protein is regulated by both Ca2+ and polyphosphoinositides. Micromolar concentrations of Ca2+ activate gelsolin to bind to the sides of actin filaments, sever, and cap the filament end. Polyphosphoinositides, in particular PIP and PIP2, release gelsolin from the filament ends. A structure-function analysis of gelsolin indicates that its N-terminal half is primarily responsible for severing actin filaments, and elucidates mechanisms by which Ca2+ and phospholipid may regulate gelsolin functions. The ultrastructure of actin filaments in the macrophage cortical cytoplasm is regulated, to a large extent, by the actin cross-linking protein, actin-binding protein (ABP) which defines filament orthogonality.

Heat-stable, calcium-inhibited cyclic nucleotide phosphodiesterase from Neurospora crassa

A heat-stable, Ca 2+-inhibited cyclic nucleotide phosphodiesterase (cPDE) (EC 3.1.4.17) from Neurospora crassa was isolated and could be separated into three aggregation forms ( ca M r values of 83 000, 151 000 and 330 000–400 000) by gel-filtration at 25° in the presence of EGTA, which suggests that the enzyme exists in monomeric, dimeric and tetrameric forms. Aggregation was temperature dependent; under similar conditions at 6°, a single form of ca M r 400 000 was present. The enzyme was not activated by calmodulin but was inhibited by micromolar concentrations of Ca 2+ and could be activated by addition of excess EGTA. Both cyclic AMP and cyclic GMP were substrates for the enzyme, and kinetic studies indicated that both bound at the same catalytic site. The enzyme had complex kinetics with two K m values. The affinity of the high K m component for both substrates was the same, whereas the low K m component had a higher affinity for cyclic GMP than for cyclic AMP. Ca 2+ was a competitive inhibitor of the high K m component, whereas inhibition of the low K m component was of the mixed type; Ca 2+ caused an increase in both K m values. The specific activity of the enzyme when cyclic AMP was used as a substrate was always higher than that with cyclic GMP, under all experimental conditions studied. A comparison of the heat-stable cPDE and cPDE activity in unheated extracts was also carried out using gel-filtration and ion-exchange chromatography. A working hypothesis is presented to account for the results.

Activity of a gelsolin-like actin modulator in rat skeletal muscle under protein catabolic conditions.

A gelsolin-like actin-modulating protein was isolated from rat skeletal muscle and characterized with respect to its interaction with actin. The protein, with a molecular mass of approx. 85 kDa, forms a stoichiometric complex with two actin molecules and is activated by micromolar concentrations of Ca2+. It effectively severs actin filaments and promotes nucleation of actin polymerization. The activity of this protein is detectable already in crude extracts by its capability to reduce the steady state viscosity of actin. Actin-modulating activities were determined in muscle extracts of rats kept under protein catabolic conditions, i.e. as generated by corticosterone treatment and starvation. In both cases we found a marked increase of modulator activity. The possibility is discussed that the increased activity of actin modulator indicates a fragmentation of actin filaments prior to the proteolytic degradation of actin.