Calcium and tip growth inNeurospora crassa

Summary We examined the ionic regulation of tip growth inNeurospora crassa by a combination of electrophysiology and confocal microscopy. To determine if transmembrane ionic fluxes are required for tip growth, we voltage clamped the membrane from –200 to +50 mV. In this voltage range, transmembrane ionic fluxes would either reverse (e.g., K⁺) or change dramatically (e.g., Ca²⁺ influx) but had no effect on hyphal growth rates. Therefore, ionic fluxes (including Ca²⁺ influx) may not be required for tip growth. However, intracellular Ca²⁺ may still play an obligatory role in tip growth. To assess this possibility, we first increased cytosolic Ca²⁺ directly by ionophoresis. Elevated Ca²⁺ induced subapical branch initiation, often multiple tips. At hyphal tips, fluorescence ratio imaging using fluo-3 and fura-red revealed a pronounced tip-high Ca²⁺ gradient within 10 m of the tip in growing hyphae which was not observed in nongrowing hyphae. Injection of the Ca²⁺ chelator 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate consistently inhibited growth concomitantly with a depletion of intracellular Ca²⁺ and dissipation of the tip-high gradient. We conclude that Ca²⁺ plays a regulatory role in tip initiation and the maintenance of tip growth. Because plasma membrane ionic fluxes do not play a role in tip growth, we suggest that the tip-high Ca²⁺ gradient is generated from intracellular Ca²⁺ stores in the ascomyceteN. crassa.Abbreviations BAPTA 1,2-bis(ortho-aminophenoxy)ethane-N,N,N,N-tetrapotassium acetate - [Ca²⁺]_i intracellular Ca²⁺ concentration - fluo-3 2,7-dichloro-6-hydroxy-3-oxo-9-xanthenyl-4-methyl-2,2-(ethylenedioxy)dianiline-N,N,N,N-tetraacetic acid     

Ca2+ triggers toxicyst discharge inDidinium nasutum

Summary A carnivorous ciliate,Didinium nasutum, captures a prey,Paramecium spp., by discharging extrusomes (i.e., toxicysts) from the proboscis. To directly examine the role of Ca²⁺ for the discharge, we injected Ca²⁺ intoD. nasutum. Injection of Ca²⁺ evoked discharge of toxicysts, if the site of the injection was the periphery region of the proboscis. After the discharge,D. nasutum, opened the proboscis and swallowed the discharged toxicysts. These observations demonstrate that a rise in cytoplasmic Ca²⁺ level is an actual cause of toxicyst discharge inD. nasutum.Abbreviations EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FITC fluorescein isothiocyanate     

Introduction of antibody (PL/IM 430) to a 100 kDa protein into permeabilised platelets inhibits intracellular sequestration of Ca2+

A monoclonal antibody (PL/IM 430), previously found to inhibit the uptake of Ca²⁺ into highly purified platelet intracellular membrane vesicles (Hack, N., Wilkinson, J. M. and Crawford, N. 1988,Biochem. J. 250, 355–361) has been introduced into saponin-permeabilised platelets. At a saponin concentration (20–25 g/ml) commensurate with total LDH release, sequestration of Ca²⁺ into intracellular non-mitochondrial stores is inhibited by the antibody (50% inhibition at 20 g/ml IgG). At higher saponin concentrations when intracellular binding of¹²⁵I-labelled mAb is maximum, inhibition of Ca²⁺ sequestration approaches 70%. The inhibition is specific, control studies with non-platelet directed mouse IgG and mAbs which immunoblot platelet antigens other than the 100 kDa protein did not affect the Ca²⁺ sequestration.No effect of the antibody were observed against IP₃-induced release of prestored Ca²⁺, either in permeabilised platelets or with isolated intracellular membrane vesicles. The mAb PL/IM 430 appears to bind only to the Ca²⁺ translocating channel protein associated with the intracellular membrane (Ca²⁺+Mg²⁺) ATPase and not to Ca²⁺ channels responsive to IP₃.Abbreviations mAb monoclonal antibody - PBS phosphate buffered saline - LDH lactate dehydrogenase     

Permeation of divalent cations through α-latrotoxin channels in lipid bilayers: steady-state current-voltage relationships

Summary -Latrotoxin, a polypeptide neurotoxin known to cause massive release of transmitter from vertebrate nerve terminals, is thought to act by forming cation-selective channels in plasma membranes. This paper describes the steady-state current carried by Ca²⁺, Sr²⁺ and Ba²⁺ through pores of -LaTx molecules incorporated in artificial bilayer membranes made of neutral lipids. Even when the solutions separated by the membrane are identical, theI-V relations rectify strongly, the current being higher when the side to which the toxin is added is positive. The polarity of the rectification is consistent with the hypothesis that the mechanism of action of the toxin is, at least in part, that of promoting inwardly directed flow of cations, and thus, accumulation of Ca²⁺ and other ions in the intracellular spaces. The dependence of theI-V characteristics on voltage and Ca²⁺ concentration is well described by a one-site, one-ion model for a channel. Three parameters of the model are deduced: the binding constant of the site for Ca²⁺,K=1.5m ^–1 (orK=7m ^–1 when activities are used instead of concentrations); the electrical distance of the site from the toxin-containing solution, =0.3; the free energy difference between the two barrier peaks, F =0.26 kT. The values of the parameters deduced by studying the channel in the presence of Ca²⁺ give theoretical curves that also fit the data with Sr²⁺ and Ba²⁺, indicating a low level of discrimination among these three cations.     

External calcium is required for activation of phospholipase C by angiotensin II in adrenal glomerulosa cells

Previous studies have shown that external calcium (Ca²⁺) is required for the effects of angiotensin II (AII) on aldosterone secretion in adrenal glomerulosa zone. Using bovine adrenal glomerulosa cells prepared by collagenase dispersion, we examined whether external Ca²⁺ is required for the activation of phospholipase C by AII. Adrenal glomerulosa cells were exposed to Ca-EGTA buffered media to provide accurate estimates of external free Ca²⁺ concentrations. Phospholipase C activation was evaluated by measurement of inositol phosphates production. At 0.1 M Ca²⁺ and less, sustained AII effects on inositol monophosphate (IP), inositol bisphosphate (IP₂) and inositol trisphosphate (IP₃) were markedly inhibited. Increasing the Ca²⁺ concentration to 50kM or greater fully restored All-induced inositol phosphates production. AII-induced increases in cytosolic Ca²⁺ measured by Quin-2 fluorescence, were diminished at lower external Ca²⁺ concentrations. Treating adrenal glomerulosa cells with Chelex-100, a strong Ca²⁺ binding resin, blocked early activation of phospholipase C by AII. Inhibition of IP₃ production was also observed when inhibitors of Ca²⁺ movement across the plasma membrane were used, viz., La²⁺, TMB-8 and nifedipine. The requirement for Ca²⁺ during AII-induced activation of phospholipase C may be explained, at least partly by a requirement for Ca²⁺ at a site between the AII receptor and Phospholipase C.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Aluminum effects on the kinetics of calcium uptake into cells of the wheat root apex

The effects of aluminum on the concentration-dependent kinetics of Ca²⁺ uptake were studied in two winter wheat (Triticum aestivum L.) cultivars, Al-tolerant Atlas 66 and Al-sensitive Scout 66. Seedlings were grown in 100 M CaCl₂ solution (pH 4.5) for 3 d. Subsequently, net Ca²⁺ fluxes in intact roots were measured using a highly sensitive technique, employing a vibrating Ca²⁺-selective microelectrode. The kinetics of Ca²⁺ uptake into cells of the root apex, for external Ca²⁺ concentrations from 20 to 300 M, were found to be quite similar for both cultivars in the absence of external Al; Ca²⁺ transport could be described by Michaelis-Menten kinetics. When roots were exposed to solutions containing levels of Al that were toxic to Al-sensitive Scout 66 but not to Atlas 66 (5 to 20 M total Al), a strong correlation was observed between Al toxicity and Al-induced inhibition of Ca²⁺ absorption by root apices. For Scout 66, exposure to Al immediately and dramatically inhibited Ca²⁺ uptake over the entire Ca²⁺ concentration range used for these experiments. Kinetic analyses of the Al-Ca interactions in Scout 66 roots were consistent with competitive inhibition of Ca²⁺ uptake by Al. For example, exposure of Scout 66 roots to increasing Al levels (from 0 to 10 M) caused the K _m for Ca²⁺ uptake to increase with each rise in Al concentration, from approx. 100 M in the absence of Al to approx. 300 M in the presence of 10 M Al, while having no effect on the V _max. The same Al exposures had little effect on the kinetics of Ca²⁺ uptake into roots of Atlas 66. The results of this study indicate that Al disruption of Ca²⁺ transport at the root apex may play an important role in the mechanisms of Al toxicity in Al-sensitive wheat cultivars, and that differential Al tolerance may be associated with the ability of Ca²⁺-transport systems in cells of the root apex to resist disruption by potentially toxic levels of Al in the soil solution.We would like to thank Dr. Lionel F. Jaffe, Director of the National Vibrating Probe Facility, Marine Biological Laboratory, Woods Hole, Mass., USA, for making his calcium-selective vibrating-mi-croelectrode system available for a portion of this work. The research presented here was supported in part by USDA/NRI Competitive Grant number 91-37100-6630 to Leon Kochian. Contribution from the USDA-ARS, U.S. Plant, Soil and Nutrition Laboratory, Cornell University, Ithaca, N.Y. This research was part of the program of the Center for Root-Soil Research, Cornell University, Ithaca, N.Y. Department of Soil, Crop and Atmosphere Science, paper No. 1741.     

Comparison of the effects of the membraneassociated Ca2+/calmodulin-dependent protein kinase on Ca2+-ATPase function in cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum

In both cardiac and slow-twitch skeletal muscle sarcoplasmic reticulum (SR) there are several systems involved in the regulation of Ca²⁺-ATPase function. These include substrate level regulation, covalent modification via phosphorylation-dephosphorylation of phospholamban by both cAMP-dependent protein kinase (PKA) and Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) as well as direct CaM kinase phosphorylation of the Ca²⁺-ATPase. Studies comparing, the effects of PKA and CaM kinase on cardiac Ca²⁺-ATPase function have yielded differing results; similar studies have not been performed in slow-twitch skeletal muscle. It has been suggested recently, however, that phospholamban is not tightly coupled to the Ca²⁺-ATPase in SR vesicles from slow-twitch skeletal muscle. Our results indicate that assay conditions strongly influence the extent of CaM kinase-dependent Ca²⁺-ATPase stimulation seen in both cardiac and slow-twitch skeletal muscle. Addition of calmodulin (0.2 M) directly to the Ca²⁺ transport assay medium results in minimal ( 112–130% of control) stimulation of Ca²⁺ uptake activity when the Ca²⁺ uptake reaction is initiated by the addition of either ATP or Ca²⁺/EGTA. On the other hand, prephosphorylation of the SR by the endogenous CaM kinase and subsequent transfer of the membranes to the Ca²⁺ transport assay medium results in stimulation of Ca²⁺ uptake activity (202% of control). These effects are observable in both cardiac and slow-twitch skeletal muscle SR. PKA stimulates Ca²⁺ uptake markedly (215% of control) when the Ca²⁺ uptake reaction is initiated by the addition of prephosphorylated SR membranes or by Ca²⁺/EGTA but minimally (130% of control) when the Ca²⁺ uptake reaction is initiated by the addition of ATP. These findings imply that (a) phospholamban is coupled to the Ca²⁺-ATPase in slow-twitch skeletal muscle SR (as in cardiac SR), and (b) the amount of Ca²⁺ uptake stimulation seen upon the addition of calmodulin or PKA depends strongly on the assay conditions employed. Our observations help to explain the wide range of effects of calmodulin or PKA addition reported in previous studies. It should be noted that, since CaM kinase is now known to phosphorylate the Ca²⁺-ATPase in addition to phospholamban, further studies are required to determine the relative contributions of phospholambanversus Ca²⁺-ATPase phosphorylation in the stimulation of Ca²⁺-ATPase function by CaM kinase. Also, earlier studies attributing all of the effects of CaM kinase stimulation of Ca²⁺ uptake and Ca²⁺-ATPase activity to phospholamban phosphorylation need to be re-examined.     

Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Veratridine triggers exocytosis in Paramecium cells by activating somatic Ca channels

Paramecium tetraurelia wild-type (7S) cells respond to 2.5 mm veratridine by immediate trichocyst exocytosis, provided [Ca²⁺] _o (extracellular Ca²⁺ concentration) is between about 10^–4 to 10^–3 m as in the culture medium. Exocytosis was analyzed by light scattering, light and electron microscopy following quenched-flow/ freeze-fracture analysis. Defined time-dependent stages occurred, i.e., from focal (10 nm) membrane fusion to resealing, all within 1 sec.Veratridine triggers exocytosis also with deciliated 7S cells and with pawn mutants (without functional ciliary Ca channels). Both chelation of Ca²⁺ _o or increasing [Ca²⁺] _o to 10^–2 m inhibit exocytotic membrane fusion. Veratridine does not release Ca²⁺ from isolated storage compartments and it is inefficient when microinjected. Substitution of Na⁺ _o for N-methylglucamine does not inhibit the trigger effect of veratridine which also cannot be mimicked by aconitine or batrachotoxin. We conclude that, in Paramecium cells, veratridine activates Ca channels (sensitive to high [Ca²⁺] _o ) in the somatic, i.e., nonciliary cell membrane and that a Ca²⁺ influx triggers exocytotic membrane fusion. The type of Ca channels involved remains to be established.We thank Dr. C. Kung (Madison, WI) for providing the pawn mutant, Drs. G. Lehle and R. Waldschütz-Schüppel (Konstanz, Germany) for their help with light scattering experiments, and Ms. E. Dassler and D. Bliestle for continuous help during the extensive photographic documentation. This work has been supported by Deutsche Forschungsgemeinschaft, Schwerpunkt Neue mikroskopische Techniken für Biologie und Medizin (grant P178/11) and SFB156/B4.     

Ca2+-binding sites and phosphatase activities in sieve element reticulum and P-protein of chick-pea phloem. A cytochemical and X-ray microanalysis survey

Summary In stem ofCicer arietinum, the loss of ribosomes attached to the rough ER cisternae during sieve element ontogeny results in the formation of sieve element reticulum (SER). By enhancing contrast of the SER, the OsFeCN postfixation/staining of material prefixed in glutaraldehyde in presence of calcium enables a good visualization of this membrane system. The pattern of staining in the SER is slightly lower when Mg²⁺ is substituted for Ca²⁺. These results support the view that the OsFeCN staining requires divalent cations and that the SER can accumulate Ca²⁺. The detection of Ca²⁺ by means of the pyroantimonate method in conjunction with X-ray microanalysis and the cytochemical localization of Ca²⁺ -ATPase in the SER cisternae provides evidence for Ca²⁺ sequestration by the SER. On the other hand, Ca²⁺-binding sites and ATPase activity are localized in P-protein. The ability to bind Ca²⁺ seems to enable the SER to function as an effective Ca²⁺ sink which may participate—with the sieve tube plasma membrane and mitochondria—in the maintenance of low Ca²⁺ concentration in phloem sap. In addition, the close association between P-protein and SER membranes exhibiting Ca²⁺-binding capabilities suggests that a Ca²⁺-mediated functional relationship may exist between the two structures. It is postulated that the SER may play a role in putative Ca²⁺ control of P-protein organization.Abbreviations SER sieve element reticulum - ER endoplasmic reticulum - P protein, phloem protein - OsFeCN method, osmium tetroxide-ferricyanide method - EDTA ethylenediamine tetraacetic acid - EGTA ethyleneglycol-bis-(-aminoethyl ether) N,N-tetraacetic acid - ATP adenosine 5-triphosphoric acid - ATPase adenosine triphosphatase - PCMB p chloromercuribenzoate - IDP inosine diphosphate     

Wound-healing motility in the green alga Ernodesmis: calcium ions and metabolic energy are required

Wounding a giant cell of the marine alga Ernodesmis verticillata (Kützing) Børgesen (Chlorophyta) induces two concomitant motility phenomena: longitudinal contraction of the protoplasm away from the wound site, and centripetal contraction of the cut end around the central vacuole. Healing is complete within 30 min of wounding. Mechanical extrusion of the protoplasm into the medium with a teasing needle is followed by contraction of the protoplasm into numerous spherical protoplasts within 60 min. Utilizing a simple defined medium, it is shown that motility is almost completely inhibited by the absence of exogenous free Ca²⁺, with 5.0 mM ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid present. This inhibition is reversible by rinsing the cells with Ca²⁺-containing medium. Similarly, extruded cytoplasm fails to exhibit motility in Ca²⁺-free medium. The threshold concentration of exogenous free Ca²⁺ is approx. 10^-7 M for wound-induced contraction. The ions Ba²⁺, Cd²⁺ and Sr²⁺ will substitute for Ca²⁺, but the rate of contraction is one-half that with Ca²⁺ present. Although darkness has no inhibitory effect, lower temperature (15°C), cyanide, or micromolar amounts of phosphorylation uncouplers reversibly slow protoplasmic motility in wounded cells and extruded cytoplasm. Carbonylcyanide m-chlorophenylhydrazone and carbonylcyanide p-trifluoromethoxyphenylhydrazone are especially potent inhibitors. These results indicate that cellular wound healing utilizes metabolic energy and requires exogenous free Ca²⁺, implying that motility in Ernodesmis is a true contractile process. Since 1.0 mM La³⁺ completely and reversibly prevents contraction, it is postulated that Ca²⁺ fluxes may actually trigger motility.Abbreviations CCCP carbonylcyanide m-chlorophenylhydrazone - DMSO dimethylsulfoxide - DNP 2,4-dinitrophenol - EGTA ethylene glycol bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FCCP carbonylcyanide p-trifluoromethoxyphenylhydrazone     

Variation in selectivity of univalent cations in slime moldPhysarum polycephalum caused by reception of polyvalent cations

Summary Specificity of reception on 11 electrolytes in the slime moldPhysarum polycephalum was investigated in the presence of polyvalent cations in media. Membrane potential and motive force of tactic movement were examined with the aid of the double chamber method, and the zeta potential at the membrane surface of the slime mold was measured by electrophoretic mobility. The results obtained are summarized as follows: (1) The presence of polyvalent cations (e.g., Ca²⁺, Mg²⁺, Sr²⁺, Ba²⁺, La³⁺, Th⁴⁺) in medium led to an increase in threshold concentration,C _th , determined from the potential measurements for Na- or Li-salts, and to a decrease inC _th for K-, Rb-, or NH₄-salts,C _th for 11 electrolytes changed discontinuously when the concentration of polyvalent cations in medium exceeded their respective thresholds. (2) TheC _th determined from chemotaxis agreed with that from the potential response both in the presence and absence of polyvalent cations. (3) Sequence of selectivity of univalent cations varied extensively in the presence of polyvalent cations. (4) Changes in the zeta potential induced by NaCl reception agreed with those in the membrane potential even in the presence of Ca²⁺ in medium. (5) TheC _th for reception of NaCl changed sharply at about 12 °C in the presence of polyvalent cations, while that for KCl was independent of the temperature.Conformational changes in surface membrane of the slime mold in response to reception of polyvalent cations were then discussed in relation to the discrimination of univalent cations.     

Studies on the transition of the cristal membrane from the orthodox to the aggregated configuration. II: Determinants of the orthodox-aggregated transition in adrenal cortex mitochondria

Bovine adrenal cortex mitochondria when isolated in a medium 0·25 M in sucrose contaminated (with calcium) have tubular cristae which are periodically expanded to spherical vesicles and contracted to flattened connecting sections. This scalloped tubular form of the cristae corresponds to the nonenergized orthodox configuration—a configuration in which the matrix space is maximally expanded. When adrenal cortex mitochondria are isolated in media in which the free calcium content is relatively low, e.g., a medium 0·25 M in sucrose and 0·1 mM in EDTA, the cristae assume the aggregated configuration—a nonenergized configuration in which the matrix space is maximally contracted. The composition of the isolation medium determined the configuration. Procedures have been described for isolating bovine adrenal cortex mitochondria (a) predominantly in the orthodox configuration, (b) predominantly in the aggregated configuration, or (c) in a mixed (11) population of configurations. The concentration of Ca²⁺ bound to the mitochondrion was found to be a determinant of the nonenergized configuration. When the level of bound Ca⁺⁺ was 20–25 moles/mg protein, the cristae of the mitochondria were entirely in the orthodox configuration. Addition of Ca²⁺ could induce the transition of cristae from the aggregated to the orthodox configuration whereas addition of Mg²⁺ could induce the transition of cristae from the orthodox to the aggregated configuration. The configurational transition could be followed by any of several methods—a change in 90° light scattering, a change in O.D._{520 m}, a change in pH, or examination by electron microscopy. The orthodox to aggregated transition is energy-independent since it proceeds even in presence of inhibitors both of electron transfer and of ATP hydrolysis. The binding of Ca²⁺ is independent of the binding of Mg²⁺; this independent binding is consistent with the opposite effects induced by Ca²⁺ and Mg²⁺, respectively. Whereas Ca²⁺ induces a proton release, a decrease in 90° light scattering and a decrease in O.D._{520 m} (when the cristae are initially in the aggregated configuration), Mg²⁺ induces equal and opposite changes (when the cristae are initially in the orthodox configuration).     

Calcium-binding and its effect on circular dichroism of plant calmodulin

The Ca²⁺-binding properties of calmodulin purified from zucchini (Cucurbita pepo L.) has been determined. A value of 3.3 mol Ca²⁺ per mol of zucchini calmodulin was measured at pH 7.5 by equilibrium chromatography. The far-and near-UV circular-dichroic spectra of the Ca²⁺-and Mg²⁺-saturated as well as from the metal-free forms of zucchini calmodulin reveal that upon Ca²⁺-binding the -helix content increases. A comparison with the spectra of vertebrate calmodulin indicates that both calmodulin have a similar secondary structure, similar Ca²⁺-induced conformational changes and the same number of Ca²⁺-binding sites.Abbreviations CAPP 10-(3-aminopropyl)-2-chloro-phenothiazine - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - EDTA ethylenediaminetetraacetic acid Dedicated to Prof. Dr. Karl Decker on the occasion of his 60th birthday     

Clomipramine and Related Structures as Inhibitors of the Skeletal Sarcoplasmic Reticulum Ca2+ Pump

The Ca²⁺-pumping activity of skeletal sarcoplasmic reticulum vesicles is half-maximallyinhibited by 120 M clomipramine, 250 M desipramine, and 500 M imipramine or trimipramine.The inhibition is attributed to the dihydrodibenzazepine moiety, since3-(dimethylamino)propionitrile, reproducing the aliphatic amine chain, has no inhibitory action. The inhibitionis shown as a marked decrease of Ca²⁺ binding at equilibrium in theabsence of ATP and asa reduction of phosphorylation of the Ca²⁺-free conformation byinorganic phosphate. Therefore,the drug effect is consistent with preferential interaction of tricyclic antidepressants withthe Ca²⁺-free conformation of the nonphosphorylated enzyme. An additional decrease in theapparent rate constant of enzyme dephosphorylation, i.e., in the release of phosphate fromATP during enzyme cycling was also noticed.     

Regulation of expression by divalent cations of a light-inducible gene in Arthrobacter photogonimos

Expression of the light-inducible (lipA) gene in Arthrobacter photogonimos is repressed by Ca²⁺ at a concentration greater than 0.1 M. Expression of lipA was induced by relatively high concentrations of Zn²⁺ Ni²⁺ or Co²⁺ in cell suspensions, an effect that was blocked by an increase in the concentration of Ca²⁺ in the medium. Zn²⁺ and other metals apparently overcame repression by Ca²⁺ by competing for a cellular binding site. Expression of lipA was also induced when the amount of free Ca²⁺ was lowered with ethylene-bis (oxyethylenenitrilo)tetraacetic acid (EGTA). Our results show that the lipA gene does not require Zn²⁺ or other divalent cation for expression and that it is regulated negatively by Ca²⁺.Accumulation of the mature product of this gene (light-inducible protein, LIP) was minimal in the presence of EGTA. Accumulation increased 10-to 20-fold when divalent cations such as Ca²⁺, Mn²⁺, Cu²⁺ or Zn²⁺ were added to cell suspensions treated with chelator. These divalent cations, which allowed the protein to achieve a protease-resistant form on the cell surface, could be substituted by protease inhibitors such as antipain, leupeptin or 1,10-phenanthroline. Our data can be explained by a biparous mechanism in which divalent cations regulate both expression of the lipA gene and accumulation of the gene product.Abbreviations LIP light-inducible protein - BAPTA 1,2-bis(o-aminophenoxy)ethanc-N,N,N,N-tetraacetic acid     

Studies on activation and inhibition of cathepsin B from buffalo liver

Cathepsin B (EC 3.4.22.1) was purified from buffalo liver. The enzyme activity against-benzoyl-dl-arginine-naphthylamme (BANA) was substantially reduced by heat (above 37C) and by nondenaturing concentrations of urea (3 M) and guanidine hydrochloride (1 M). Cathepsin B was significantly activated by 1.5 mM EDTA alone. The activation of the enzyme was further enhanced in the presence of thiol compounds, e.g., cysteine thioglycolic acid, 2,3-dimercapto-1-propenol, and dithioerythritol (DTE). The minimum concentration of the thiol compound required for optimal activation of cathepsin B was found to be lowest (0.2 mM) for DTE. The BANA hydrolyzing activity of cathepsin B was substantially reduced by Cu²⁺ (20–200M) and Ca²⁺ (30–250 mM) as well as by thiol blocking reagents, e.g., iodoacetate, 5,5-dithiobis(2-nitro-benzoic acid) (DTNB), andp-hydroxymercuribenzoate (pHMB). The enzyme activity was completely abolished when the molar ratio of the reagent: cathepsin B was close to 1. The number of free sulfhydryl groups in cathepsin B was determined to be 2 by titration against DTNB and pHMB. Modification of one free thiol group of cathepsin B resulted in complete loss of BANA hydrolyzing activity.     

Excitability of Paramecium and the significance of negative surface charges

Summary On the basis of a model presented in a previous paper (Hook and Hildebrand, 1979) the influence of external cation concentrations [K⁺]₀, [Ca²⁺]₀ and of membrane voltage V_m (i.e. the actual potential difference between the two membrane faces) on the locomotor behavior of Paramecium is theoretically analyzed. In an extended model system we discuss the negative feedback of intraciliary calcium [Ca²⁺]_i on the excitability of the ciliary membrane. While a fast blocking of Ca channels is mediated by increased [Ca²⁺]_i and accounts for the short duration of action potentials, a slow [Ca²⁺ ]_i-dependent denaturation of channel molecules is assumed to determine excitability changes of Paramecium on a long time scale.It is emphasized that the duration of long-lasting ciliary reversal which reflects the excitability is not a direct function of the cation ratio J_u [K⁺]₀/[Ca²⁺] ₀ ^1/2 but rather of the membrane potential V_m.Introduction of negative surface charges can well explain why for a series of different [K⁺]₀, [Ca²⁺]₀ but constant J_a value the excitability is unchanged despite corresponding shifts in measured membrane potentials.     

2,4,6-trinitrobenzenesulfonic acid modification of the carboxyl-terminal region (C-domain) of calreticulin

The role of the primary amino groups of lysine sidechains in Ca²⁺ binding to calreticulin was evaluated by chemical modification of the amino group with 2,4,6-trinitrobenzenesulfonic acid (TNBS). TNBS binding to calreticulin could be described by two steps: (i) a fast reaction, with low affinity, and (ii) a slow reaction with a relatively high affinity. Inclusion of Ca²⁺ and/or Mg²⁺ decreased both the amount of TNBS bound to calreticulin and the apparent affinity constant of the slower reaction. In contrast, the properties of the faster reaction for TNBS binding were not sensitive to Ca²⁺ and/or Mg²⁺. Analysis of TNBS binding to the carboxyl-terminal (C-domain) and aminoterminal (N-domain) of calreticulin revealed that theC-domain andN-domain are responsible for the slow and fast component of the TNBS binding, respectively. In keeping with this, in the presence of Ca²⁺, TNBS binding to theC-domain was significantly reduced, whereas modification of theN-domain was unaffected. TNBS modification of calreticulin significantly decreased Ca²⁺ binding to the low affinity/high capacity Ca²⁺ binding site(s) which are localized to theC-domain but had no effect on the high affinity/low capacity Ca²⁺ binding localized to theN-domain.In theC-domain of calreticulin, which contains the low affinity/high capacity Ca²⁺ binding sites, acidic residues are interspersed at regular intervals with one or more positively charged lysine and arginine residues. Our results indicate that the aminogroups of the lysine sidechains in theC-domain of calreticulin have a role in the low affinity/high capacity Ca²⁺ binding that is characteristic of this region of the protein and which is proposed to contribute significantly to the capacity of the endoplasmic reticulum Ca²⁺ store. (Mol Cell Biochem130: 19–28, 1994)Abbreviations TNBS 2,4,6-Trinitrobenzenesulfonic Acid - GST Glutathione S-Transferase - SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis - EDTA Ethylenediaminetetraacetic Acid - EGTA Ethylene Glycol bis(-aminoethylether)-N,N,N,N-tetraacetic Acid - MOPS 4-Morpholinepropanesulfonic Acid