Calcium-activated protease in the giant algaChara australis

Summary There is a protease, which is activated by Ca²⁺ (about 100 M), works at neutral pH and exists in the cytoplasm inChara australis. This protease may correspond to calpain, the calcium-activated neutral protease, which has been studied in animal cells. This is the first report showing the existence of a calcium-activated protease in plant cells.     

Formation of organic products in self-radiolyzed calcium carbonate

Summary Calcium carbonate labeled with carbon-14 was self-irradiated by means of the -decay of its carbon-14. A number of products containing one or two carbon atoms were identified by high performance liquid chromatography. Formic and oxalic acids were produced in relatively high yields, while glyoxylic, glycolic, and acetic acids, as well as formaldehyde and methanol, were formed in lower yields. These results support the suggestion that carbonates subjected to ionizing radiation could have been a source of carbon for organic synthesis on the primitive earth.     

A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

Calcium-Induced Desensitization of Acetylcholine Release from Synaptosomes or Proteoliposomes Equipped with Mediatophore, a Presynaptic Membrane Protein

A "fatigue" of acetylcholine (ACh) release is described in cholinergic synaptosomes stimulated with the calcium ionophore A23187 or gramicidin. A small conditioning calcium entry, which did not trigger a large ACh release, led to a decrease of transmitter release elicited by a second large calcium influx. This fatigue was half-maximal at approximately 30 microM external calcium and developed in a few minutes. In contrast, activation of release by calcium was very rapid and was half-maximal at approximately 0.5 mM external calcium. Activation and desensitization of release could be attributed to the recently identified presynaptic membrane protein, the "mediatophore." Proteoliposomes equipped with purified mediatophore showed a calcium-dependent activation and "fatigue" of ACh release similar to that of synaptosomes. It was found that the ionophore A23187 rapidly equilibrated internal and external calcium concentrations in proteoliposomes. Thus, the external calcium concentration gave the internal concentration required for activation or desensitization of proteoliposomal ACh release. The mediatophore showed remarkable calcium binding properties (20 sites/molecule) with a KD of 25 microM. The physiological implications of desensitization on the organization of release sites are discussed.     

Calcium Dependence of Vasoactive Intestinal Peptide-Stimulated Cyclic AMP Accumulation in Rat Hippocampal Slices

Previous work has shown that incubation of hippocampal slices in medium without added calcium markedly attenuates the capacity of vasoactive intestinal peptide (VIP) to elevate cyclic AMP levels. The present studies examined the mechanism that confers calcium dependence on VIP stimulation of cyclic AMP accumulation in hippocampal slices. Calcium dependence was apparent immediately on slice preparation and was reversible only if calcium ions were added back very early during slice incubation (within 5 min). The cyclic AMP response to VIP was not abolished by preincubating slices in 100 microM adenosine, suggesting that calcium-dependent, VIP-induced release of adenosine does not mediate VIP elevation of cyclic AMP. VIP-stimulated cyclic AMP accumulation was not decreased by agents that block calcium influx (verapamil, nifedipine, magnesium ions), or by calmodulin antagonists (trifluoperazine, calmidozolium). In fact both verapamil (100 microM) and magnesium (14 mM) augmented VIP stimulation of cyclic AMP generation. Incubation of slices with the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX) did not affect VIP activation of cyclic AMP accumulation if slices were incubated without added calcium, but MIX did enhance VIP elevation of cyclic AMP content in slices incubated with calcium. Thus calcium dependence of the cyclic AMP response to VIP in hippocampal slices is unlikely to result from VIP-dependent calcium influx, from interactions with calmodulin, or from calcium-inhibited phosphodiesterase(s).(ABSTRACT TRUNCATED AT 250 WORDS)     

Calcium-Dependent and-Independent Release of Glutamate from Synaptosomes Monitored by Continuous Fluorometry

An enzyme-linked fluorometric assay is described for the continuous monitoring of the unidirectional efflux of glutamate from guinea-pig synaptosomes. Glutamate efflux from freshly suspended, polarized synaptosomes occurs at 0.35-0.39 nmol min-1 mg of protein-1 and is not significantly affected by external Ca2+. KCl depolarization (30 mMKCl) in the absence of Ca2+ doubles this rate, whereas in the presence of Ca2+, the initial kinetics of the assay are consistent with the release in the first 5 s of 0.6 nmol mg of protein-1. The final extent of Ca2+-dependent release amounts to 1.9 nmol mg of protein-1, or 8.5% of the total intrasynaptosomal glutamate content. Preincubation of synaptosomes at 30 degrees C for 2 h before depolarization leads to a decrease in Ca2+-independent release and an increase in Ca2+-dependent release, consistent with an intrasynaptosomal relocation of the amino acid.     

Solubilization and Characterization of the Nitrendipine Receptor in Rat Brain

The nitrendipine receptor associated with the voltage-dependent calcium channel in rat brain was solubilized by detergent extraction and sonication. The detergent solution used for extraction consisted of 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 0.25% (wt/vol) polyoxyethylene 20 cetyl ether (Brij 58), and 0.025% (wt/vol) polyoxyethylene 17 cetyl stearyl ether (Lubrol WX) in the presence of 30% (wt/vol) glycerol as a stabilizer. The molecular weight of the receptor was estimated to be 1,800K by Sephacryl S-500 gel filtration and 800K by sucrose density gradient sedimentation. The equilibrium dissociation constant of [3H]nitrendipine to the solubilized receptors was 5.6 nM, which is approximately 10 times that of the membrane-bound receptor. The binding of nitrendipine to the receptor was inhibited noncompetitively by the structurally unrelated calcium channel inhibitors verapamil and prenylamine; their concentrations for 50% inhibition were both 1.0 X 10(-7) M, and they caused maximal inhibitions of 70 and 100%, respectively.     

Tubular invaginations in cerebral endothelium and their relation to smooth-surfaced cisternae in hagfish (Myxine glutinosa)

Summary The organization of vesicular profiles in the endothelium of cerebral capillaries of the hagfish, Myxine glutinosa, has been reinvestigated. Judged from random thin sections the endothelial cells contain numerous vesicles and tubules, in contrast to brain endothelia of most other vertebrates. However, three-dimensional reconstructions based on ultrathin serial sections (thickness 18 nm) showed that the profiles represent a system of irregular tubular invaginations of the cell membrane, comparable to the vesicular invaginations demonstrated in extracerebral capillary endothelia of frogs and rats. In addition, smooth-surfaced cisternae were present in close relation to the invaginations. The function of endothelial invaginations is unknown. They do not transport macromolecules, because the blood-brain barrier is practically impermeable to proteins. However, since the system of the invaginations and smooth-surfaced cisternae is structurally similar to the system of caveolae and sarcoplasmic reticulum in smooth muscle cells, a common function seems likely. It is proposed that endothelial invaginations and smooth-surfaced cisternae are involved in regulation of cytosolic Ca⁺⁺-concentration.     

The calcium requirement for functional vesicle development and nitrogen fixation by Frankia strains EAN1pec and CpI1

A calcium requirement was shown for both vesicle development and nitrogenase activity by Frankia strains EAN1_pec and CpI1. Washing cells with EGTA or EDTA inhibited both vesicle development and nitrogenase activity. The inhibition of both was reversed by the addition of calcium. A variety of agents known to affect calcium-dependent biological processes, such as a Ca-ATPase inhibitor, Ca-channel blockers, Ca-ionophores, calmodulin antagonists and the local anaesthetics, tetracaine and dibucaine, inhibited nitrogenase activity. Respiratory studies showed that a CN-insensitive respiration process occurred only under nitrogen derepressing conditions. Respiration by NH₄Cl-grown cells was completely inhibited by KCN while N₂-grown cells were inhibited by only 70%. Removal of calcium ions by EGTA or by the addition of dibucaine or tetracaine blocked the CN-insensitive respiration. This CN-insensitive respiration may be involved in protecting nitrogenase inside the vesicles from oxygen.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethyleneglycol-bis-( amino-ethyl ether) N,N¹-tetraacetic acid - GI germination inhibitor - MOPS 3-[N-morpholino] propane sulfonic acid - PCMBS p-chloromercuribenzene sulphonate - TMB 8,8-(diethylamino)-octyl-3,4,5-trimethoxybenzoate     

Calcium requirement of phytochrome-mediated fern-spore germination: No direct phytochrome-calcium interaction in the phytochrome-initiated transduction chain

Phytochrome-mediated germination of fern spores of Dryopteris paleacea Sw. was initiated by a saturating red-light (R) irradiation after 20 h of imbibition. For its realization external Ca²⁺ was required, with a threshold at a submicromolar concentration, and an optimum was reached around 10^-4 M. At concentrations 10^-1 M only a reduced response was obtained, based probably on an unspecific osmotic or ionic effect. The germination response was inhibited by La³⁺, an antagonist of Ca²⁺. From these results it is concluded that Ca²⁺ influx from the medium into the spores may be an important event in phytochrome-mediated germination. In the absence of Ca²⁺ the R-stimulated system remained capable of responding to Ca²⁺, added as late as 40 h after R. Moreover, Ca²⁺ was effective even if added after the active form of phytochrome, Pfr, had been abolished by far-red (FR) 24 h after R. Thus, the primary effect of Pfr, that initiates the transduction chain, does not require calcium. Coupling of Pfr to subsequent dark reactions has been investigated by R-FR irradiations with various dark intervals. The resulting escape kinetics were characterized by a lag phase (6 h) and half-maximal escape from FR reversibility (19 h). These kinetics were not significantly changed by the presence or absence of calcium. Thus, direct interaction of Pfr and calcium is not a step in the transduction chain initiated by the active form of photochrome.Abbreviations EGTA ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - FR far-red light - Pr red-light-absorbing form of phytochrome - Pfr far red-light-absorbing form of phytochrome - Pipes piperazine-1,4-bis(2-ethanesulfonic acid) - R red light A preliminary report of this work was presented at the XIV Int. Bot. Congr., Berlin (West), Germany, Book of Abstracts, 2-116a-5 (1987)