Glutamate-Induced Loss of Ca2+/Calmodulin-Dependent Protein Kinase II Activity in Cultured Rat Hippocampal Neurons

Abstract: The exposure of cultured rat hippocampal neurons to 500 µ M glutamate for 20 min induced a 55% decrease in the total Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity. The Ca²⁺-independent activity and autophosphorylation of CaM kinase II decreased to the same extent as the changes observed in total CaM kinase II activity, and these decreases in activities were prevented by pretreatment with MK-801, an N -methyl- d -aspartate (NMDA)-type receptor antagonist, and the removal of extracellular calcium but not by antagonists against other types of glutamate receptors and protease inhibitors. Similarly, the decrease in the CaM kinase II activity was induced by a Ca²⁺ ionophore, ionomycin. Immunoblot analysis with the anti-CaM kinase II antibody revealed a significant decrease in the amount of the enzyme in the soluble fraction, in contrast with the inverse increase in the insoluble fraction; thus, the translocation was probably induced during treatment of the cells with glutamate. These results suggest that glutamate released during brain ischemia induces a loss of CaM kinase II activity in hippocampal neurons, by stimulation of the NMDA receptor, and that inactivation of the enzyme may possibly be involved in the cascade of the glutamate neurotoxicity following brain ischemia.     

Inactivation and Subcellular Redistribution of Ca2+/ Calmodulin-Dependent Protein Kinase II Following Spinal Cord Ischemia

Abstract: Reversible spinal cord ischemia in rabbits induced a rapid loss of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) activity measured as incorporation of phosphate into exogenous substrates. About 70% of the activity was lost from the cytosolic fraction of spinal cord homogenates after 15 min of ischemia preceding irreversible paraplegia, which takes 25 min in this model. The loss of enzyme activity correlated with a loss of in situ renaturable autophosphorylation activity and a loss of CaM kinase II α and β subunits in the cytosol detected by immunoblotting. CaM kinase II activity in the particulate fraction also decreased but the protein levels of the a and β subunits increased. Thus ischemia resulted in an inactivation of CaM kinase II and a sequential or concurrent subcellular redistribution of the enzyme. However, denaturation and renaturation in situ of the CaM kinase subunits immobilized on membranes partly reversed the apparent inactivation of the enzyme in the particulate fraction. CaM kinase II activity was restored after reperfusion following short (≤25 min) durations of ischemia but not after longer durations (60 min) that result in irreversible paraplegia. The ischemia-induced inactivation of CaM kinase II, which phosphorylates proteins regulating many cellular processes, may be important in the cascade of events leading to delayed neuronal cell death.     

Calmodulin/Ca2+-ATPase interaction at the Arabidopsis thaliana plasma membrane is dependent on calmodulin isoform showing isoform-specific Ca2+ dependencies

Arabidopsis thaliana plasma membrane (PM) Ca²⁺-ATPase is a type IIB P-type ATPase, which binds calmodulin (CaM) to an autoinhibitory N-terminal domain. Here, we took advantage of the fact that PM isolated from cultured cells mainly contains At -ACA8, the first cloned A. thaliana PM Ca²⁺-ATPase, to analyse its interaction with CaM in detail. Analysis of the ability of different peptides designed from At -ACA8 N-terminus to compete with the native protein for binding of bovine brain CaM (bbCaM) showed that peptide ⁴¹I-T⁶³ had the same affinity of the native protein [apparent dissociation constant (KD) at 10 µ M free Ca²⁺ about 25 n M ], thus localizing At -ACA8 CaM-binding site within this sequence. The interaction of At -ACA8 N-terminus with bbCaM, as determined by surface plasmon resonance, was rapid, and slowly but was fully reversible. Analysis of Ca²⁺-ATPase activation as a function of the concentration of different isoforms of A. thaliana CaM showed that Ca²⁺-ATPase is activated to similar extent by bbCaM and by different isoforms of homologous CaM. However, the affinity for the divergent A. thaliana isoform CaM8 was lower than that for canonical CaM isoforms such as A. thaliana CaM2, CaM4 and CaM6 or bbCaM. The apparent KD for CaM isoforms of the native enzyme increased with the decrease of free Ca²⁺ concentration, suggesting that enzyme conformation is affected by Ca²⁺. Binding of CaM isoforms to At -ACA8 N-terminus was affected differently by free Ca²⁺ concentration, suggesting that plant CaMs may have different affinities for Ca²⁺.     

Identification and purification of the calcium‐regulated Ca2+‐ATPase from the endoplasmic reticulum of a higher plant mechanoreceptor organ

Erythrosin b, a potent inhibitor of the Ca²⁺‐ATPases and the Ca²⁺‐release channel (BCC1) in mechanosensitive tissue of Bryonia dioica Jacq., effectively suppresses a tendril's reaction to touch, suggesting that Ca²⁺‐transporters are involved in signal transduction in this organ. The Ca²⁺‐ATPase located in the endoplasmic reticulum (ER) represents a multiregulated enzyme that is stimulated by calmodulin (CaM), KCl and lysophospholipids. Limited proteolysis of ER‐membranes by trypsin results in an irreversible activation of the Ca²⁺‐ATPase and loss of the CaM sensitivity, presumably through removal of an autoinhibitory domain where CaM binds. Mild trypsination mimics the effects of CaM on V_max and the affinity for Ca²⁺ and ATP. Irrespective of a trypsin treatment, the enzyme can be additionally stimulated by KCl and lysolipids, indicating that the sites of interaction for these effectors are not located in the domain removed by the protease. CaM‐stimulated ATPase activity was purified from microsomal and ER fractions using a combination of CaM‐affinity and anion‐exchange chromatography. The isolated polypeptide was enzymatically active, showed a calcium‐dependent mobility‐shift in SDS‐PAGE from 109 kDa in the absence of Ca²⁺ to 104 kDa in the presence of 10 m M CaCl₂ and could be radiolabeled with [³⁵S]‐CaM. The characteristics of the purified enzyme remained closely similar to those of the ER‐bound Ca²⁺‐transporting activity, including the enzymatic data, CaM stimulation, and the sensitivity towards a range of inhibitors.     

Sea urchin fertilization stimulates CaM kinase-II (multifunctional [type II] Ca2+/CaM kinase) activity and association with p34cdc2

Upon fertilization, the sea urchin egg synthesizes proteins which impart a Ca²⁺ dependence to M-phase onset. A potential target of this Ca²⁺ dependence may be CaM kinase-II (the multifunctional [type II] Ca²⁺/calmodulin [CaM]-dependent protein kinase) which is necessary for nuclear envelope breakdown in fertilized sea urchin eggs. This study was intended to determine whether sea urchin CaMK-II is activated after fertilization and whether it interacts with other known M-phase regulators, such as p34^cdc2. We report that total CaMK-II activity, measured by solution assays, increases after fertilization, peaking just prior to cleavage. Interestingly, total CaMK-II activity continues to fluctuate, peaking again prior to second and third cleavage. Gel assays also reveal enhanced levels of the 56 and 62 kDa potential CaMK-II phosphoproteins after fertilization. Finally, CaMK-II activity and only the 62 kDa phosphoprotein physically associate with p34^cdc2, but again only after fertilization. These changes in CaMK-II activity and p34^cdc2-association after fertilization may ensure that Ca²⁺ signals are targeted to the M-phase machinery at the appropriate developmental times.     

Ca2+-dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat ( Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [³²P]-ATP into phosphoproteins was maximal in the presence of 1 m M CaCl₂ and 5 m M MgCl₂Ca²⁺ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin-layer electrophoresis showed that the Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca²⁺ dependent protein phosphorylktion was inhibited by phenothiazine-derived drugs. Addition of S-adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca²⁺-dependent protein phosphorylation.     

Ca2+ -dependent protein phosphorylation in germinated pollen of Nicotiana alata, an ornamental tobacco

The 40 000 g supernatant and 40 000 g pellet from extracts of germinated pollen of Nicotiana alata Link et Otto contain protein kinase activity which catalyzes the phosphorylation of histones, casein and a range of endogenous polypeptides. Phosphorylation of certain low-molecular-weight, casein-derived polypeptides is activated at low (12–37 μ M ) and partially inhibited at higher (540 μ M ) concentrations of free Ca²⁺. Histone phosphorylation is largely Ca²⁺-dependent and is activated by 540 μM free Ca²⁺. No activation of protein phosphorylation by micromolar concentrations of calmodulin is found, but phenothiazine-derived calmodulin antagonists markedly stimulate protein phosphorylation.     

Persistent Translocation of Ca2+/Calmodulin-Dependent Protein Kinase II to Synaptic Junctions in the Vulnerable Hippocampal CA1 Region Following Transient Ischemia

Abstract: The influence of brain ischemia on the subcellular distribution and activity of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) was studied in various cortical rat brain regions during and after cerebral ischemia. Total CaM kinase II immunoreactivity (IR) and calmodulin binding in the crude synaptosomal fraction of all regions studied increase but decrease in the microsomal and cytosolic fractions, indicative of a translocation of CaM kinase II to synaptosomes. The translocation of CaM kinase II to synaptic junctions occurs but not to synaptic vesicles. The translocation in neocortex and CA3/DG (dentate gyrus) is transient, whereas in the hippocampal CA1 region, it persists for at least 1 day of reperfusion. The Ca²⁺/calmodulin-dependent activity of CaM kinase II in the subsynaptosomal fractions of neocortex is persistently decreased by up to 85%, despite the increase in CaM kinase II IR. The decrease in activity is more pronounced than the decline in IR, suggesting that CaM kinase II is covalently modified in the postischemic phase. The persistent translocation of CaM kinase II in the vulnerable ischemic CA1 region indicates that a pathological process is sustained in the area after the reperfusion phase and this may be of significance for ischemic brain injury.     

Ca2+/Calmodulin-Dependent Protein Kinase II Inhibitor KN-62 Inhibits Adrenal Medullary Chromaffin Cell Functions Independent of Its Action on the Kinase

Abstract: KN-62, an inhibitor of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), inhibited significantly catecholamine secretion and tyrosine hydroxylase activity stimulated by acetylcholine in cultured bovine adrenal medullary cells. KN-62, however, showed an additional inhibitory effect on acetylcholine-induced ⁴⁵Ca²⁺ influx, which is essential for functional responses. Carbachol-stimulated ²²Na⁺ influx, veratridine-induced ²²Na⁺ influx, and 56 m M K⁺-evoked ⁴⁵Ca²⁺ influx were also attenuated by KN-62. Inhibitions by KN-62 of these ion influxes were correlated closely with those of catecholamine secretion. KN-04, which is a structural analogue of KN-62 but does not inhibit CaM kinase II activity, elicited inhibitory effects on the three kinds of stimulant-evoked ion influxes with an inhibitory potency similar to KN-62. These results suggest that KN-62 inhibits catecholamine secretion and tyrosine hydroxylase activation due to mainly its ion channel blockade on the plasma membrane rather than the inhibition of CaM kinase II activity in the cells.     

Calcium/Calmodulin-Dependent Kinase II Phosphorylates Drosophila Visual Arrestin

Abstract: Light activation of rhodopsin in the Drosophila photoreceptor induces a G protein-coupled signaling cascade that results in the influx of Ca²⁺ into the photoreceptor cells. Immediately following light activation, phosphorylation of a photoreceptor-specific protein, phosrestin I, is detected. Strong sequence similarity to mammalian arrestin and electroretinograms of phosrestin mutants suggest that phosrestin I is involved in light inactivation. We are interested in identifying the protein kinase responsible for the phosphorylation of phosrestin I to link the transmembrane signaling to the light-adaptive response. Type II Ca²⁺/calmodulin-dependent kinase is one of the major classes of protein kinases that regulate cellular responses to transmembrane signals. We show here that partially purified phosrestin I kinase activity can be immunodepleted and immunodetected with antibodies to Ca²⁺/calmodulin-dependent kinase II and that the kinase activity exhibits regulatory properties that are unique to Ca²⁺/calmodulin-dependent kinase II such as Ca²⁺ independence after autophosphorylation and inhibition by synthetic peptides containing the Ca²⁺/calmodulin-dependent kinase II autoinhibitory domain. We also show that Ca²⁺/calmodulin-dependent kinase II activity is present in Drosophila eye preparations. These results are consistent with our hypothesis that Ca²⁺/calmodulin-dependent kinase II phosphorylates phosrestin I. We suggest that Ca²⁺/calmodulin-dependent kinase II plays a regulatory role in Drosophila photoreceptor light adaptation.     

Histamine-Evoked Chromaffin Cell Scinderin Redistribution, F-Actin Disassembly, and Secretion: In the Absence of Cortical F-Actin Disassembly, an Increase in Intracellular Ca2+ Fails to Trigger Exocytosis

Abstract: Histamine is a known chromaffin cell secretagogue that induces Ca²⁺-dependent release of catecholamines. However, conflicting evidence exists as to the source of Ca²⁺ utilized in histamine-evoked secretion. Here we report that histamine-H₁ receptor activation induces redistribution of scinderin, a Ca²⁺-dependent F-actin severing protein, cortical F-actin disassembly, and catecholamine release. Histamine evoked similar patterns of distribution of scinderin and filamentous actin. The rapid responses to histamine occurred in the absence of extracellular Ca²⁺ and were triggered by release of Ca²⁺ from intracellular stores. The trigger for the release of Ca²⁺ was inositol 1,4,5-trisphosphate because U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP₃ and intracellular Ca²⁺ levels, scinderin redistribution, cortical F-actin disassembly, and catecholamine release in response to histamine. Thapsigargin, an agent known to mobilize intracellular Ca²⁺, blocked the rise in intracellular Ca²⁺ concentration, scinderin redistribution, F-actin disassembly, and catecholamine secretion in response to histamine. Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca²⁺ from intracellular stores. This suggests that protein kinase C is involved in histamine-induced responses. The results also show that in the absence of F-actin disassembly, rises in intracellular Ca²⁺ concentration are not by themselves capable of triggering catecholamine release.     

Effects of saponin extracts from Solanum elaeagnifotium fruits on ion uptake by clover seedlings

Solanum elaeagnifolium Cav. fruits contain high concentrations of steroidal saponins. Treatment of 3-day-old clover seedlings with aqueous fruit extracts modified Ca²⁺ uptake without significantly altering K⁺ and H₂PO₄⁻ uptake. The extracts increased Ca²⁺ uptake in the concentration range of 0.2 to 20 m M Ca²⁺. Uptake curves could be represented by two phases. In the lower phase (0.2-1.0 m M Ca²⁺), this change could be related to an increase in V_max. Pretreatment of seedlings with saponin extracts significantly reduced ATP-dependent Ca²⁺ uptake and Ca²⁺-dependent ATPase activity in a fraction isolated from root homogenates by centrifugation at 1500 g for 15 min. Saponins purified from S. eleagnifolium extracts by thin-layer chromatography modified in vitro the Ca²⁺-ATPase activity of this fraction, indicating that the steroid may act directly on Ca²⁺ transport across membranes.     

Ca2+ and Phospholipid-Dependent Protein Kinase Activity and Phosphorylation of Endogenous Proteins in Bovine Adrenal Medulla

Abstract: Soluble and membrane fractions of bovine adrenal medulla contain several substrates for the Ca²⁺/ phospholipid-dependent and cyclic AMP-dependent protein kinases. The phosphorylation of soluble proteins (36 and 17.7 kilodaltons) and a membrane protein (22.5 kilo-daltons) showed an absolute requirement for the presence of both Ca²⁺ and phosphatidylserine; other substrates showed less stringent phosphorylation requirements and many of these proteins were specific for each of the protein kinases. The Ca²⁺/phospholipid-dependent phosphorylation was rapid, with effects seen as early as at 30 s of incubation. Measurement of enzyme activities with histone HI as an exogenous substrate demonstrated that the Ca²⁺/phospholipid-dependent protein kinase was equally distributed between the soluble and membrane fractions whereas the cyclic AMP-dependent enzyme was predominantly membrane-bound in adrenal medulla and chromaffin cells. The activity of the soluble Ca²⁺/phos-pholipid-dependent protein kinase of adrenal medulla was found to be about 50% of the enzyme level present in rat brain, a tissue previously shown to contain a very high enzyme activity. These results suggest a prominent role for the Ca²⁺/phospholipid-dependent protein kinase in chromaffin cell function.     

Stimulation of plant plasma membrane Ca2+-ATPase activity by acidic phospholipids

The effect of phospholipids on the activity of the plasma membrane (PM) Ca²⁺-ATPase was evaluated in PM isolated from germinating radish ( Raphanus sativus L. cv. Tondo Rosso Quarantino) seeds after removal of endogenous calmodulin (CaM) by washing the PM vesicles with EDTA. Acidic phospholipids stimulated the basal Ca²⁺-ATPase activity in the following order of efficiency: phosphatidylinositol 4,5-diphosphate (PIP2)≈phosphatidylinositol 4-monophosphate>phosphatidylinositol≈phosphatidylserine≈phosphatidic acid. Neutral phospholipids as phosphatidylcholine and phosphatidylethanolamine were essentially ineffective. When the assays were performed in the presence of optimal free Ca²⁺ concentrations (10 μ M ) acidic phospholipids did not affect the Ca²⁺-ATPase activated by CaM or by a controlled trypsin treatment of the PM, which cleaved the CaM-binding domain of the enzyme. Analysis of the dependence of Ca²⁺-ATPase activity on free Ca²⁺ concentration showed that acidic phospholipids increased V_max and lowered the apparent K_m for free Ca²⁺ below the value measured upon tryptic cleavage of the CaM-binding domain; in particular, PIP2 was shown to lower the apparent K_m for free Ca²⁺ of the Ca²⁺-ATPase also in trypsin-treated PM. These results indicate that acidic phospholipids activate the plant PM Ca²⁺-ATPase through a mechanism only partially overlapping that of CaM, and thus involving a phospholipid-binding site in the Ca²⁺-ATPase distinct from the CaM-binding domain. The physiological implications of these results are discussed.     

Agents that Promote Protein Phosphorylation Inhibit the Activity of the Na+/Ca2+ Exchanger and Prolong Ca2+ Transients in Bovine Chromaffin Cells

Abstract: The Na⁺/Ca²⁺ exchanger is an important element in the maintenance of calcium homeostasis in bovine chromaffin cells. The Na⁺/Ca²⁺ exchanger from other cell types has been extensively studied, but little is known about its regulation in the cell. We have investigated the role of reversible protein phosphorylation in the activity of the Na⁺/Ca²⁺ exchanger of these cells. Cells treated with 1 m M dibutyryl cyclic AMP (dbcAMP), 1 µ M phorbol 12,13-dibutyrate, 1 µ M okadaic acid, or 100 n M calyculin A showed lowered Na⁺/Ca²⁺ exchange activity and prolonged cytosolic Ca²⁺ transients caused by depolarization. A combination of 10 n M okadaic acid and 1 µ M dbcAMP synergistically inhibited Na⁺/Ca²⁺ exchange activity. Conversely, 50 µ M 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine, a protein kinase inhibitor, enhanced Na⁺/Ca²⁺ exchange activity. Moreover, we used cyclic AMP-dependent protein kinase and calcium phospholipid-dependent protein kinase catalytic subunits to phosphorylate isolated membrane vesicles and found that the Na⁺/Ca²⁺ exchange activity was inhibited by this treatment. These results indicate that reversible protein phosphorylation modulates the activity of the Na⁺/Ca²⁺ exchanger and suggest that modulation of the exchanger may play a role in the regulation of secretion.     

Basic Protein in Brain Myelin Is Phosphorylated by Endogenous Phospholipid-Sensitive Ca2+-Dependent Protein Kinase

Abstract: Phosphorylation of myelin basic protein (MBP) in rat or rabbit brain myelin was markedly stimulated by Ca²⁺, and this reaction was not essentially augmented by exogenous phosphatidylserine or calmodulin or both. Solubilization of myelin with 0.4% Triton X-100 plus 4 m M EGTA, with or without further fractionation, showed that Ca²⁺-dependent phosphorylation of MBP required phosphatidylserine, but not calmodulin. DEAE-cellulose chromatography of solubilized myelin revealed a pronounced peak of protein kinase activity stimulated by a combination of Ca²⁺ and phosphatidylserine; a protein kinase stimulated by Ca²⁺ plus calmodulin was not detected. These findings clearly indicate an involvement of phospholipid-sensitive Ca²⁺-dependent protein kinase in phosphorylation of brain MBP, although a possible role for the calmodulin-sensitive species of Ca²⁺-dependent protein kinase in this reaction could not be excluded or established. Phosphorylation of MBP in solubilized rat myelin catalyzed by the phospholipid-sensitive enzyme was inhibited by adriamycin, palmitoylcarnitine, trifluoperazine, melittin, polymyxin B, and N -(6-aminohexyl)-5-chloro-l-naphthalenesulfonamide (W–7).     

Effect of Hypothyroidism on the Subcellular Distribution of Ca2+/Calmodulin-Stimulated Protein Kinase II in Chicken Brain During Posthatch Development

Abstract: In developing chicken brain Ca²⁺/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T₃) at 42 days. The concentrations of α- and β-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca²⁺/calmodulin- or Zn²⁺-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca²⁺/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.     

Overexpression of Ca2+/Calmodulin-Dependent Protein Kinase II Inhibits Neurite Outgrowth of PC12 Cells

Abstract: To investigate the physiological role of Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) in neuronal differentiation, we transfected the cDNA of the α subunit of mouse CaM kinase II (CaM kinase IIα) into PC12 cells and established clonal cell lines that constitutively express the transfected CaM kinase IIα gene. The expression of CaM kinase IIα was confirmed by northern blot and immunoblot analyses. Northern blot analysis showed that the γ and δ subunits of CaM kinase II are mainly expressed in PC12 cells. Treatment of the cells with ionomycin activated CaM kinase IIα through autophosphorylation and generation of the Ca²⁺/calmodulin-independent form. It is interesting that the neurite outgrowth induced by dibutyryl cyclic AMP was inhibited in these cell lines in accordance with the activities of overexpressed CaM kinase IIα. The activity of cyclic AMP-dependent protein kinase showed similar levels among these cell lines. These results suggest that CaM kinase II is involved in the modulation of the neurite outgrowth induced by activation of the cyclic AMP system.     

Effects of cold on NAD+ kinase activity in winter rape plants

Low temperature (2°C) caused an increase in the activity of NAD⁺ kinase (EC 2.7.1.23) in leaves of winter rape plants ( Brassica napus L. var. oleifera L. cv. Górezański). The enzyme activity markedly increased between day 4 and 11 of plant exposure to cold, then tended to decrease. Changes in activity of NAD⁺ kinase coincided with the previously observed changes in the levels of pyridine nucleotides, NADP(H) (U. Maciejewska and A. Kacperska, Physiol. Plant. 69: 687–691, 1987). As a result of cold treatment, Ca²⁺–calmodulin–dependent and Ca²⁺–calmodulin–independent NAD⁺ kinase activities increased to almost the same extent. It seems therefore, that the cold–induced activation of NAD⁺ kinase does not depend on the Ca²⁺–calmodulin complex.     

A calcium- and phospholipid-dependent protein kinase from rice (Oryza sativa) leaves

Protein kinases in plants have not been examined in detail, but protein phosphorylation has been shown to be essential for regulating plant growth via the signal transduction system. A Ca²⁺- and phospholipid-dependent protein kinase, possibly involved in the intracellular signal transduction system from rice leaves, was partially purified by sequential chromatography on DE52, Phenyl Superose and Superose 12. This protein kinase phosphorylated the substrate, histone III-S, in the presence of Ca²⁺ and phosphatidylserine. The apparent molecular mass of the Ca²⁺- and phosphatidylserine-dependent protein kinase (Ca²⁺/PS PK), determined by phosphorylation in SDS-polyacrylamide gel containing histone III-S, was 50 kDa. The protein kinase differed from Ca²⁺-dependent protein kinase (CDPK) in rice leaves in that Ca²⁺/PS PK showed phospholipid dependency and the molecular mass of Ca²⁺/PS PK exceeded that of CDPK. Investigations were carried out on changes in Ca²⁺/PS PK and CDPK activity in the cytosolic and membrane fractions during germination. The maximum activity of Ca²⁺/PS PK in the cytosolic fraction was observed before imbibition and that of CDPK in the membrane fraction was noted at 6 days following imbibition. Protein kinases are likely to regulate plant growth through protein phosphorylation.