Ca(2+)-dependent in vivo protein phosphorylation and encystment induction in the ciliated protozoan Colpoda cucullus

Encystment induction of Colpoda cucullus is promoted by an increase in external Ca²⁺ and overpopulation of Colpoda vegetative cells. Using phos-tag detection assays, the present study revealed that the in vivo phosphorylation level in several proteins [33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa, 49 kDa, etc.] was raised when the vegetative cells were stimulated by overpopulation to encyst in a medium containing 0.1 mM Ca²⁺ or without the addition of Ca²⁺. Both overpopulation-mediated encystment induction and protein phosphorylation were suppressed by the addition of EGTA. Ca²⁺/overpopulation-stimulated encystment induction and protein phosphorylation were also suppressed by the addition of BAPTA-AM. These results suggest that the Ca²⁺ inflow promoted by cell-to-cell stimulation due to overpopulation may activate signaling pathways involving protein phosphorylation and encystment induction. In the presence of cAMP-AM, the phosphorylation levels of 33 kDa, 37 kDa, 37.5 kDa, 43 kDa, 47 kDa and 49 kDa proteins were enhanced, and encystment induction was promoted. Enzyme immunoassays (EIAs) showed that intracellular cAMP concentration was raised prior to encystment when the cells were stimulated by overpopulation. These results suggest that cAMP/PKA-dependent protein phosphorylation, which is an event on Ca²⁺-triggered signaling pathways, may be involved in encystment induction.     

Preparation of Chitooligosaccharide-alditols by Hydrogenation with a Ruthenium Catalyst

The existence of glycosylated DNA-binding proteins was demonstrated in a whole cell extract from a filamentous fungus, Aspergillus oryzae. The proteins were specifically eluted from a DNA-cellulose column by the eluate containing shared double-stranded DNA and were detected by wheat germ agglutinin (WGA)-probing. The apparent molecular masses of these proteins on SDS-PAGE were 140 kDa, 115 kDa, 105kDa, 68 kDa, and 60 kDa. The labeling of the proteins by uridine 5′-diphosphate(UDP)-[¹⁴C]galactose using galactosyltransferase showed the same electrophoretic pattern with the WGA-probing. The [¹⁴C]- galactose-labeled saccharides were released from the proteins by mild-base treatment but not by N-glycopeptidase F digestion, indicating the O-glycosidic linkage of the saccharide chain attachment to proteins. The [¹⁴C]galactose-labeled saccharides co-migrated with galactose-(β1 → 4)-N-acetylglucosaminitoI on a silica gel plate. Thus, it was seen that several proteins which had the DNA-binding activity were modified by N-acetylglucosamine monosaccharide through an O-glycosidic linkage in A. oryzae.     

Studies on Two Closely Related Species of Octocorallians: Biochemical and Molecular Characteristics of the Organic Matrices of Endoskeletal Sclerites

Two species of alcyonarian corals, Lobophytum crassum and Sinularia polydactyla, are closely related to each other. It is reported that the calcified organic substances in the skeletons of both contain a protein–polysaccharide complex playing a key role in the regulation of biocalcification. However, information on the matrix proteins of endoskeletal sclerite has been lacking. Hence we studied the proteinaceous organic matrices of sclerites for both species, to analyze the sequences and the functional properties of the proteins present. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed four bands of proteins with apparent molecular masses of 102, 67, 48, and 37 kDa for L. crassum and seven bands of 109, 83, 70, 63, 41, 30, and 22 kDa for S. polydactyla. A major protein band of about 67 kDa in L. crassum and two bands of proteins of about 70 and 63 kDa in S. polydactyla yielded N-terminal amino acid sequences. Periodic acid-Schiff staining indicated that the 67-kDa protein in L. crassum, and 83- and 63-kDa proteins in S. polydactyla were glycosylated. For detection of calcium binding proteins, a Ca²⁺ overlay analysis was conducted in the extract via ⁴⁵Ca autoradiography. The 102- and 67-kDa calcium binding proteins in L. crassum, and the 109- and 63-kDa Ca²⁺ binding proteins in S. polydactyla were found to be radioactive. An assay for carbonic anhydrase (CA), which is thought to play an important role in the process of calcification, revealed specific activities. Newly derived protein sequences were subjected to standard sequence analysis involving identification of similarities to other proteins in databases. The significantly different protein expressions and compositional analysis of sequences between two species were demonstrated.     

Soluble proteins control growth of skeleton crystals in three coralline demosponges

Summary Calcified demosponges (coralline sponges, sclero-sponges), the first metazoa producing a carbonate skeleton, used to be important reef building organisms in the past. The relatives of this group investigated here,Spirastrella (Acanthochaetetes) wellsi, Astrosclera willeyana andVaceletia cf.crypta, are restricted to cryptic niches of modern Pacific coral reefs and may be considered as “living fossils’. They are characterized by a basic biologically controlled metazoan biomineralization process. Each of the investigated taxa forms its calcareous basal skeleton in a highly specialized way. Moreover, each taxon secretes distinct Ca²⁺-binding macromolecules which were entrapped within the calcium carbonate crystals during skeleton formation. Therefore these Ca²⁺-binding macromolecules were also described as intracrystalline macromolecules. When isolated and separated by SDS polyacrylamide gel electrophoresis, the organic skeleton matrix of the three species revealed to be composed of a respective distinct array of EDTA-soluble proteins. A single protein of 41 kDa was detected inS. wellsi, two proteins of 38 and 120 kDa inA. willeyana, and four proteins of 18 kDa, 30 kDa, 33 kDa, and 37 kDa inVaceletia sp. When run on IEF gel, the Ca²⁺-binding proteins gave staining bands at pH values between 5.25 and 5.65. As proved by anin vitro mineralization assay, the extracted proteins effectively inhibit CaCO₃ and SrCO₃ precipitation, respectively, in a saturated solution. Biochemical properties and behavior of the extracted proteins strongly suggest that they are involved in crystal nucleation and skeleton carbonate formation within the calcified sponges studied here.     

Protein phosphorylation in human peripheral nerve: Altered phosphorylation of a 25-kDa glycoprotein in leprosy

Protein phosphorylation in a low speed supernatant of human peripheral nerve (tibial and sural) homogenate was investigated. The major phosphorylated proteins had molecular mass in the range of 70, 55, 45, and 25 kDa. Mg²⁺ or Mn²⁺ was essential for maximum phosphorylation although Zn²⁺, Co²⁺, and Ca²⁺, could partially support phosphorylation. External protein substrates casein and histone were also phosphorylated. The protein phosphatase inhibitor orthovanadate enhanced the phosphorylation of the 45 and 25 kDa proteins significantly. Concanavalin A-Sepharose chromatography of the phosphorylated peripheral nerve proteins showed that the 25 kDa protein was a glycoprotein. Protein phosphorylation of peripheral nerves from leprosy affected individuals was compared with normals. The phosphorylation of 25 kDa protein was decreased in most of the patients with leprosy.     

The autoantigen La/SSB is a calmodulinmbinding protein

The work reported here has been directed to the identification of new nuclear calmodulin-binding proteins. To achieve this goal, nuclei from rat hepatocytes were purified and a fraction enriched in DNA- and RNA-binding proteins was extracted using DNase I and RNase A. Calmodulin-binding proteins present in this nuclear subfraction were purified by chromatography using first a DEAE-Sephacel column and subsequently a calmodulin-Sepharose column. Four major polypeptides of 118, 107, 48 and 45 kDa were found to bind to the calmodulin column in a Ca²⁺-dependent way. [¹²⁵I]-calmodulin overlay analysis confirmed that the proteins of 118, 48 and 45 kDa are calmodulin-binding proteins. These proteins bind single-stranded and also double-stranded DNA. A partial amino acid sequence obtained from the 48 kDa protein revealed a 100% identity with the La/SSB protein, an autoantigen implicated in several autoimmune diseases, such as lupus erythematosus and Sjögren's syndrome. Two-dimensional gel electrophoresis, Western blot analysis and experiments of binding to poly(U), also supports the identity of p48 as La/SSB. CaM and La/SSB protein colocalize in the heterochromatinic regions within the nucleus of rat hepatocytes. Preincubation of La/SSB with calmodulin in the presence of Ca²⁺ resulted in an increase in the binding of ssDNA to La/SSB, suggesting that calmodulin can play a role in the regulation of the association of La/SSB with DNA.     

Regulation of endogenously catalyzed ADP-ribosylation in adipocyte plasma membranes by Ca and calmodulin

The effects of Ca²⁺ and calmodulin on endogenously catalyzed ADP-ribosylation were investigated in adipocyte plasma membranes. Four specific proteins of 70, 65, 61 and 52 kDa were labeled with [³²P]ADP-ribose and ADP-ribosylation of the proteins was highly dependent upon the conditions employed. ADP-ribosylation of the 70 kDa protein was observed only in membranes supplemented with Ca²⁺. Maximal incorporation of [³²P] into the protein was achieved with free Ca²⁺ concentrations of 90 μM. Calcium-stimulated ADP-ribosylation of the 70 kDa protein was inhibited by calmodulin. Half-maximal inhibition was observed in membranes incubated with 1.2 μM calmodulin. The effect of calmodulin was characterized by an inhibition of the incorporation of [³²P]ADP-ribose as opposed to a stimulation of its removal. ADP-ribosylation of the 61 kDa protein was not altered by added Ca²⁺ and/or calmodulin whereas ADP-ribosylation of the 65 kDa protein was partially (50%) inhibited by free Ca²⁺ concentrations between 10⁻⁶ – 10⁻⁵ M. These results provide evidence that the adipocyte plasma membrane contains ADP-ribosyltransferase activities and demonstrate that ADP-ribosylation of a 70 kDa protein is regulated by Ca²⁺ and calmodulin.     

Manganese enhances the phosphorylation of membrane-associated proteins isolated from barley roots

Microsomal membranes isolated from barley roots (Hordeum vulgare L. cv. CM72) contained endogenous protein phosphorylation activities that were greatly enhanced by Mn²⁺. Mg²⁺ions also stimulated protein phosphorylation, but to a lesser extent than Mn²⁺. Ca²⁺ enhanced Mg²⁺, but not Mn²⁺-dependent phosphorylation. It is proposed that this strong enhancement by Mn²⁺ may be due to a greater affinity of Mn²⁺ than either Ca²⁺ or Mg²⁺ for both the Ca²⁺ and Mg²⁺ binding sites of certain kinases. Some Mn²⁺ stimulated kinase activity was eliminated from the membrane by washing with 0.2 mol/L KCl. The KCl extract contained histone and casein kinase activities, and 4 major phosphoproteins that were phosphorylated on serine and threonine residues. Phosphorylation of a 52 kDa polypeptide corresponded with the characteristics of the histone kinase activity and may represent the autophosphorylation of a CDPK-type kinase. Phosphorylation of a 36 kDa polypeptide was Ca²⁺ stimulated and may represent the autophosphorylation of a different type of unknown kinase. Polypeptides of 18 and 15 kDa had characteristics that suggest they were autophosphorylating subunits of a membrane bound nucleotide di-phosphokinase.     

Cloning and overexpression of a maltase gene from Schizosaccharomyces pombe in Escherichia coli and characterization of the recombinant maltase

The Schizosaccharomyces pombe maltase structural gene (SPMAL1⁺) was amplified from genomic DNA of S. pombe by PCR. An open reading frame of 1740 bp, encoding a putative 579 amino-acid protein with a calculated molecular mass of 67.7 kDa was characterized in the genomic DNA insert of plasmid pQE30. The specific maltase activity in the induced transformants was 21 times higher than that in wild-type. However, the estimated molecular mass of the purified recombinant maltase was 44.3 kDa by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified recombinant maltase were 40 °C and 6, respectively. The recombinant maltase was weakly activated by Mg²⁺, Ca²⁺, Na⁺, and Ba²⁺, but was strongly inhibited by Hg²⁺, Ag⁺ and Cu²⁺, EDTA, and PMSF. The purified maltase could actively hydrolyse ρ-nitrophenyl glucoside (PNPG), maltose, dextrin, and soluble starch. The results demonstrate that maltase from S. pombe was different from that from other yeasts, and might be usefully exploited in the future by the biotechnology industry or lead to the development of new molecular genetic tools.     

Purification and Characterization of Intracellular Proteinases in Pleurotus ostreatus Fruiting Bodies

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL.

ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn²⁺-depleted ProB can regain the activity with Zn²⁺, Co²⁺, or Mn²⁺. Specific cleavage of Pro₂₉-LYS₃₀ in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the α-amino group of lysyl residues.

ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn²⁺ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.     

Determination of some nuclear deoxyribonucleases in X-irradiated rat thymocytes

Summary The spectrum of nuclear nucleases in control and irradiated (4 Gy) thymocytes has been investigated. Using the method of SDS electrophoresis of nuclear proteins in³H - DNA-polyacrylamide gels a number of polypeptides of MW. 35, 32, 17.7, 17.2 and 16.4 kDa possessing nuclease activity were found. The 35 kDa enzyme is only active in the presence of Ca²⁺ and Mg²⁺ ions. In response to cycloheximide injection (3 mg/100 g body weight) and irradiation, we did not detect the 35 kDa nuclease activity. Nucleases of 32, 17.7, 17.2 and 16.4 kDa are active in the presence of Ca²⁺ ions. The activities of these nucleases increases 60 min after irradiation. These nucleases were also found in the fraction of polydeoxyribonucleotide (PDN).     

Purification and Properties of a Nuclease Preferential for Thymidine Residue from Neurospora crassa Mycelia

From the mycelia of Neurospora crassa (wild type No. 6068) multiple forms of a nuclease which had very close isoelectric points (pI = 9.6 (peak I), 9.4 (peak II)) were isolated by ampholine electrofocusing column chromatography (pH 8.5 ~ 10). The nuclease was about 300-fold purified from the crude extract. The two fractions of Peak I, II were indistinguishable in their enzymatic properties and were considered as manifestation of the same enzyme with minor physicochemical differences. The molecular weight was around 41,000 as estimated by the gel filtration method. The enzyme could hydrolyze both DNA and RNA in the order of heat-denatured DNA > native DNA DNA ≧ RNA. RNA competitively inhibited DNA degradation with this enzyme. The enzyme was therefore regarded as a nuclease. The pH optimum was around pH 6.5 toward native DNA, pH 6.7 toward heat-denatured DNA and pH 7.9 toward RNA. The temperature optimum was around 40°C toward these substrates and most of the activities were lost by heating at 55°C for 15 min. The enzyme required Mg²⁺ for action toward heat-denatured DNA and Mg²⁺, Mn²⁺ or Co²⁺ toward native DNA. In the presence of EDTA, the activities toward both types of DNA were lost and recovered by addition of the respective activating metallic ions. p-CMB inhibited this nuclease, but β-mercapto-ethanol and glutathione had no effect. Polyamìnes showed no activation of the nuclease for DNA degradation.     

Isolation of an Arabidopsis cDNA sequence encoding a 22 kDa calcium-binding protein (CaBP-22) related to calmodulin

Complementary DNA sequences were isolated from a library of cloned Arabidopsis leaf mRNA sequences in gt10 that encoded a 21.7 kDa polypeptide (CaBP-22), which shared 66% amino acid sequence identity with Arabidopsis calmodulin. The putative Ca²⁺-binding domains of CaBP-22 and calmodulin, however, were more conserved and shared 79% sequence identity. Ca²⁺ binding by CaBP-22, which was inferred from its amino acid sequence similarity with calmodulin, was demonstrated indirectly by Ca²⁺-induced mobility shifting of in vitro translated CaBP-22 during SDS-polyacrylamide gel electrophoresis. CaBP-22 is encoded by a ca. 0.9 kb mRNA that was detected by northern blotting of leaf poly(A)⁺ RNA; this mRNA was slightly larger than the 809 bp CaBP-22 cDNA insert, indicating that the deduced amino acid sequence of CaBP-22 is near full-length. CaBP-22 mRNA was detected in RNA fractions isolated from leaves of both soil-grown and hydroponically grown Arabidopsis, but below the limits of detection in RNA isolated from roots, and developing siliques. Thus, CaBP-22 represents a new member of the EF-hand family of Ca²⁺-binding proteins with no known animal homologue and may participate in transducing Ca²⁺ signals to a specific subset of response elements.     

Behavior of organelle nuclei (nucleoids) in generative and vegetative cells during maturation of pollen inLilium longiflorum andPelargonium zonale

Summary The behavior of organelle nuclei during maturation of the male gametes ofLilium longiflorum andPelargonium zonale was examined by fluorescence microscopy after staining with 4,6-diamidino-2-phenylindole (DAPI) and Southern hybridization. The organelle nuclei in both generative and vegetative cells inL. longiflorum were preferentially degraded during the maturation of the male gametes. In the mature pollen grains ofL. longiflorum, there were absolutely no organelle nuclei visible in the cytoplasm of the generative cells. In the vegetative cells, almost all the organelle nuclei were degraded. However, in contrast to the situation in generative cells, the last vestiges of organelle nuclei in vegetative cells did not disappear completely. They remained in evidence in the vegetative cells during germination of the pollen tubes. InP. zonale, however, no evidence of degradation of organelle nuclei was ever observed. As a result, a very large number of organelle nuclei remained in the sperm cells during maturation of the pollen grains. When the total DNA isolated from the pollen or pollen tubes was analyzed by Southern hybridization with a probe that contained therbc L gene, for detection of the plastid DNA and a probe that contained thecox I gene, for detection of the mitochondrial DNA, the same results were obtained. Therefore, the maternal inheritance of the organelle genes inL. longiflorum is caused by the degradation of the organelle DNA in the generative cells while the biparental inheritance of the organelle genes inP. zonale is the result of the preservation of the organelle DNA in the generative and sperm cells. To characterize the degradation of the organelle nuclei, nucleolytic activities in mature pollen were analyzed by an in situ assay on an SDS-DNA-gel after electrophoresis. The results revealed that a 40kDa Ca²⁺-dependent nuclease and a 23 kDa Zn²⁺ -dependent nuclease were present specifically among the pollen proteins ofL. longiflorum. By contrast, no nucleolytic activity was detected in a similar analysis of pollen proteins ofP. zonale.     

Characterization of plasma membrane domains of mouse EL4 lymphoma cells obtained by affinity chromatography on concanavalin A-Sepharose

Purified plasma membranes of mouse EL₄ lymphoma cells were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (MF1) eluted freely from the affinity column, the second (MF2) adhered specifically to Con A-Sepharose. Both membrane subfractions proved to be of plasma membrane origin, as evidenced by the following criteria. (i) The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. (ii) When isolated plasma membranes were labelled with tritiated NaBH₄, both subfractions exhibited identical specific radioactivities. (iii) After enzymatic radioiodination of the cells, the total content of labelled proteins was very similar in isolated plasma membranes and in both subfractions. (iv) Some plasma membrane marker enzymes exhibited nearly identical specific activities in plasma membranes, MF1 or MF2 including γ-glutamyl transpeptidase, 5′-nucleotidase and Mg²⁺-ATPase. Both subfractions exhibited characteristic differences. Thus the specific activities of (Na⁺ + K⁺)-ATPase, Ca²⁺-ATPase and lysophosphatidylcholine acyltransferase were several-fold enriched in MF2 compared to MF1. SDS-polyacrylamide gel electrophoresis revealed a different polypeptide composition of the two subfractions. Polypeptides of apparent molecular mass of 116, 95, 42, 39, 30 and 28 kDa were highly enriched in MF2, whereas MF1 contained another set of proteins, of apparent molecular mass of 70, 55 and 24 kDa. The phospholipid fatty acid composition of the subfractions proved to be different, as well, MF2 contained more saturated fatty acids than MF1. The data suggest the existence of plasma membrane domains in the plasma membranes of the mouse EL₄ lymphoma cells, containing a set of polypeptides, among others membrane bound enzymes, embedded in a different phospholipid milieu.     

Possible involvement of Ca<Superscript>2+</Superscript>-dependent protein kinases in spore germination of the fern<Emphasis Type="Italic">Osmunda Japonica</Emphasis>

Fern gametophyte is a good model system to investigate signal transduction in plant cells. In this work, we examined whether CDPKs are involved in the mechanisms of spore germination of the fernOsmunda japonica. A protein extract from the spores included four CDPK isoforms with relative molecular weights of 56, 53, 49, and 47 kDa, as detected by immunoblot analysis, and they showed CDPK-like activities, as detected by in-gel protein-kinase assay. It was also found that the inhibitors effective on CDPKs, such as a general protein kinase inhibitor, K252a, and a calmodulin antagonist, W-7, largely suppressed the spore germination, and that many proteins of the spores were phosphorylated in vivo in a calcium dependent manner in the period when the spores require external Ca²⁺ for the germination. Furthermore, we showed that Sr²⁺ and Mn²⁺, which could substitute for Ca²⁺ in the spore germination, were also able to activate theOsmunda CDPKs. From these results, we concluded that CDPKs would participate in the spore germination ofO. japonica.     

Water channels in the plant plasma membrane cloned by immunoselection from a mammalian expression system

Expression in mammalian COS cells and an efficient microtiter-based strategy for immunoselection was used in a novel approach to identify genes encoding plant membrane proteins. COS cells were transfected with an Arabidopsis thaliana root cDNA library constructed in a bacterial mammalian shuttle vector and screened with an antiserum raised against purified deglycosylated integral plasma membrane proteins from A. thaliana roots. Antibodies directed against a prominent 27 kDa antigen led to the identification of five different genes. They comprised two subfamilies related to the major intrinsic protein (MIP) superfamily and were named plasma membrane intrinsic proteins, PIP1 and PIP2, since the cellular localization of PIP1 and most probably PIP2 proteins in the plasma membrane was independently confirmed by their co-segregation with marker enzymes during aequeous two-phase partitioning. Surprisingly, expression in Xenopus laevis oocytes revealed that all five PIP mRNAs coded for Hg²⁺-sensitive water transport facilitating activities. There had been no previous evidence of the existence of water channels in the plasma membrane of plant cells and the high diffusional water permeability of the lipid bilayer was considered to be sufficient for water exchange. Nevertheless, Northern and Western analyses showed that the PIP genes are constitutively and possibly even redundantly expressed from the small A. thaliana genome.     

Ca2+-dependent protein kinase from alfalfa (Medicago varia): Partial purification and autophosphorylation

A calcium-dependent protein kinase (CDPK) was purified to 1400-fold from the soluble fraction of alfalfa (Medicago varia) cells by ammonium sulfate fractionation, Sephacryl-300, DEAE-Sephacel, Phenyl-Sepharose and Hydroxylapatite column chromatography. The enzyme is mainly monomeric. During the course of the purification steps a 50 kDa phosphoprotein doublet and a 56 kDa phosphoprotein copurified with the CDPK activity. Mobility shift of these proteins have been shown by SDS PAGE in Ca²⁺ free conditions. Tests on enzyme activity after separation by native gel electrophoresis revealed two protein kinase activities in our enzyme preparation and the phosphorylation of the 50 kDa and 56 kDa proteins. We suggest that these proteins are the autophosphorylated forms of calcium dependent protein kinases. Preincubation of the CDPK in ATP resulted in a marked increase in enzyme activity, but did not alter the Ca²⁺ sensitivity of the protein kinase.     

Phosphorylation of a putative myosin light chain inChara by calcium-dependent protein kinase

Summary Ca²⁺-dependent protein kinase (CDPK) has been proposed to mediate inhibition by Ca²⁺ of cytoplasmic streaming in the green algaChara. We have identified the in vivo substrate(s) of CDPK inChara by using vacuolar perfusion of individual internodal cells with [-³²P]ATP. Phosphorylation of several polypeptides is enhanced when perfusions are performed at 10^–4M free Ca²⁺ compared to <10^–9M free Ca²⁺. The Ca²⁺-stimulated phosphorylation of these proteins is inhibited by the presence of a monoclonal antibody to soybean CDPK. One of these proteins is 16 to 18kDa and is recognized by an antibody against gizzard myosin light chains. These results demonstrate that inChara, several polypeptides are phophorylated by CDPK and one of these proteins has been tentatively identified as a myosin light chain. These observations support the hypothesis that Ca²⁺-regulated phosphorylation of myosin is involved in the regulation of cytoplasmic streaming.Abbreviations CDPK calcium-dependent protein kinase - mAb monoclonal antibody