Biomedical subjects

F L Huang

Publications and source records attributed to F L Huang.

At least 37 records · Page 2Linked to original sources

PubMed1997

Molecular cloning, structural analysis, and expression of carp ZP2 gene.

The cDNAs encoding carp ZP2 homologous to winter flounder and mammalian ZP2 were cloned. Carp ZP2 contains a tandemly repetitive domain and a nonrepetitive domain. A repeat is composed of 13 amino-acid residues whose consensus sequence is QQTSQQFQPQKPA/V. The length of the repetitive domain is highly variable, but that of the nonrepetitive domain is fairly constant among various cDNAs. The termination codons of various cDNAs appear at three different positions. Three groups of cDNAs were therefore categorized. Groups I-III encode a nonrepetitive domain of 356, 255, and 10 residues, respectively. A carp ZP2 gene corresponding to group II cDNA was cloned. It spans 2.4 kb and consists of eight exons and seven introns. Carp ZP2 mRNA was detected only in oocytes but not in other tissues. Carp ZP2 is heterogenous in size. The molecular weight ranges from 40-80 kDa. It is present in vitellogenic but not in previtellogenic oocytes, nor in other tissues. Carp ZP2 content in oocytes increases as vitellogenesis proceeds.

Amino Acid Sequence↗

PubMed1996

Molecular cloning and characterization of a novel casein kinase II substrate, HASPP28, from rat brain.

HASPP28 (heat- and acid-stable phosphoprotein of 28 kDa) has been purified to near homogeneity from the acid-stable protein fraction of rat brain extract. Based on the N-terminal 40 amino acid sequence, a pair of highly degenerate primers was used to generate a 107-bp probe from rat brain RNA by RT-PCR. From the rat brain lambda gt11 library, this probe identified two positive clones that together provided a cDNA of 837 bp with an open reading frame of 546 bp. This cDNA was extended by 3'RACE to 1.2 kb that included a polyadenylation signal and a poly(A) tail. The 180-amino-acid sequence derived from the open reading frame, which did not correspond to any known protein, was predicted to have phosphorylation sites for protein kinase C, casein kinase II (CKII), and protein kinase A. Indeed, both the purified rat brain HASPP28 and the recombinant HASPP28 (rHASPP28) can be phosphorylated by these kinases. Northern blot analysis indicated that HASPP28 was present in all rat tissues tested, including those from the brain, lung, spleen, kidney, liver, heart, and muscle, in decreasing order of abundance. Phosphopeptide analysis of rHASPP28 phosphorylated in vitro by various kinases showed different tryptic patterns on two-dimensional mapping and isoelectric focusing gels. From [32P]PO4-labeled N1E115 neuroblastoma cells, HASPP28 can be immunoprecipitated with a polyclonal antiserum raised against rHASPP28. The immunoprecipitated protein showed a phosphopeptide pattern similar to that of rHASPP28 phosphorylated by CK II in vitro. Furthermore, the immunoprecipitates from cells treated with phorbol 12-myristate 13-acetate or 8-bromo-cAMP did not show any increased phosphorylation over those of untreated ones, and the phosphopeptide patterns of the immunoprecipitates again were similar to that of CK II phosphorylated protein. These results suggest that HASPP28 is a novel phosphoprotein that can be phosphorylated by several kinases in vitro. In intact cells, CK II seems to be solely responsible for the phosphorylation of HASPP28.

Amino Acid Sequence↗

PubMed1996

Molecular cloning, structural analysis, and expression of carp ZP3 gene.

Two types of cDNAs coding for a major component of carp egg membrane were clones from a carp ovarian cDNA library. They encode polypeptides of 422-424 amino acid residues whose sequences are homologous to those of medaka and mammalian ZP3. Similar to the mammalian ZP3 genes, carp ZP3 gene also consists of eight exons and seven introns. Carp ZP3 genes are 2.9 kb in length and present in multiple forms. Carp ZP3 is a glycoprotein of 45 kDa. It was transcribed and translated exclusively in oocytes, in contrast with medaka ZP3, which was synthesized in liver. The transcription of carp ZP3 starts very early in oogenesis, but translation occurs during vitellogenesis, as it is present in vitellogenic but not in previtellogenic oocytes. ZP3 content in oocytes increases as vitellogenesis proceeds.

Amino Acid Sequence↗

PubMed1996

Purification and molecular cloning of carp ovarian cystatin.

The ovarian fluid of carp consists of many components. Using the antiserum against carp serum, Western blot analysis of ovarian fluid was done in order to distinguish substances synthesized by the ovary from those derived from the serum. Several ovary-specific substances were detected including a protein of 12 kDa (p12), which was purified to homogeneity. Purified p12 displays a single band in SDS-PAGE under nonreducing condition and it can inhibit the enzymatic activity of papain with an apparent inhibition constant of 0.01 nM. The primary structure of p12 was partially determined by Edman degradation and fully elucidated by molecular cloning. A cDNA of 531 bp encoding p12 was obtained. The precursor of p12 has 129 residues, including a signal peptide of 18 residues and a mature protein of 111 residues. The N- and C-terminus of p12 are threonine and methionine, respectively. The p12 shares many common features of the family 2 cystatins of other species, including the similarity of the protein size (in the range of 110 to 120 residues), the presence of 4 cysteine residues and the occurrence of invariant residues throughout the molecule.

Amino Acid Sequence↗

PubMed1996

Expression, characterization, and genomic structure of carp JAK1 kinase gene.

A 3.7-kb cDNA encodes the carp JAK1 kinase of 1,156 amino acid residues. The overall amino acid sequence identity between carp JAK1 and murine JAK1, JAK2, JAK3, and human TYK2 is 57%, 35.5%, 31.3%, and 42.4%, respectively. In addition, carp JAK1 shows higher sequence homology to mammalian JAK1 in both the kinase-like (JH2) and kinase (JH1) domains (approximately 70% identity). Therefore, carp JAK1 is a homolog of mammalian JAK1. To investigate the possible function of JH2 domain, full-length, and various truncated forms of carp JAK1 were produced in the baculovirus system. Our results demonstrate that c-JH1 and c-JH2 associate with each other and c-JH2 can be tyrosine-phosphorylated by c-JAK1 and by c-JH(1 + 2). The JAK1 gene was also isolated from a carp genomic library and characterized. This gene is divided into 24 exons spanning at least 31 kb of genomic DNA. Exon 1 contains the 5'-untranslated region and exon 2 contains the putative translation initiation site. The 2.5-kb DNA region upstream of the transcription initiation site contains numerous potential binding sites for transcription factors including NF-IL6, HNF-5, AP1, GHF-5, and E2A. When this DNA fragment was placed upstream of the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into a carp CF cell line, it could drive the synthesis of CAT enzyme 16 times more efficiently than the promoterless pCAT-Basic. Deletion analysis defined a positive regulatory region between -1,023 and -528. A smaller region (-181 to +59) without any typical TATA-box sequences, G + C-rich sequences, or other binding sequences for known transcription factors still had promoter activity. Constructs without this region did not have detectable promoter activity. This suggests that this region of DNA may play an important role in the expression of carp JAK1 gene.

Amino Acid Sequence↗

PubMed1995

Identification of a rapidly dephosphorylating 95-kDa protein as elongation factor 2 during 8-Br-cAMP treatment of N1E115 neuroblastoma cells.

Treatment of 8-Br-cAMP promotes neurite outgrowth and neuronal differentiation in N1E115 mouse neuroblastoma cells. Prior or simultaneous treatment of PMA blocks 8-Br-cAMP-mediated neurite outgrowth. Phosphorylation of cellular proteins during these treatments was examined in a permeabilized cell system. While PMA promotes phosphorylation of the heat-stable protein kinase C substrates MARCKS and neuromodulin, 8-Br-cAMP hastens the dephosphorylation of a protein of M(r)95k (p95). Extensively purified, N-terminal sequenced, and judged from its phosphorylation properties, p95 was identified as the eukaryotic elongation factor-2 (eEF-2), whose dephosphorylation has been reported to be related to an increase in protein synthesis. It is likely 8-Br-cAMP stimulates dephosphorylation of eEf-2, promotes protein synthesis that eventually leads to neuronal differentiation in N1E115 cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1995

Structure and regulation of the gene encoding the neuron-specific protein kinase C substrate neurogranin (RC3 protein).

A 13-kilobase pair genomic DNA encoding a 78-amino acid brain-specific calmodulin-binding protein kinase C (PKC) substrate, neurogranin (Ng/RC3; also known as RC3 or p17), has been sequenced. The Ng/RC3 gene is composed of four exons and three introns, with the protein-coding region located in the first and second exons. This gene was found to have multiple transcriptional start sites clustered within 20 base pairs (bp); it lacks the TATA, GC, and CCAAT boxes in the proximal upstream region of the start sites. The promoter activity was characterized by transfection of 293 cells with nested deletion mutants of the 5'-flanking region fused to the luciferase reporter gene. A minimal construct containing bp +11 to +256 was nearly as active as that covering bp -1508 to +256, whereas a shorter one covering bp +40 to +256 had a greatly reduced activity. Between bp +11 and +40 lies a 12-nucleotide sequence (CCCCGCCCACCC) containing overlapping binding sites for AP2 (CCGCCCACCC) and SP1 (CCCGCC); this region may be important for conferring the basal transcriptional activity of the Ng/RC3 gene. The expression of a Ng/RC3-luciferase fusion construct (-1508/+256) in transfected 293 cells was stimulated by phorbol 12-myristate 13-acetate (PMA), but not by cAMP, arachidonic acid, vitamin D, retinoic acid, or thyroxines T3 and T4. PMA caused a 2-4-fold stimulation of all the reporter gene constructs ranging from +11/+256 to -1508/+256. The stimulatory effects of PMA could be magnified by cotransfection with both Ca(2+)-dependent and -independent phorbol ester-binding PKC-alpha, -beta I, -beta II, -gamma, -delta, and -epsilon cDNAs, but not by non-phorbol ester-binding PKC-zeta cDNA. The Ng/RC3 and PKC-gamma genes have a similar expression pattern in the brain during development. These two genes share at least four conserved sequence segments 1.5 kilobase pair upstream from their transcriptional start sites and a gross similarity in that they possess several AT-rich segments within bp -550 to -950. A near homogeneous 20-kDa DNA-binding protein purified from rat brain was able to bind to these AT-rich regions of both Ng/RC3 and PKC-gamma genes with footprints containing ATTA, ATAA, and AATA sequences.

Amino Acid Sequence↗

PubMed1995

Differential responses of protein kinase C substrates (MARCKS, neuromodulin, and neurogranin) phosphorylation to calmodulin and S100.

Phosphorylation of three physiological substrates of protein kinase C (PKC), MARCKS, neuromodulin (Nm), and neurogranin (Ng), was analyzed to determine their relative efficacy as substrates of PKC alpha, beta, and gamma and sensitivities to inhibition by calmodulin (CaM) and S100. Comparison of the Vmax/Km of the phosphorylation of each individual substrate indicated the order of efficacy as PKC substrate was MARCKS > Nm > Ng. Phosphorylation of these proteins in a mixture by PKC beta and gamma was indistinguishable from that when each individual substrate was phosphorylated by these two isozymes. In contrast, the rates of PKC alpha-catalyzed phosphorylation of Nm and Ng in a mixture also containing MARCKS were significantly reduced as compared to that when Nm or Ng was individually phosphorylated by this isozyme. When these substrates were present in a mixture, both CaM and S100 inhibited the PKC-catalyzed phosphorylation of MARCKS to a higher degree than that of Nm or Ng. Protease-activated catalytic fragment of PKC (PKM) was used to determine the effects of Ca2+ and phospholipid on the CaM and S100-mediated inhibition of PKC substrate phosphorylation. CaM and S100 inhibited the PKM-catalyzed phosphorylation of MARCKS only in the presence of Ca2+ and addition of phosphatidylserine (PS)/dioleoylglycerol (DG) did not influence the inhibitory effect. Phosphorylation of Nm or Ng by PKM was inhibited by CaM to a higher degree in the absence than in the presence of Ca2+. S100 was ineffective in inhibiting the phosphorylation of Nm and Ng without Ca2+ and only poorly effective in the presence of Ca2+. The CaM-mediated inhibition of Nm or Ng phosphorylation by PKM was also not affected by PS/DG either with or without Ca2+. The results presented here demonstrate that MARCKS is a preferred substrate of PKC and its phosphorylation by PKC is most sensitive to inhibition by regulatory proteins such as CaM and S100.

Animals↗

PubMed1995

A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: I. Protein purification and characterization.

Two isoforms of a protease inhibitor of the serpin family (p62) have been purified from bighead carp perimeningeal fluid. Both isoforms migrate with an apparent molecular mass of 62 kDa on reducing and nonreducing sodium dodecyl sulfate-polyacrylamide gels. Both proteins inhibited the activities of bovine trypsin, bovine chymotrypsin, and porcine pancreatic elastase. They also formed complexes with these proteases that were resistant to sodium dodecyl sulfate treatment. p62 exists in the extracts of all tissues examined, including brain, head kidney, kidney, liver, muscle, ovary, pituitary, and spleen. It is also present in serum, ovarian fluid, and milt as well as perimeningeal fluid. The protease inhibitor is a glycoprotein, and its carbohydrate moiety could be removed by endoglycosidase F. Because p62 resembles mammalian alpha 1-antitrypsin in many aspects, it is likely a fish equivalent of alpha 1-antitrypsin.

Animals↗

PubMed1995

A protease inhibitor of the serpin family is a major protein in carp perimeningeal fluid: II. cDNA cloning, sequence analysis, and Escherichia coli expression.

A cDNA clone, pCP9, has been isolated from a common carp liver cDNA library by immunoscreening with polyclonal antiserum raised against purified bighead carp alpha 1-antitrypsin. This clone is 1,396 bp in length and has an open reading frame encoding a protein of 410 amino acid residues. The deduced amino acid sequence shows moderate homology to human alpha 1-antitrypsin (38%), guinea pig contrapsin (35%), human alpha 1-antichymotrypsin (34%), and human proteinase C inhibitor (31%), all members of the serine protease inhibitor (serpin) family. To confirm further that the cDNA clone was derived from the authentic carp alpha 1-antitrypsin gene, the presumptive mature protein of pCP9 was expressed in Escherichia coli. The molecular mass of the recombinant protein matched that predicted from the nucleotide sequence. This recombinant protein, which was recognized by antiserum against native alpha 1-antitrypsin, was capable of formation of serpin-enzyme complexes with trypsin, chymotrypsin, and elastase. Therefore, we conclude that the protein encoded by the pCP9 clone is indeed carp alpha 1-antitrypsin. Expression of alpha 1-antitrypsin in brain was confirmed by reverse transcription and polymerase chain reaction performed on mRNA derived from both common carp and bighead carp brain.

Amino Acid Sequence↗

PubMed1995

Opposing neuroendocrine actions of the natriuretic peptides: C-type and A-type natriuretic peptides do not interact with the same hypothalamic cells controlling prolactin secretion.

The two major members of the family of natriuretic peptides (NPs) in brain, A-type natriuretic peptide (ANP) and C-type natriuretic peptide (CNP) exert opposing actions on the neuroendocrine regulation of prolactin (PRL) secretion. We have targeted for compromise and destruction cells within the diencephalon which bear receptors for ANP (NRP-A receptors), CNP (NRP-B receptors), or both peptides (NPR-C receptors) using novel cytotoxin cell targeting methodology in order to determine if the neuroendocrine effects of these two peptides are exerted on similar cell systems. In animals pretreated with ANP conjugated to the cytotoxic A chain of ricin, central administration of a dose of ANP which is known to inhibit PRL secretion did not alter PRL levels in plasma; however, subsequent administration of CNP elicited the stimulation of PRL secretion. In rats pretreated with CNP-ricin A chain conjugate, a treatment we hypothesize targets for destruction CNP responsive cells, ANP injection did inhibit PRL secretion, while the stimulatory effect of CNP was absent. These results suggest that the neuroendocrine effects of these two natriuretic peptides on PRL secretion are expressed on different cellular elements of the hypothalamo-pituitary axis. Furthermore, they reveal that neither peptide acts directly on the tuberoinfundibular dopamine system since pretreatment with either cytotoxin conjugate failed to alter basal PRL levels. Thus ANP and CNP do not appear to express opposing actions on the same cell systems, suggesting the recruitment of each peptide individually by differing, unique stimuli for PRL release.

Animals↗

PubMed1994

Molecular cloning and sequencing of a carp cDNA encoding mitogen-activated protein kinase kinase.

We have cloned a full-length cDNA encoding the mitogen-activated protein kinase kinase (MKK) from a carp liver cDNA library. The cDNA contains 1970 bp with a single open reading frame encoding a 397 amino acid protein. By comparing with known MKK sequences from other species, carp MKK is 78%, 80%, 76% and 58% identical to rat MKK1, rat MKK2, Xenopus MKK and Drosophila MKK.

Amino Acid Sequence↗

PubMed1994

The complete nucleotide sequence and gene organization of carp (Cyprinus carpio) mitochondrial genome.

The complete sequence of the carp mitochondrial genome of 16,575 base pairs has been determined. The carp mitochondrial genome encodes the same set of genes (13 proteins, 2 rRNAs, and 22 tRNAs) as do other vertebrate mitochondrial DNAs. Comparison of this teleostean mitochondrial genome with those of other vertebrates reveals a similar gene order and compact genomic organization. The codon usage of proteins of carp mitochondrial genome is similar to that of other vertebrates. The phylogenetic relationship for mitochondrial protein genes is more apparent than that for the mitochondrial tRNA and rRNA genes.

Amino Acid Sequence↗

PubMed1994

Peripheral deafferentation alters calcitonin gene-related peptide mRNA expression in visceral sensory neurons of the nodose and petrosal ganglia.

Visceral sensory neurons of the glossopharyngeal and vagus nerves are located in the petrosal and nodose ganglia, respectively. Our previous studies showed that peripheral axotomy which removes afferent input to visceral sensory perikarya decreased the number of calcitonin gene-related peptide (CGRP)-immunoreactive (ir) neurons in the petrosal but not the nodose ganglion. To evaluate axotomy-induced changes in CGRP mRNA expression, we used in situ hybridization histochemistry with 35S-labeled oligonucleotide probes. CGRP mRNA-containing neurons were studied 1, 3, 7 and 14 days after peripheral deafferentation of the left nodose and petrosal ganglia via transection of the left cervical vagus, superior laryngeal, glossopharyngeal and carotid sinus nerves. The numbers of CGRP mRNA-containing neurons in the deafferented petrosal ganglion were significantly reduced at 3, 7 and 14 days compared to either intact or sham-operated control ganglia. However, the density of hybridization product in the positively-labeled petrosal ganglion cells was not significantly changed. The numbers of CGRP mRNA-containing neurons in the deafferented nodose ganglion were significantly reduced at 3 and 7 days. These data suggest that axotomy-induced changes in CGRP-ir neurons of the petrosal ganglion correlate with changes in CGRP mRNA and probably result from altered CGRP gene expression. In addition, in situ hybridization histochemistry revealed changes in CGRP neurons of the nodose ganglion which were not apparent with immunocytochemistry.

Afferent Pathways↗

PubMed1994

L-type Ca2+ channel is involved in the regulation of spontaneous transmitter release at developing neuromuscular synapses.

Involvement of an L-type Ca2+ channel in the regulation of spontaneous transmitter release was studied in Xenopus nerve-muscle cultures. The frequency of spontaneous synaptic currents, which reflects impulse-independent acetylcholine release from the nerve terminals, showed a marked increase in high-K+ medium or after treatment with a phorbol ester, 12-O-tetradecanoyl-phorbol 13-acetate, a drug that activates protein kinase C and depolarizes the presynaptic neuron. The potentiation effect of high K+ and 12-O-tetradecanoyl-phorbol 13-acetate requires Ca2+ influx through the L-type Ca2+ channel in the plasma membrane, since it was significantly reduced by the presence of nifedipine, verapamil or diltiazem and enhanced by Bay K 8644, an L-type Ca2+ channel agonist. It was shown recently that adenosine 5'-triphosphate markedly potentiates the spontaneous acetylcholine release at these synapses through the binding of P2-purinoceptors and the activation of protein kinase C. We found in the present study that potentiation effects of adenosine 5'-triphosphate are inhibited by L-type Ca2+ channel blockers, suggesting that the L-type Ca2+ channel is responsible for the positive regulation of spontaneous acetylcholine secretion by adenosine 5'-triphosphate at the developing neuromuscular synapses. Our data suggest that modulation of the L-type Ca2+ channel in embryonic motor nerve terminals is important for the regulation of spontaneous transmitter release.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗

PubMed1994

Potentiation by endogenously released ATP of spontaneous transmitter secretion at developing neuromuscular synapses in Xenopus cell cultures.

1. Previously we have shown that extracellular application of ATP, a substance co-stored and co-released with acetylcholine (ACh) in the peripheral nervous system, markedly potentiated the frequency of spontaneous synaptic currents (SSCs) produced by ACh. In the present study, we have further characterized the purinoceptor which mediates the potentiation effect of ATP and the role of endogenously released ATP. 2. Pretreatment with a P2-purinoceptor antagonist, suramin (0.3 mM), but not a P1-purinoceptor antagonist, 8-phenyltheophylline (0.1 mM), prevented the potentiating effect of ATP. 3. We studied the role of endogenously released ATP by examining the effect of a specific P2-purinoceptor antagonist on the frequency of spontaneous synaptic events at high-activity synapses (> or = 3 Hz) and found that suramin, but not 8-phenyltheophylline markedly reduced the frequency of SSCs at these high-activity synapses. In addition, desensitizing the P2-purinoceptor with alpha,beta-methylene ATP also produced similar effects to suramin. 4. Extracellular application of the L-type Ca2+ channel blockers, verapamil, nifedipine or diltiazem (10 microM each) reduced SSC frequency of high-activity synapses, while the N-type Ca2+ channel blocker, omega-conotoxin had no appreciable effect. The potentiating effect of ATP was further prevented by pretreatment with the L-type Ca2+ channel blockers. On the other hand, Bay K 8644, which is a depolarization-dependent L-type Ca2+ channel agonist, potentiated SSC frequency at these high-activity synapses. 5. These results suggest that endogenous release of ATP at developing neuromuscular synapses is responsible for the maintenance of high levels of spontaneous ACh release, which is known to play a crucial role in regulating postsynaptic differentiation.

Acetylcholine↗

PubMed1993

Protein kinase C phosphorylates Ser152, Ser156 and Ser163 but not Ser160 of MARCKS in rat brain.

MARCKS is one of the major physiological substrates of PKC and was reported to be phosphorylated by PKC at 4 serine residues that are within the CaM-binding region (Graff et al., J. Biol. Chem. 264, 11912, 1989). Using MARCKS from rat brain and a synthetic peptide of 25 amino acids containing all 4 of the serine residues, we investigate the differences in phosphorylation by PKC isozymes I, II and III. Tryptic peptide analysis of PKC phosphorylated MARCKS or peptide, we found 32P was in peptides of (K)S152FK, (R)FS156FK and LS160GFS163FK. Further digestion of LSGFSFK with alpha-chymotrypsin revealed that 32P incorporation occurred only at Ser163 but not at Ser160. The initial rates and stoichiomatry of phosphorylation of Ser152 and Ser156 were twice as those of Ser163 using either one of the three PKC isozymes. These results indicate that in vitro, PKC phosphorylates MARCKS only at three sites, but not at Ser160 as that reported previously, and there was no preferential phosphorylation of MARCKS by either PKC isozyme I, II or III.

Amino Acid Sequence↗

PubMed1993

Characterization of a 7.5-kDa protein kinase C substrate (RC3 protein, neurogranin) from rat brain.

A 7.5-kDa heat- and acid-stable rat brain protein kinase C (PKC) substrate was purified to near homogeneity by a two-step procedure using DEAE-cellulose and hydroxylapatite column chromatography. This 78-amino-acid protein has a sequence identical to that deduced from rat brain RC3 cDNA identified with a cortex-minus-cerebellum subtracted cDNA probe (J. B. Watson et al., J. Neurosci. Res. 26, 397-408, 1990) and exhibits extensive sequence identity to bovine brain neurogranin (J. Baudier et al., J. Biol. Chem. 266, 229-237, 1991). On sodium dodecyl sulfate-polyacrylamide gel electrophoresis this protein, RC3, migrated as a M(r) 15-18K species in the presence of reducing agent and as heterogeneous species of M(r) 13-28K in the absence of reducing agent. Phosphorylation of RC3 by PKC alpha, beta, or gamma was stimulated by Ca2+, phospholipid, and diacylglycerol. A single site, Ser36, which is adjacent to the predicted calmodulin (CaM)-binding domain, was phosphorylated by these enzymes. Phosphorylation of RC3 by PKC or PKM, a protease-degraded PKC, was inhibited by CaM. The effect of CaM apparently targets at RC3, as phosphorylation of protamine sulfate by PKM was not inhibited by CaM. In the absence of Ca2+, RC3 formed a stoichiometric complex with CaM as evidenced by an increase in the M(r) determined by gel filtration chromatography. In the presence of Ca2+, the affinity of RC3 toward CaM is greatly reduced and Ca2+/CaM becomes less inhibitory of the PKM-catalyzed phosphorylation of RC3. Phosphorylation of RC3 by PKM prevented the interaction of this protein with CaM even in the absence of Ca2+. A 20-amino-acid synthetic peptide (AS-20F-W) containing the PKC phosphorylation site and CaM-binding domain of RC3 (Ala29-Ser48) with a substitution of Phe37 with tryptophan was used to monitor the interaction of this peptide with CaM by spectrofluorometry. In the absence of Ca2+, CaM caused negligible change in tryptophan fluorescence of the peptide; however, an enhancement and blue-shift of the emission fluorescence was observed in the presence of Ca2+. It seems that this synthetic peptide, as well as RC3 holoprotein, interacts with CaM through electrostatic interaction in the absence of Ca2+ but through hydrophobic interaction in the presence of Ca2+. In rat brain homogenate, RC3 formed a stable complex with CaM in the presence of Ca2+, as demonstrated by immunoblot analysis following gel filtration chromatography.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗