Developmental and Functional Studies of Parvalbumin and Calbindin D28K in Hypothalamic Neurons Grown in Serum-Free Medium

The Ca2+-binding proteins parvalbumin (Mr = 12K) and calbindin D28K [previously designated vitamin D-dependent Ca2+-binding protein (Mr = 28K)] are neuronal markers, but their functional roles in mammalian brain are unknown. The expression of these two proteins was studied by immunocytochemical methods in serum-free cultures of hypothalamic cells from 16-day-old fetal mice. Parvalbumin is first detected in all immature neurons, but during differentiation, the number of parvalbumin-immunoreactive neurons greatly declines to a level reminiscent of that observed in vivo, where only a subpopulation of neurons stains for parvalbumin. In contrast, calbindin D28K was expressed throughout the period investigated only in a distinct subpopulation of neurons. Depolarization of fully differentiated hypothalamic neurons in culture resulted in a dramatic decrease of parvalbumin immunoreactivity but not of calbindin D28K immunoreactivity. The parvalbumin staining was restored on repolarization. Because the anti-parvalbumin serum seems to recognize only the metal-bound form of parvalbumin, the loss of immunoreactivity may signal a release of Ca2+ from intracellular parvalbumin during depolarization of the cells. We suggest that parvalbumin might be involved in Ca2+-dependent processes associated with neurotransmitter release.     

Site-specific replacement of amino acid residues within the CD binding loop of rat oncomodulin

Relative to the same site in oncomodulin, the CD ion-binding domain of rat parvalbumin exhibits much greater affinity for Ca2+ and Mg2+. As part of an effort to understand the structural basis for these differences, site-specific variants of oncomodulin have been prepared in which the amino acid residues at positions 52, 54, 57, 59, and 60 have been replaced with the residues present at the corresponding positions in rat parvalbumin. The proteins resulting from the single-site substitutions at residues 52, 54, and 57 are indistinguishable from the wild-type protein on the basis Eu3+ luminescence spectroscopy, and none of the three variants displays increased affinity for Ca2+. By contrast, the substitutions at residues 59 and 60 perturb both the Eu3+ luminescence parameters and the Ca2+ and Mg2+ affinities, and these differences are amplified when both replacements are simultaneously incorporated into the protein. The Eu3+ 7F0----5D0 spectrum of the double variant (D59E/G60E) at pH 5.0, with a maximum at 5796 A and pronounced shoulder at 5791 A, strongly resembles that obtained with pike parvalbumin. Consistent with this increased parvalbumin-like character, KCa is decreased from 0.78 microM (for the wild-type protein) to 0.41 microM, and KMg is decreased from 3.5 to 0.74 mM. Nevertheless, the affinity of the CD ion-binding domain in D59E/G60E for Ca2+ remains almost 2 orders of magnitude lower than the corresponding site in rat parvalbumin, strongly suggesting that residues besides those present in the binding loop are involved in dictating the metal ion-binding properties of the oncomodulin CD site.     

Remodeling of the AB site of rat parvalbumin and oncomodulin into a canonical EF-hand.

Parvalbumin (PV) and the homologous protein oncomodulin (OM) contain three EF-hand motifs, but the first site (AB) cannot bind Ca2+. Here we aimed to recreate the putative ancestral proteins [D19-28E]PV and [D19-28E]OM by replacing the 10-residue-long nonfunctional loop in the AB site by a 12-residue canonical loop. To create an optical conformational probe we also expressed the homologs with a F102W replacement. Unexpectedly, in none of the proteins did the mutation reactivate the AB site. The AB-remodeled parvalbumins bind two Ca2+ ions with strong positive cooperativity (nH = 2) and moderate affinity ([Ca2+]0.5 = 2 microM), compared with [Ca2+]0.5 = 37 nM and nH = 1 for the wild-type protein. Increasing Mg2+ concentrations changed nH from 2 to 0.65, but without modification of the [Ca2+]0. 5-value. CD revealed that the Ca2+ and Mg2+ forms of the remodeled parvalbumins lost one-third of their alpha helix content compared with the Ca2+ form of wild-type parvalbumin. However, the microenvironment of single Trp residues in the hydrophobic cores, monitored using intrinsic fluorescence and difference optical density, is the same. The metal-free remodeled parvalbumins possess unfolded conformations. The AB-remodeled oncomodulins also bind two Ca2+ with [Ca2+]0.5 = 43 microM and nH = 1.45. Mg2+ does not affect Ca2+ binding. Again the Ca2+ forms display two-thirds of the alpha-helical content in the wild-type, while their core is still strongly hydrophobic as monitored by Trp and Tyr fluorescence. The metal-free oncomodulins are partially unfolded and seem not to possess a hydrophobic core. Our data indicate that AB-remodeled parvalbumin has the potential to regulate cell functions, whereas it is unlikely that [D19-28E]OM can play a regulatory role in vivo. The predicted evolution of the AB site from a canonical to an abortive EF-hand may have been dictated by the need for stronger interaction with Mg2+ and Ca2+, and a high conformational stability of the metal-free forms.     

Association between human erythrocyte calmodulin and the cytoplasmic surface of human erythrocyte membranes

This report describes Ca2+-dependent binding of 125I-labeled calmodulin (125I-CaM) to erythrocyte membranes and identification of two new CaM-binding proteins. Erythrocyte CaM labeled with 125I-Bolton Hunter reagent fully activated erythrocyte (Ca2+ + Mg2+)-ATPase. 125I-CaM bound to CaM depleted membranes in a Ca2+-dependent manner with a Ka of 6 x 10(-8) M Ca2+ and maximum binding at 4 x 10(-7) M Ca2+. Only the cytoplasmic surface of the membrane bound 125I-CaM. Binding was inhibited by unlabeled CaM and by trifluoperazine. Reduction of the free Ca2+ concentration or addition of trifluoperazine caused a slow reversal of binding. Nanomolar 125I-CaM required several hours to reach binding equilibrium, but the rate was much faster at higher concentrations. Scatchard plots of binding were curvilinear, and a class of high affinity sites was identified with a KD of 0.5 nM and estimated capacity of 400 sites per cell equivalent for inside-out vesicles (IOVs). The high affinity sites of IOVs most likely correspond to Ca2+ transporter since: (a) Ka of activation of (Ca2+ + Mg2+)-ATPase and KD for binding were nearly identical, and (b) partial digestion of IOVs with alpha-chymotrypsin produced activation of the (Ca2+ + Mg2+)-ATPase with loss of the high affinity sites. 125I-CaM bound in solution to a class of binding proteins (KD approximately 55 nM, 7.3 pmol per mg of ghost protein) which were extracted from ghosts by low ionic strength incubation. Soluble binding proteins were covalently cross-linked to 125I-CaM with Lomant's reagent, and 2 bands of 8,000 and 40,000 Mr (Mr of CaM subtracted) and spectrin dimer were observed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis autoradiography. The 8,000 and 40,000 Mr proteins represent a previously unrecognized class of CaM-binding sites which may mediate unexplained Ca2+-induced effects in the erythrocyte.     

Purification and characterization of the parvalbumin from monkey skeletal muscle

1. A high affinity Ca2+ binding and low mol. wt protein, parvalbumin, was purified from monkey skeletal muscle. 2. As compared with other animals, only one component and a lower content of monkey parvalbumin were found. 3. This may suggest that both the component and the content of parvalbumin decreases with biological evolution. 4. The parvalbumin was found to have a mol. wt of 11,400, a pI of 5.1, a high aspartic acid and lysine content, maximum absorption at around 260 nm, a blocked amino-terminal, an immunological distinction, 2 mol Ca2+ binding/mol, and a conformational change by Ca2+ binding. 5. Parvalbumin was shown to have alpha type properties.     

Parvalbumin in rat kidney. Purification and localization

The Ca2+-binding parvalbumin has been purified for the first time from rat kidney. Its biochemical and immunological properties were indistinguishable from the muscle counterpart. By immunohistochemical methods parvalbumin was localized in part of the distal tubule and proximal collecting duct, similar to the vitamin D-dependent Ca2+-binding protein, calbindin-28K. Parvalbumin was found to be independent of the vitamin D status of the animal since its concentration remained unchanged in kidney extracts of normal, rachitic and vitamin D-replete rats. Both proteins may be involved in the regulation of intracellular Ca2+ in kidney.     

Perch muscle parvalbumin: general characterization and magnesium-binding properties.

1. Parvalbumin exists in two major forms, amounting to 2.5 and 3.2 g per kg of fresh muscle. 2. The composition divergence index between both forms, calculated from their amino acid composition, indicates 81% sequence identity; however both isoparvalbumins are immunologically distinct. 3. Beside Ca2+, perch parvalbumin binds 2 g atoms Mg2+ per mol with a dissociation constant of 10(-5) M. 4. Determination of the affinity for magnesium is of particular importance as there are indications that, in vivo, parvalbumin can remain in the Mg2+-state during the contraction-relaxation cycle instead of switching from the Ca2+-to the Mg2+-state and vice versa.     

Identification and quantification in single muscle fibers of four isoforms of parvalbumin in the iliofibularis muscle of Xenopus laevis

The major parvalbumins present in the iliofibularis muscle of Xenopus laevis were identified and the total parvalbumin content of different types of single fibers of this muscle was determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate (SDS). The criteria used in the identification of proteins as parvalbumins were: a relative molecular mass (Mr) between 10,000 and 14,000, an isoelectric point (pI) between 4.0 and 5.0, and a Ca2+-dependent mobility when run on a polyacrylamide gel in the absence of SDS. Four proteins were thus identified as parvalbumins: PA1, Mr 14,000, pI 4.90; PA2, Mr 11,000, pI 4.90; PA3, Mr 11,000, pI 4.95; and PA4, Mr 11,000, pI 4.25. An ultraviolet absorbance spectrum characteristic of parvalbumins was recorded for a purified preparation of these four proteins. Because the apparent Mr of rabbit parvalbumin in the gel system used was 14,000, whereas the true value is 12,100, it is not excluded that the Mr of component PA1 of 14,000 is an overestimation. The total parvalbumin content of muscles and single muscle fibers was determined using the supernatant obtained after centrifugation of tissue homogenates. Analysis of the protein pattern after electrophoresis in the presence of SDS of this fraction indicated that the Mr 14,000 and 11,000 protein bands contained virtually only parvalbumin. Quantification of the total parvalbumin content of relatively fast (type 1) and slow (type 2) contracting and relaxing single muscle fibers, using laser densitometric analysis of minigels, yielded mean values (mg protein/g wet wt., +/- S.D.) of 5.2 +/- 0.8 for nine type 1 fibers, and 1.9 +/- 1.0 for five type 2 fibers. Both fiber types contained about 2.5-times as much of the Mr 14,000 isoform relative to the combined Mr 11,000 isoforms.     

Purification and characterization of a calcium-calmodulin-dependent phospholamban kinase from canine myocardium

A Ca2+-calmodulin-dependent protein kinase was purified to apparent homogeneity from the cytosolic fraction of canine myocardium, with phospholamban as substrate. Purification involved sequential chromatography on DEAE-cellulose, calmodulin-agarose, DEAE-Bio-Gel A, and phosphocellulose. This procedure resulted in a 987-fold purification with a 5.4% yield. The purified enzyme migrated as a single band on native polyacrylamide gels, and it exhibited an apparent molecular weight of 550,000 upon gel filtration. Gel electrophoresis under denaturing conditions revealed a single protein band with Mr 55,000. The purified kinase could be autophosphorylated in a Ca2+-calmodulin-dependent manner, and under optimal conditions, 6 mol of Pi was incorporated per mole of 55,000-dalton subunit. The activity of the enzyme was dependent on Ca2+, calmodulin, and ATP.Mg2+. Other ions which could partially substitute for Ca2+ in the presence of Mg2+ and saturating calmodulin concentrations were Sr2+ greater than Mn2+ greater than Zn2+ greater than Fe2+. The substrate specificity of the purified Ca2+-calmodulin-dependent protein kinase for cardiac proteins was determined by using phospholamban, troponin I, sarcoplasmic reticulum membranes, myofibrils, highly enriched sarcolemma, and mitochondria. The protein kinase could only phosphorylate phospholamban and troponin I either in their purified forms or in sarcoplasmic reticulum membranes and myofibrils, respectively. Exogenous proteins which could also be phosphorylated by the purified protein kinase were skeletal muscle glycogen synthase greater than gizzard myosin light chain greater than brain myelin basic protein greater than casein. However, phospholamban appeared to be phosphorylated with a higher rate as well as affinity than glycogen synthase.(ABSTRACT TRUNCATED AT 250 WORDS)     

Parvalbumin in non-muscle tissues of the rat. Quantitation and immunohistochemical localization

Parvalbumin, a high affinity Ca2+-binding protein, is known to be expressed only in muscles and brain in the rat. We have investigated its distribution and characteristics in other rat tissues by several biochemical and immunohistochemical methods. Evidence for the presence of parvalbumin in teeth, bone, skin, prostate, seminal vesicles, testes, and ovary is given by two-dimensional polyacrylamide gel electrophoresis, immunoblotting ("Western technique") of one-dimensional gels, and its concentration measured by reverse phase high performance liquid chromatography. The distribution within several parvalbumin-positive organs was monitored by the immunohistochemical peroxidase-antiperoxidase method. In teeth, only ameloblasts reacted with anti-rat parvalbumin serum and in bone the calcified extracellular cartilage was the target of the immunoreaction. The panniculus carnosus was the exclusive site of parvalbumin in the skin. Besides the already known parvalbumin distribution in the brain, parvalbumin is also expressed in distinct cell types of the peripheral nervous system. Leydig cells were found to be the only parvalbumin location in testes. These observations lead us to conclude that parvalbumin in contrast to the multifunctional and constitutive calmodulin must function in Ca2+-dependent processes related to specific cell types.     

Calelectrins are a ubiquitous family of Ca2+-binding proteins purified by Ca2+-dependent hydrophobic affinity chromatography by a mechanism distinct from that of calmodulin

The calelectrins, a heterogeneous group of three new Ca2+-binding proteins of M 67 000, 35 000 and 32 500, copurify with calmodulin during Ca2+-dependent hydrophobic affinity chromatography (Südhof et al., Biochemistry, in press, 1984). This property is exploited for the rapid purification of all three calelectrins including for the first time the Mr 35 000, from commercially available acetone powders from several bovine tissues (heart, liver, brain, pancreas and testis). The nature of the Ca2+-dependent interaction of the calelectrins with hydrophobic affinity matrices has been investigated. As with calmodulin, the Ca2+-binding sites of all three purified calelectrins can be probed with Tb3+ which binds to them in a stoichiometric, saturable and Ca2+-displaceable manner. However, using several hydrophobic fluorescence probes which bind to the proteins, contrary to calmodulin no Ca2+-dependent exposure of hydrophobic sites could be detected in any of the three purified proteins. Therefore the Ca2+-dependent purification of the calelectrins on hydrophobic affinity columns seems not to involve the surface exposure of hydrophobic sites and the calelectrins have in this respect little similarity to calmodulin.     

Ca2+-binding parvalbumin in rat testis. Characterization, localization, and expression during development

Parvalbumin, a Ca2+-binding protein, was isolated from rat testis. This is the first demonstration of the protein in endocrine glands. By using a rat parvalbumin cDNA probe, parvalbumin mRNA was demonstrated in the testis, indicating that the protein is synthesized in this tissue and that testis parvalbumin is a product of the same gene as the one encoding for muscle parvalbumin. Parvalbumin was localized by immunohistochemical methods in the Leydig cells and in the acrosome region of maturing spermatids (stages 1-15). The expression of parvalbumin during testis development was followed. High parvalbumin protein and mRNA levels were found at stages of highest Leydig cell activity, i.e. at late fetal stages until birth and again around postnatal day 50. This suggests that parvalbumin may be involved in the production of testosterone in Leydig cells, a process which is highly dependent on calcium.     

Coordination of cardiac sarcoplasmic reticulum and myofibrillar function by protein phosphorylation

Adrenergic stimulation alters functional dynamics of the heart by mechanisms most likely involving cyclic AMP (cAMP)-dependent protein phosphorylation. In vitro studies indicate that the myofibrils and sarcoplasmic reticulum (SR) may act as effectors of the adrenergic stimulation. cAMP-dependent phosphorylation of troponin I (TnI), one of the regulatory proteins of cardiac myofibrils, results in a decreased steady-state affinity of troponin C (TnC) for calcium, an increase in the off-rate for Ca2+ exchange with TnC, and a rightward shift of the relation between free Ca2+ and myofibrillar force or ATPase. Phosphorylation of phospholamban, a regulatory protein of cardiac SR, results in an increased velocity of Ca2+ transport by SR vesicles, an increased affinity of the transport protein for Ca2+, and an increased turnover of elementary steps of the ATPase reaction. These in vitro findings support the hypothesis that the inotropic response of the heart to catecholamine stimulation involves phosphorylation of TnI and phospholamban. Our in vivo studies with perfused rabbit hearts show that during the peak of the inotropic response to isoproterenol there is a simultaneous phosphorylation of TnI and an 11,000-dalton protein in the SR, most likely the monomeric form of phospholamban.     

Localization of a fibrinogen calcium binding site between gamma-subunit positions 311 and 336 by terbium fluorescence

Calcium is required for effective fibrin polymerization. The high affinity Ca2+ binding capacity of fibrinogen was directly localized to the gamma-chain by autoradiography of nitrocellulose membrane blots of fibrinogen subunits incubated with 45Ca2+. Terbium (Tb3+) competitively inhibited 45Ca2+ binding to fibrinogen during equilibrium dialysis, accelerated fibrin polymerization, and limited fibrinogen fragment D digestion by plasmin. The intrinsic fluorescence of Ca2+-depleted fibrinogen was maximally enhanced by Ca2+ and Tb3+, but not by Mg2+, at about 3 mol of cation/mol of fibrinogen. Protein-bound Tb3+ fluorescence at 545 nm was maximally enhanced by resonance energy transfer from tryptophan (excitation at 290 nm) at about 2 mol of Tb3+mol of fibrinogen and about 1 mol of Tb3+/mol of plasmic fragment D94 (Mr 94,000). Fibrinogen fragments D78 (Mr 78,000) and E did not show effective enhancement of Tb3+ fluorescence, suggesting that the Ca2+ site is located within gamma 303 to gamma 411, the peptide which is absent in fragment D78 but present in D94. When CNBr fragments of the carboxyamidated gamma-subunit were assayed for enhancement of Tb3+ fluorescence, peptide CBi (gamma 311-336) bound 1 mol of Tb3+/mol of CBi. Thus, the Ca2+ site is located within this peptide. The sequence between gamma 315 and gamma 329 is homologous to the calmodulin and parvalbumin Ca2+ binding sites.     

Sodium and potassium binding to parvalbumins measured by means of intrinsic protein fluorescence

The binding of Na+ and K+ to whiting parvalbumin (pI 4.4) and pike parvalbumins (pI 4.2 and 5.0) results in a shift of the tryptophan fluorescence spectrum towards shorter wavelengths by 2-4 nm for the whiting protein and in a rise of the tyrosine and phenylalanine fluorescence quantum yield for the pike proteins. The effective binding constants of Na+ and K+ to parvalbumins are within the range of 10 M-1 to 100 M-1. Physiological concentrations of Na+ and K+ lower the affinity of whiting parvalbumin for Ca2+ and Mg2+ by almost an order of magnitude.     

Calmodulin-stimulated protein methylation in rat liver cytosol

The in vitro methylation of three liver cytosolic proteins was found to be selectively stimulated by calmodulin. This effect was also seen, although to a much smaller degree, in kidney and lung, but not in testes, brain, or spleen. The three methylated proteins affected by calmodulin have apparent Mr = 29,000, 32,000, and 45,000. The stimulation of methylation by calmodulin was greatest for the Mr 29,000 protein; there was an equal degree of methylation of the other two proteins. Dialysis of liver cytosolic fractions also stimulated the methylation of these proteins; the methylation of the Mr 32,000 and 45,000 proteins was stimulated to a greater extent by dialysis than by calmodulin. The degree of stimulation of methylation of the Mr 29,000 protein by calmodulin and dialysis was equivalent, but the addition of calmodulin to dialyzed liver cytosolic fractions gave additive effects on the stimulation of methylation of the Mr 29,000 protein, but not of either the Mr 32,000 or 45,000 proteins. Troponin C stimulated the methylation of the Mr 29,000 protein, but not the Mr 32,000 or 45,000 proteins, whereas parvalbumin stimulated methylation of the Mr 32,000 protein, but not the Mr 29,000 or 45,000 proteins. The effects of calmodulin and dialysis on protein methylation are cation-dependent and substrate-specific; methylation of the Mr 29,000 was supported by Mn2+, Ca2+, and Co2+, and to a lesser degree by Mg2+, Ni2+, and Zn2+. Methylation of the Mr 32,000 protein was supported only by Mn2+ and Mg2+ and methylation of the Mr 45,000 protein by Mn2+, Mg2+, Ca2+, Ni2+, and Zn2+, and to a much smaller extent by Fe2+. In extracts of fetal liver, stimulation of protein methylation by calmodulin or dialysis was restricted to the Mr 45,000 protein. In regenerating liver, stimulation of the methylation of all three proteins was observed, but the stimulation provided by dialysis plus calmodulin was much less than that observed in preparations from intact adult liver, suggesting a possible negative correlation between the rate of cell division and calmodulin-dependent methylation of these hepatic proteins. These results are consistent with the presence in liver of a minimum of three distinct N-methyltransferases and a dialyzable inhibitor which antagonizes calmodulin-dependent protein methylation.     

Electrospray ionization mass spectrometry: analysis of the Ca2+-binding properties of human recombinant alpha-parvalbumin and nine mutant proteins

A set of 10 different recombinant human parvalbumins was used to establish a method for the investigation of the Ca2+-binding properties of proteins by electrospray ionization mass spectrometry (ESI-MS). Human PVWT was found to bind 2 mol Ca2+ ions/mol of protein, whereas its mutants (PVE101V, PVD90A, PVE62V, PVD51A, PVD90A,E101V, PVE62V,E101V, PVD51A,D90A, PVD51A,E62V, PVD51A,E62V, D90A,E101V) containing inactivating substitutions in the Ca2+-binding loops bind 0 or 1 Ca2+ ion per protein molecule, depending on the degree of inactivation. These findings fully agree with previously reported results obtained by flow dialysis experiments. The RP-HPLC desalted metal-free proteins were analyzed in 10 mM ammonium acetate at pH 7.0. The experimental conditions were optimized with the recombinant parvalbumin test system before analyzing the Ca2+-binding properties of rat and murine parvalbumins in muscle tissue extracts. ESI-MS revealed that (i) rat and murine alpha-parvalbumins each bind specifically two Ca2+ ions per protein molecule and (ii) both extracted parvalbumins were found to be posttranslationally modified; each protein is acetylated at the N-terminus. Finally, during our investigations of the murine parvalbumin a sequencing error was detected at the C-terminus where the amino acid at position 109 is Ser and not Thr as mentioned in the SwissProt data base (Accession No. P32848). This work demonstrates the great potential of the ESI-MS technique as a sensitive, specific, and rapid method for direct identification and determination of the stoichiometry of Ca2+-binding proteins and other metalloproteins.     

Ca2+ binding to skeletal muscle troponin C in skeletal and cardiac myofibrils

Ca2+ binding to skeletal muscle troponin C in skeletal or cardiac myofibrils was measured by the centrifugation method using 45Ca. The specific Ca2+ binding to troponin C was obtained by subtracting the amount of Ca2+ bound to the CDTA-treated myofibrils (troponin C-depleted myofibrils) from that to the myofibrils reconstituted with troponin C. Results of Ca2+ binding measurement at various Ca2+ concentrations showed that skeletal troponin C had two classes of binding sites with different affinity for Ca2+. The Ca2+ binding of low-affinity sites in cardiac myofibrils was about eight times lower than that in skeletal myofibrils, while the high-affinity sites of troponin C in skeletal or cardiac myofibrils showed almost the same affinity for Ca2+. The Ca2+ sensitivity of the ATPase activity of skeletal troponin C-reconstituted cardiac myofibrils was also about eight times lower than that of skeletal myofibrils reconstituted with troponin C. These findings indicated that the difference in the sensitivity to Ca2+ of the ATPase activity between skeletal and cardiac CDTA-treated myofibrils reconstituted with skeletal troponin C was mostly due to the change in the affinity for Ca2+ of the low-affinity sites on the troponin C molecule.     

Independent Protein Kinases Associated with the Rat Cerebral Synaptic Junction: Comparison with Cyclic AMP-Dependent and Ca2+/Calmodulin-Dependent Protein Kinases in the Synaptic Junction

Independent protein kinases in the synaptic junction (SJ) isolated from rat cerebrum were characterized. SJ showed a protein kinase activity, phosphorylating intrinsic proteins, even in the absence of cyclic AMP or Ca2+ plus calmodulin (CaM) exogenously added. The activity was affected neither by Ca2+ concentrations in the physiological fluctuation range nor by the addition of specific ligands such as glutamate, aspartate, acetylcholine, and concanavalin A. The activity was not due to cyclic AMP-dependent protein kinase in SJ, since the activity was not inhibited by an inhibitor protein for cyclic AMP-dependent protein kinase, and since synapsin I was not specifically phosphorylated whereas cyclic AMP-dependent kinase appeared to phosphorylate selectively the protein in SJ. Phosphorylation of SJ proteins by the independent kinases was about one-third of that of the Ca2+/CaM-dependent protein kinase intrinsic to SJ. The apparent Km for ATP was estimated to be 700 microM. Proteins of 16K Mr and 117K Mr were specifically phosphorylated under the basic condition (in the absence of the substances known to activate specifically protein kinases), as well as six other proteins both under the basic conditions and in the presence of Ca2+ and CaM. The phosphorylation of 150K Mr, 60K Mr, 51K Mr, and 16K Mr SJ proteins was enhanced after prephosphorylation of SJ proteins by intrinsic kinase in the presence of Ca2+ and CaM.(ABSTRACT TRUNCATED AT 250 WORDS)