The DHICA Oxidase Activity of the Melanosomal Tyrosinases LEMT and HEMT

Although melanins can be formed in vitro by the unique action of tyrosinase on L-tyrosine, it is now well accepted that other enzymes termed tyrosinase-related proteins are involved in mammalian melanogenesis. However, some aspects of their roles in the regulation of the pathway are still unknown. The action of dopachrome tautomerase on L-dopachrome yields DHICA, a stable dihydroxyindole with a low rate of spontaneous oxidation. However, DHICA is efficiently incorporated to the pigment, as judged by the high content of carboxylated indole units in natural melanins. Therefore, the fate of this melanogenic intermediate and the mechanisms of its incorporation to the melanin polymer are major issues in the study of melanogenesis. We have recently shown that mouse melanosomes contain two electrophoretically distinguishable tyrosinase isoenzymes, LEMT and HEMT, that can be purified and completely resolved (Jiménez-Cervantes et al., 1993a). Herein, we have compared the ability of these tyrosinases to catalyze DHICA oxidation. Although highly purified LEMT shows a very low specific activity for dopa oxidation in comparison to HEMT, it is able to catalyze DHICA oxidation. However, the DHICA oxidase activity of HEMT was very low, if significant. The ability of purified LEMT to catalyze DHICA oxidation was abolished by heat, trypsin, or phenylthiourea treatments. LEMT acting on DHICA caused the formation of a brownish soluble color similar to DHICA-melanin. Immunoprecipitation of the DHICA oxidase activity of LEMT by specific antibodies suggests that this activity corresponds to TRP1. These results indicate that LEMT, most probably identical to the product of the b locus, is a tyrosinase having a specific DHICA oxidase activity. Opposite to HEMT, the true tyrosinase encoded by the albino locus, its role in melanogenesis would be related to the incorporation of DHICA into eumelanin rather than to the first steps of the pathway.     

Calcium transport by rat brain mitochondria and oxidation of 2-oxoglutarate

Contrary to previous reports brain mitochondria have a substantial capacity for net Ca²⁺ uptake (approx. 1.2 μeq. Ca²⁺ per mg protein) providing succinate is the oxidizable substrate. ATP stimulates calcium uptake (to 1.8 μeq. per mg protein), but is not required. The accumulation of Ca²⁺ with NAD-linked substrates is, however, significantly less. With 2-oxoglutarate, very limited Ca²⁺ uptake occurs before respiration is inhibited. At low concentrations (10 μM), Ca²⁺ stimulates the 2-oxoglutarate dehydrogenase activity of detergent solubilized mitochondria. Millimolar [Ca²⁺] is required for inhibition. Therefore, Ca²⁺ inhibition of 2-oxoglutarate oxidation can explain the low maximum uptake with this substrate, but probably not by directly effecting the dehydrogenase. Hence, the oxidation of 2-oxoglutarate can be either enhanced or suppressed depending upon the net Ca²⁺ accumulated by brain mitochondria.     

Freeze-fracture study of water-soluble,standard proteins and of detergent-solubilized forms of sarcoplasmic reticulum Ca2+-ATPase

Conventional freeze-fracture electron microscopy was used to study water-soluble proteins and different forms of Ca²⁺-ATPase-detergent complexes. Freeze-fracture images of solutions containing proteins larger than myoglobin showed the presence of distinct, randomly dispersed particles on smooth fracture surfaces. The distribution of sizes of these particles was close to Gaussian, with a mean size which was correlated to the Stokes diameter. Monomeric Ca²⁺-ATPase from sarcoplasmic reticulum, solubilized by deoxycholate or a non-ionic detergent, showed a bimodal distribution of particles sizes. Even more complex distributions were found for dimeric and trimeric preparations of Ca²⁺-ATPase. The results can be interpreted on the assumption that the Ca²⁺-ATPase molecule is elongated, with an overall length of about 110 Å and a width in its largest part of about 75 Å. It is concluded on the basis of the presented results that freeze-fracture electron microscopy can be successfully used for morphological studies of protein molecules in solution.     

A New Method of Preparing Ca2+-ATPase from Sarcoplasmic Reticulum: Extraction with Octylglucoside

A fast method for preparing Ca²⁺-ATPase from rabbit muscle sarcoplasmic reticulum was devised. The method involves extracting extrinsic membrane proteins with the non-ionic detergent octylglucoside at high salt concentration. A Ca²⁺ ATPase of consistently high specific activity (about 25 μmoles/mg·min) is found in the insoluble residue. The method was optimized with respect to the concentrations of detergent and salt, pH, and other extraction conditions. By the criteria of the protein pattern in SDS-polyacrylamide gel electrophoresis, dependence of the hydrolytic activity on the presence of Ca²⁺, and the phosphoprotein formation, the preparation is identical with the Ca²⁺-ATPase isolated previously by MacLennan [10] and other authors. The main advantages of the new method are its rapidity, its reliability, and the high specific activity of the purified enzyme.     

Cloning and Molecular Characterization of a SDS‐Activated Tyrosinase from Marinomonas mediterranea

The sequence of the tyrosinase gene cloned from Marinomonas mediterranea is reported. It is the second tyrosinase cloned from a Gram negative bacterium. Its size is higher than that of Gram positive tyrosinases from Streptomyces, and more similar to the eukaryotic enzymes. Its sequence shares the features of copper‐binding sites found in all tyrosinases. Based in the comparison of tyrosinases from all types of organisms, an extension of the characteristic signatures existing at Prosite is proposed. This tyrosinase shares with some plant and amphibian tyrosinases a strong specific activation by submicellar concentrations of SDS. Intrinsic fluorescence and kinetic properties indicate that the activation is caused by an SDS‐dependent conformational change that facilitates the substrate accessibility to the dicopper active site.     

Thapsigargin affinity purification of intracellular P2A-type Ca ATPases

The ubiquitous sarco(endo)plasmic reticulum (SR/ER) Ca²⁺ ATPase (SERCA2b) and secretory-pathway Ca²⁺ ATPase (SPCA1a) belong both to the P_2A-type ATPase subgroup of Ca²⁺ transporters and play a crucial role in the Ca²⁺ homeostasis of respectively the ER and Golgi apparatus. They are ubiquitously expressed, but their low abundance precludes purification for crystallization. We have developed a new strategy for purification of recombinant hSERCA2b and hSPCA1a that is based on overexpression in yeast followed by a two-step affinity chromatography method biasing towards properly folded protein. In a first step, these proteins were purified with the aid of an analogue of the SERCA inhibitor thapsigargin (Tg) coupled to a matrix. Wild-type (WT) hSERCA2b bound efficiently to the gel, but its elution was hampered by the high affinity of SERCA2b for Tg. Therefore, a mutant was generated carrying minor modifications in the Tg-binding site showing a lower affinity for Tg. In a second step, reactive dye chromatography was performed to further purify and concentrate the properly folded pumps and to exchange the detergent to one more suitable for crystallization. A similar strategy was successfully applied to purify WT SPCA1a. This study shows that it is possible to purify functionally active intracellular Ca²⁺ ATPases using successive thapsigargin and reactive dye affinity chromatography for future structural studies. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

A rapid separation of bovine brain S-100a and S-100b proteins and related conformation studies

S-100 protein absorbs to the calmodulin antagonist W-7 coupled to epoxy-activated Sepharose 6B in the presence of Ca²⁺ and is eluted by ethylene glycol bis(β-aminoethyl ether)-N,N′-tetraacetic acid buffer. S-100a and S-100b were separated and isolated by Ca²⁺-dependent affinity chromatography on W-7 Sepharose. The Ca²⁺-induced conformational changes of S-100a and S-100b were examined using circular dichroism, ultraviolet difference spectra, and a fluorescence probe. Differences in Ca²⁺-dependent conformational changes between S-100a and S-100b became apparent. Circular dichroism studies revealed that both S-100a and S-100b undergo a conformational change upon binding of Ca²⁺ in the aromatic and far-uv range. In the presence or absence of Ca²⁺, the aromatic CD spectrum of S-100a differed completely from that of S-100b, possibly due to the single tryptophan residue of S-100a. Far-uv studies indicate that α-helical contents of both S-100a and S-100b decreased with addition of Ca²⁺. Ca²⁺-induced conformational changes of S-100a and S-100b were also detected by uv difference spectra. The spectrum of S-100a also differed from that of S-100b. Fluorescence studies using 2-p-toluidinylnaphthalene-6-sulfonate (TNS), a hydrophobic probe for protein, revealed a slight difference in conformational changes of these two components. The interaction of TNS and S-100b was observed with concentrations above 3 μm Ca²⁺; on the other hand, S-100a required concentrations above 8 μm. This finding was supported by the difference in the binding affinities of S-100a and S-100b to the W-7 (N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide)-Sepharose column; both S-100a and S-100b bound the column in the presence of Ca²⁺ but S-100a was eluted prior to S-100b. These results suggest that S-100a and S-100b differ in their dependence on Ca²⁺ and that the affinity-chromatographic separation of S-100a from S-100b on the W-7-Sepharose column makes feasible a rapid purification of these two components.     

Role of Ca2+ in the allosteric regulation of platelet actomyosin

The role of Ca²⁺ in regulation of platelet actomyosin ATPase activity has been investigated. The results suggest that Ca²⁺ has at least two roles in the reaction mechanism; (a) it forms a complex with ATP to form the substrate, CaATP and (b) it forms a complex with the protein to activate the enzyme. Both the substrate and free Ca²⁺ bind cooperatively to the protein. The binding of free Ca²⁺ stimulates the enzymic activity and causes a change in the apparent K_m value. The apparent K_m value for CaATP is 0.15mM in the absence of free Ca²⁺ and 0.07mM in the presence of 2.5mM Ca²⁺. Thus Ca²⁺ appears to act as a positive allosteric effector.     

Ca2+- and phospholipid-binding properties ofTorpedo electric organ calelectrin

The Ca²⁺-regulated lipid-binding properties of the H- and L-forms of calelectrin present in the electric organ ofTorpedo marmorata have been compared. Binding of H-calelectrin required Ca²⁺ in millimolar concentrations, whereas that of L-calelectrin occurred in the micromolar range. Dissociation of H-calelectrin previously bound to lipids in the presence of 2 mM Ca²⁺ took place only when the Ca²⁺ concentration was reduced to micromolar concentrations. Binding was most effective to acidic phospholipids such as phosphatidylserine. Both forms of calelectrin promoted the aggregation of membrane vesicles in the presence of Ca²⁺, Mg²⁺, Na⁺ and K⁺ inhibited the Ca²⁺-induced binding to phospholipid, decreasing in effectiveness in that order. Binding was also inhibited by high pH. The surface activity and hdyrophobicity index showed that H-calelectrin is a hydrophilic molecule. It may represent a less active, more highly phosphorylated ‘down-regulated’ form of L-calelectrin. The role of calcium in H-calelectrin binding to lipid appeared to be consistent with the formation of a ternary complex of the protein, an acidic lipid and Ca²⁺, rather than with a direct interaction of lipid with hydrophobic sequences in H-calelectrin whose accessibility is Ca²⁺-regulated.     

Modulation of the kinetic characteristics of the sarcoplasmic reticulum ATPase by membrane fluidity

(Ca²⁺ + Mg²⁺)-ATPase from sarcoplasmic reticulum has been reconstituted with dipalmitoylphosphatidylcholine, and the activating effect of ATP and Ca²⁺ on this enzyme has been studied at different temperatures. It has been found that two kinetic forms of the enzyme are interconverted at about 31°C, and this is possibly related to a phase change in the phospholipid which is more directly associated with the protein. Above 31°C the enzyme is less dependent on ATP activation at high ATP concentrations but shows positive cooperativity for Ca²⁺ activation. On the other hand, below 31°C, the reconstituted enzyme is more dependent on ATP for activation at high ATP concentrations than the purified ATPase and does not show cooperativity for Ca²⁺ activation.     

Translational suppression by Ca2+ ionophores: Reversibility and roles of Ca2+ mobilization, Ca2+ influx, and nucleotide depletion

The divalent cation selective ionophores A23187 and ionomycin were compared for their effects on the Ca²⁺ contents, nucleotide contents, and protein synthetic rates of several types of cultured cells. Both ionophores reduced amino acid incorporation by approximately 85% at low concentrations (50–300 nmol/L) in cultured mammalian cells without reducing ATP or GTP contents. At these concentrations A23187 and ionomycin each promoted substantial Ca²⁺ efflux, whereas at higher concentrations a large influx of the cation was observed. Ca²⁺ influx occurred at lower ionophore concentrations and to greater extents in C6 glioma and P3X63Ag8 myeloma than in GH₃ pituitary cells. The ATP and GTP contents of the cells and their ability to adhere to growth surfaces declined sharply at ionophore concentrations producing increased Ca²⁺ influx. Prominent reductions of nucleotide contents occurred in EGTA-containing media that were further accentuated by extracellular Ca²⁺. Ionomycin produced more Ca²⁺ influx and nucleotide decline than comparable concentrations of A23187. The inhibition of amino acid incorporation and mobilization of cell-associated Ca²⁺ by ionomycin were readily reversed in GH₃ cells by fatty acid-free bovine serum albumin, whereas the effects of A23187 were only partially reversed. Amino acid incorporation was further suppressed by ionophore concentrations depleting nucleotide contents. Mitochondrial uncouplers potentiated Ca²⁺ accumulation in response to both ionophores. At cytotoxic concentrations Lubrol PX abolished protein synthesis but did not cause Ca²⁺ influx. Nucleotide depletion at high ionophore concentrations is proposed to result from increased plasmalemmal Ca²⁺-ATPase activity and dissipation of mitochondrial proton gradients and to cause intracellular Ca²⁺ accumulation. Increased Ca²⁺ contents in response to Ca²⁺ ionophores are proposed as an indicator of ionophore-induced cytotoxicity.Abbreviations BSA bovine serum albumin - EGTA [ethylenebis(oxyethylenenitrilo)]tetraacetic acid - PKR double-stranded RNA-regulated protein kinase - ER endoplasmic reticulum - eIF eukaryotic initiation factor     

Metal-controlled interdomain cooperativity in parvalbumins

Conformational behavior of five homologous proteins, parvalbumins (PAs) from northern pike (α and β isoforms), Baltic cod, and rat (α and β isoforms), was studied by scanning calorimetry, circular dichroism, and bis-ANS fluorescence. The mechanism of the temperature-induced denaturation of these proteins depends dramatically on both the peculiarities of their amino acid sequences and on their interaction with metal ions. For example, the pike α-PA melting can be described by two successive two-state transitions with mid-temperatures of 90 and 120 °C, suggesting the presence of two thermodynamic domains. The intermediate state populated at the end of the first transition was shown to bind Ca²⁺ ions, and was characterized by the largely preserved secondary structure and increased solvent exposure of hydrophobic groups. Mg²⁺- and Na⁺-loaded forms of pike α-PA demonstrated a single two-state transition. Therefore, the mechanism of the PA thermal denaturation is controlled by metal binding. It ranged from the absence of detectable first-order transition (apo-form of pike PA), to the two-state transition (e.g., Mg²⁺- and Na⁺-loaded forms of pike α-PA), to the more complex mechanisms (Ca²⁺-loaded PAs) involving at least one partially folded intermediate. Analysis of isolated cavities in the protein structures revealed that the interface between the CD and EF subdomains of Ca²⁺-loaded pike α-PA is much more loosely packed compared with PAs manifesting single heat-sorption peak. The impairment of interactions between CD and EF subdomains may cause a loss of structural cooperativity and appearance of two separate thermodynamic domains. One more peculiar feature of pike α-PA is that depending on its interactions with metal ions, it can be an intrinsically disordered protein (apo-form), an ordered protein of mesophilic (Na⁺-bound state), thermophilic (Mg²⁺-form), or even of the hyperthermophilic origin (Ca²⁺-form).     

Association Properties and Unfolding of a βγ-Crystallin Domain of a Vibrio-Specific Protein

The βγ-crystallin superfamily possesses a large number of versatile members, of which only a few members other than lens βγ-crystallins have been studied. Understanding the non-crystallin functions as well as origin of crystallin-like properties of such proteins is possible by exploring novel members from diverse sources. We describe a novel βγ-crystallin domain with S-type (Spherulin 3a type) Greek key motifs in protein vibrillin from a pathogenic bacterium Vibrio cholerae. This domain is a part of a large Vibrio-specific protein prevalent in Vibrio species (found in at least fourteen different strains sequenced so far). The domain contains two canonical N/D-N/D-X-X-S/T-S Ca²⁺-binding motifs, and bind Ca²⁺. Unlike spherulin 3a and other microbial homologues studied so far, βγ-crystallin domain of vibrillin self-associates forming oligomers of various sizes including dimers. The fractionated dimers readily form octamers in concentration-dependent manner, suggesting an association between these two major forms. The domain associates/dissociates forming dimers at the cost of monomeric populations in the presence of Ca²⁺. No such effect of Ca²⁺ has been observed in oligomeric species. The equilibrium unfolding of both forms follows a similar pattern, with the formation of an unfolding intermediate at sub-molar concentrations of denaturant. These properties exhibited by this βγ-crystallin domain are not shown by any other domain studied so far, demonstrating the diversity in domain properties.     

The Structural Basis for Phospholamban Inhibition of the Calcium Pump in Sarcoplasmic Reticulum

P-type ATPases are a large family of enzymes that actively transport ions across biological membranes by interconverting between high (E1) and low (E2) ion-affinity states; these transmembrane transporters carry out critical processes in nearly all forms of life. In striated muscle, the archetype P-type ATPase, SERCA (sarco(endo)plasmic reticulum Ca²⁺-ATPase), pumps contractile-dependent Ca²⁺ ions into the lumen of sarcoplasmic reticulum, which initiates myocyte relaxation and refills the sarcoplasmic reticulum in preparation for the next contraction. In cardiac muscle, SERCA is regulated by phospholamban (PLB), a small inhibitory phosphoprotein that decreases the Ca²⁺ affinity of SERCA and attenuates contractile strength. cAMP-dependent phosphorylation of PLB reverses Ca²⁺-ATPase inhibition with powerful contractile effects. Here we present the long sought crystal structure of the PLB-SERCA complex at 2.8-Å resolution. The structure was solved in the absence of Ca²⁺ in a novel detergent system employing alkyl mannosides. The structure shows PLB bound to a previously undescribed conformation of SERCA in which the Ca²⁺ binding sites are collapsed and devoid of divalent cations (E2-PLB). This new structure represents one of the key unsolved conformational states of SERCA and provides a structural explanation for how dephosphorylated PLB decreases Ca²⁺ affinity and depresses cardiac contractility.     

Akt regulates L-type Ca2+ channel activity by modulating Cavα1 protein stability

The insulin IGF-1–PI3K–Akt signaling pathway has been suggested to improve cardiac inotropism and increase Ca²⁺ handling through the effects of the protein kinase Akt. However, the underlying molecular mechanisms remain largely unknown. In this study, we provide evidence for an unanticipated regulatory function of Akt controlling L-type Ca²⁺ channel (LTCC) protein density. The pore-forming channel subunit Ca_vα1 contains highly conserved PEST sequences (signals for rapid protein degradation), and in-frame deletion of these PEST sequences results in increased Ca_vα1 protein levels. Our findings show that Akt-dependent phosphorylation of Ca_vβ2, the LTCC chaperone for Ca_vα1, antagonizes Ca_vα1 protein degradation by preventing Ca_vα1 PEST sequence recognition, leading to increased LTCC density and the consequent modulation of Ca²⁺ channel function. This novel mechanism by which Akt modulates LTCC stability could profoundly influence cardiac myocyte Ca²⁺ entry, Ca²⁺ handling, and contractility.     

Characteristics of Ca2+/calmodulin- and Ca2+/phosphatidylserine-stimulated phosphoproteins in rat striatum

The characteristics of endogenous Ca²⁺/calmodulin (CaM)- and Ca²⁺/phosphatidylserine (PS)-stimulated phosphorylated proteins in the striatum of rat were partially determined and compared in this study. The Ca²⁺/CaM-dependent phosphoproteins were associated with serine and threonine residues. The sensitivity of these proteins for phosphorylation by Ca²⁺/CaM was not affected by pretreatment of tissue with Ca²⁺ chelating agent, EGTA or with non-ionic detergent, Triton X-114. Triton X-114 phase separation experiments revealed that these Ca²⁺/CaM-dependent phosphoproteins were partitioned in the detergent rich phase suggesting that they are integral proteins of the striatal membrane. On the other hand, the Ca²⁺/PS-dependent phosphorylated proteins were primarily associated with the serine residue. Phosphorylation of these proteins by Ca²⁺/PS were abolished after the treatment with EGTA or Triton X-114. These results suggest that Ca²⁺/PS-dependent striatal phosphoproteins are biochemically unstable in maintaining their state of phosphorylation.     

GM3与Ca2+-ATP酶的重建及其冷冻断裂电镜观察

应用生物膜的分离与重建技术, 将GM3、大豆磷脂与肌质网Ca²⁺-ATP酶共同重建在脂质体上, 酶活力明显增加. 经负染、冷冻断裂复型后电镜等形态学方法证实形成的脂酶体囊泡封闭性好,脂酶体上Ca²⁺-ATP酶蛋白颗粒均匀、直径增大.     

Calcium regulation in wild populations of a freshwater cartilaginous fish, the lake sturgeon Acipenser fulvescens

Lake sturgeon, Acipenser fulvescens, are one of a few species of cartilaginous fishes that complete their life cycle entirely in freshwater. Sturgeons maintain very low concentrations of circulating calcium (Ca²⁺) compared with other vertebrates, and therefore, face unique challenges in regard to Ca²⁺ regulation, which are likely to be magnified during vitellogenic stages of the reproductive cycle. In the present study, Ca²⁺ concentrations and associated hormones of female and male lake sturgeon were examined in two wild populations, and were related to reproductive stage. In both populations, free, bound and total Ca²⁺ were low, peaking in mid-late vitellogenic females. Internal Ca²⁺ and phosphate (PO₄³⁻) concentrations were inversely related to environmental concentrations, suggesting that these ions are preferentially retained and that mechanisms for mobilization are up-regulated under diminished environmental concentrations. Plasma 17β-estradiol, 11-ketotestosterone and testosterone, peaked in mid-late vitellogenic females, while the androgens peaked in spawning males. Urine Ca²⁺ was more tightly regulated than other divalent ions and decreased in spawning fish. Therefore, the increases in free plasma Ca²⁺, the very low circulating concentrations of free and total Ca²⁺, and the increase in PO₄³⁻ and bound Ca²⁺ in low Ca²⁺ environments indicate unique adaptations to Ca²⁺ regulation in the lake sturgeon.     

The functional unit of calcium-plus-magnesium-ion-dependent adenosine triphosphatase from sarcoplasmic reticulum. The aggregational state of the deoxycholate-solubilized protein in an enzymically active form

Vesicles consisting of (Ca²⁺+Mg²⁺)-dependent ATPase (adenosine triphosphatase), and lipid were prepared from sarcoplasmic reticulum of rabbit skeletal muscle. As with non-ionic detergents [le Maire, Møller & Tanford (1976) Biochemistry 15, 2336–2342] the (Ca²⁺+Mg²⁺)-dependent ATPase after solubilization by deoxycholate showed a pronounced tendency to form oligomers in gel-chromatographic experiments, when eluted in the presence of deoxycholate and phosphatidylcholine. To evaluate the functional significance of oligomer formation the properties of enzymically active preparations of ATPase, solubilized by deoxycholate, were studied. Such preparations were obtained at a protein concentration of 2.5mg/ml in the presence of a high salt concentration (0.4m-KCl) and sucrose (0.3m) in the solubilization medium. Analytical ultracentrifugation of solubilized ATPase showed one protein boundary moving at the same rate as gel-chromatographically prepared monomeric ATPase (s_20,w=6.0S). From simultaneous measurements of the diffusion coefficient an apparent molecular weight of 133000 was calculated, consistent with solubilization of ATPase in predominantly monomeric form. The enzymic activity of deoxycholate-solubilized ATPase when measured directly in the solubilization medium at optimal Ca²⁺ and MgATP concentrations was about 35–50% of that of vesicular ATPase. The dependence of enzymic activity on MgATP concentration indicated that the solubilized ATPase retained high-affinity binding of MgATP, but the presence of high concentrations of the nucleotide did not stimulate activity further, in contrast with that of vesicular ATPase. The dependence of enzymic activity on the free Ca²⁺ concentration was essentially the same for both solubilized and vesicular forms, indicating that interaction of ATPase with more than one molecule of Ca²⁺ is required for enzyme activity. Solubilized enzyme at 20°C was phosphorylated to about the same degree as vesicular ATPase. It is concluded that the catalytic activity of monomeric ATPase retains most of the features of vesicular ATPase and that extensive oligomer formation in gel-chromatographic experiments in the presence of deoxycholate probably reflects processes taking place during inactivation and delipidation of the protein.     

Protein Kinase C�� Negatively Regulates Store-independent Ca2+ Entry and Phosphatidylserine Exposure Downstream of Glycoprotein VI in Platelets

Platelet activation must be tightly controlled to provide an effective, but not excessive, response to vascular injury. Cytosolic calcium is a critical regulator of platelet function, including granule secretion, integrin activation, and phosphatidylserine (PS) exposure. Here we report that the novel protein kinase C isoform, PKCθ, plays an important role in negatively regulating Ca²⁺ signaling downstream of the major collagen receptor, glycoprotein VI (GPVI). This limits PS exposure and so may prevent excessive platelet procoagulant activity. Stimulation of GPVI resulted in significantly higher and more sustained Ca²⁺ signals in PKCθ^−/− platelets. PKCθ acts at multiple distinct sites. PKCθ limits secretion, reducing autocrine ADP signaling that enhances Ca²⁺ release from intracellular Ca²⁺ stores. PKCθ thereby indirectly regulates activation of store-operated Ca²⁺ entry. However, PKCθ also directly and negatively regulates store-independent Ca²⁺ entry. This pathway, activated by the diacylglycerol analogue, 1-oleoyl-2-acetyl-sn-glycerol, was enhanced in PKCθ^−/− platelets, independently of ADP secretion. Moreover, LOE-908, which blocks 1-oleoyl-2-acetyl-sn-glycerol-induced Ca²⁺ entry but not store-operated Ca²⁺ entry, blocked the enhanced GPVI-dependent Ca²⁺ signaling and PS exposure seen in PKCθ^−/− platelets. We propose that PKCθ normally acts to restrict store-independent Ca²⁺ entry during GPVI signaling, which results in reduced PS exposure, limiting platelet procoagulant activity during thrombus formation.