Anti-SSTR2 peptide based targeted delivery of potent PLGA encapsulated 3,3'-diindolylmethane nanoparticles through blood brain barrier prevents glioma progression.

Current therapy for Glioblastoma is insufficient because of the presence of blood brain barrier. It limits the transport of essential drugs to the tumor sites. To overcome this limitation we strategized the delivery of an anticancer compound 3,3'-diindolylmethane by encapsulation in poly (lactic-co-glycolic acid) nanoparticles. These nanoparticles were tagged with a novel peptide against somatostatin receptor 2 (SSTR2), a potential target in glioma. The nanoformulation (27-87nm) had loading and encapsulation efficiency of 7.2% and 70% respectively. It was successfully internalized inside the glioma cells resulting in apoptosis. Furthermore, an in vivo bio-distribution study revealed the selective accumulation of the nanoformulation into rat brain tumor sites by crossing the blood brain barrier. This resulted in abrogation of epidermal growth factor receptor pathway activation in glioma cells. Our novel nanopreparation therefore shows great promise to serve as a template for targeted delivery of other therapeutics in treating GBM.

Benzo(a)pyrene Induced p53 Mediated Male Germ Cell Apoptosis: Synergistic Protective Effects of Curcumin and Resveratrol.

Benzo(a)pyrene (B(a)P) is an environmental toxicant that induces male germ cell apoptosis. Curcumin and resveratrol are phytochemicals with cytoprotective and anti-oxidative properties. At the same time resveratrol is also a natural Aryl hydrocarbon Receptor (AhR) antagonist. Our present study in isolated testicular germ cell population from adult male Wistar rats, highlighted the synergistic protective effect of curcumin and resveratrol against B(a)P induced p53 mediated germ cell apoptosis. Curcumin-resveratrol significantly prevented B(a)P induced decrease in sperm cell count and motility, as well as increased serum testosterone level. Curcumin-resveratrol co-treatment actively protected B(a)P induced testicular germ cell apoptosis. Curcumin-resveratrol co-treatment decreased the expression of pro-apoptotic proteins like cleaved caspase 3, 8 and 9, cleaved PARP, Apaf1, FasL, tBid. Curcumin-resveratrol co-treatment decreased Bax/Bcl2 ratio, mitochondria to cytosolic translocation of cytochrome c and activated the survival protein Akt. Curcumin-resveratrol decreased the expression of p53 dependent apoptotic genes like Fas, FasL, Bax, Bcl2, and Apaf1. B(a)P induced testicular reactive oxygen species (ROS) generation and oxidative stress were significantly ameliorated with curcumin and resveratrol. Curcumin-resveratrol co-treatment prevented B(a)P induced nuclear translocation of AhR and CYP1A1 (Cytochrome P4501A1) expression. The combinatorial treatment significantly inhibited B(a)P induced ERK 1/2, p38 MAPK and JNK 1/2 activation. B(a)P treatment increased the expression of p53 and its phosphorylation (p53 ser 15). Curcumin-resveratrol co-treatment significantly decreased p53 level and its phosphorylation (p53 ser 15). The study concludes that curcumin-resveratrol synergistically modulated MAPKs and p53, prevented oxidative stress, regulated the expression of pro and anti-apoptotic proteins as well as the proteins involved in B(a)P metabolism thus protected germ cells from B(a)P induced apoptosis.

Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells.

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3'-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3'-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.

Formulation and antitumorigenic activities of nanoencapsulated nifetepimine: A promising approach in treating triple negative breast carcinoma.

Triple negative breast cancer (TNBC) is one of the most common invasive malignancies among women, associated with poor prognosis. Standard chemotherapy targets all dividing cells, resulting in dose-limiting toxicities. In this study, we demonstrated a strategy of encapsulating a hydrophobic synthetic compound, nifetepimine, having anticancer properties, in poly (lactic-co-glycolic acid) nanoparticles to increase selectivity of drug to cancerous cells with minimum toxicity towards normal cells. Nanoencapsulated nifetepimine (30-100nm) having loading and encapsulation efficiency of 7.45% and 75% respectively, was successfully internalized inside TNBC cells upon sustained release resulting in apoptosis. An in vivo bio-distribution study indicated that nanonifetepimine selectively accumulated into breast tumor sites of mice, primarily due to prolonged blood circulation time and binding of nifetepimine to epidermal growth factor receptor that remains overexpressed in most of the TNBC tumors. Moreover, we observed significant reduction in breast tumor volume with improved survival implying high tumor targetability of nanonifetepimine.

Resveratrol ameliorates benzo(a)pyrene-induced testicular dysfunction and apoptosis: involvement of p38 MAPK/ATF2/iNOS signaling.

Benzo(a)pyrene [B(a)P] is an environmental toxicant that alters the steroidogenic profile of testis and induces testicular dysfunction. In the present study, we have investigated the molecular signaling of B(a)P and the ameliorative potential of the natural aryl hydrocarbon receptor (AhR) antagonist and antioxidant, resveratrol, on B(a)P-induced male reproductive toxicity. Studies showed that B(a)P treatment resulted in p38 MAPK activation and increased inducible nitric oxide synthase (iNOS) production along with testicular apoptosis and steroidogenic dysfunction. Resveratrol cotreatment maintained testicular redox potential, increased serum testosterone level and enhanced expression of major testicular steroidogenic proteins (CYPIIA1, StAR, 3ßHSD, 17ßHSD) and prevented subsequent onset of apoptosis. Resveratrol cotreatment resulted inhibition of testicular cytochrome P4501A1 (CYP1A1) expression, which is the major B(a)P metabolizing agent for BPDE-DNA adduct formation. Resveratrol also significantly decreased the B(a)P-induced AhR protein level, its nuclear translocation and subsequent promoter activation, thereby decreased the expression of CYP1A1. Resveratrol also down-regulated B(a)P-induced testicular iNOS production through suppressing the activation of p38 MAPK and ATF2, thus improved the oxidative status of the testis and prevented apoptosis. Our findings cumulatively suggest that resveratrol inhibits conversion of B(a)P into BPDE by modulating the transcriptional regulation of CYP1A1 and acting as an antioxidant thus prevents B(a)P-induced oxidative stress and testicular apoptosis.

Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells.

Triple-negative breast cancer (TNBC), is a specific subtype of epithelial breast tumors that are immuno-histochemically negative for the protein expression of the estrogen receptor (ER), the progesterone receptor (PR) and lack over expression/gene amplification of HER2. This subtype of breast cancers is highly metastatic, shows poor prognosis and hence represents an important clinical challenge to researchers worldwide. Thus alternative approaches of drug development for TNBC have gained utmost importance in the present times. Dietary indole and its derivatives have gained prominence as anti-cancer agents and new therapeutic approaches are being developed to target them against TNBC. But a major drawback with 3, 3'di Indolyl methane (DIM) is their poor bioavailability and high effective concentration against TNBC. However, the Aryl methyl ring substituted analogs of DIM display interesting anti-cancer activity in breast cancer cells. In the current study we report the synthesis of a novel synthetic aryl methyl ring substituted analog of DIM, named as Phemindole as an effective anti-tumor agent against TNBC cells. Furthermore, we enumerated that Phemindole caused reactive oxygen species mediated mitochondrial-dependent apoptosis in MDAMB-231 cells. Furthermore, Phemindole mediated Store Operated Calcium Entry (SOCE) retardation favored inactivation of STIM1 and henceforth activated ER stress to induce apoptosis in TNBC cells. Simultaneously, Phemindole was also found to restrict the in vitro cell migration through its anti mitotic property and pFAK regulation. Studies extended to ex ovo and in vivo mice models further validated the efficacy of Phemindole. Thus our results cumulatively propose Phemindole as a new chemotherapeutic regime which might be effective to target the deadly aspects of the TNBC.

Cross-talk between endoplasmic reticulum (ER) stress and the MEK/ERK pathway potentiates apoptosis in human triple negative breast carcinoma cells: role of a dihydropyrimidone, nifetepimine.

Triple negative breast cancers (TNBC) are among the most aggressive and therapy-resistant breast tumors and currently possess almost no molecular targets for therapeutic options in this horizon. In the present study we discerned the molecular mechanisms of potential interaction between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway in inducing apoptosis in TNBC cells. Here we observed that induction of ER stress alone was not sufficient to trigger significant apoptosis but simultaneous inhibition of the MEK/ERK pathway enhanced ER stress-induced apoptosis via a caspase-dependent mechanism. Our study also demonstrated nifetepimine, a dihydropyrimidone derivative as a potent anti-cancer agent in TNBC cells. Nifetepimine down-regulated the MEK/ERK pathway in MDAMB-231 and MDAMB-468 cells and resulted in blockage of ER stress-mediated GRP78 up-regulation. Detailed mechanistic studies also revealed that nifetepimine by down-regulating pERK expression also declined the promoter binding activity of TFII-I to the GRP78 promoter and in turn regulated GRP78 transcription. Studies further extended to in vivo Swiss albino and SCID mice models also revalidated the anti-carcinogenic property of nifetepimine. Thus our findings cumulatively suggest that nifetepimine couples two distinct signaling pathways to induce the apoptotic death cascade in TNBC cells and raises the possibility for the use of nifetepimine as a potent anti-cancer agent with strong immune-restoring properties for therapeutic intervention for this group of cancer bearers.

Regulation of PKC mediated signaling by calcium during visceral leishmaniasis.

Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca2+ pumps and channels. Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.

Nifetepimine, a dihydropyrimidone, ensures CD4+ T cell survival in a tumor microenvironment by maneuvering sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA).

Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca(2+)-ATPase (SERCA) have long been known to regulate intracellular Ca(2+) homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4(+) T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4(+) T cell apoptosis. Prostaglandin E(2) produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4(+) T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4(+) T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.

Regioselective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones: screening of their biological activities against Ca(2+)-ATPase.

A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base Cs(2)CO(3) and alkyl halides at room temperature has been reported. The selectivity of this methodology is excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca(2+)-ATPase, which revealed that the parent compounds can act as Ca(2+)-ATPase inhibitors whereas the N1-alkylated derivatives are inefficient for this purpose.

Elucidation of the involvement of p14, a sperm protein during maturation, capacitation and acrosome reaction of caprine spermatozoa.

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

Efficient synthesis of (6-deoxy-glycopyranosid-6-yl) sulfone derivatives and their effect on Ca2+-ATPase.

Synthesis of a series of novel (6-deoxy-glycopyranosid-6-yl) sulfone derivatives has been achieved using a general synthetic strategy. Yields were excellent in every case. The synthetic compounds were evaluated for their biological potential against Ca2+-ATPase, an important enzyme involves in transporting Ca2+ across the cell membranes.

Eicosapentaenoic and docosahexaenoic acids enriched polyunsaturated fatty acids from the coastal marine fish of Bay of Bengal and their therapeutic value.

Eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) enriched polyunsaturated fatty acids (PUFA) significantly present in marine fish oil emerge as preventive agents for combating many health problems specially in chronic or metabolic disorders. The fish in the coastal area of Bay of Bengal has remained unexplored with respect to EPA/DHA enriched PUFA content in its oils, although it may be a potential source in harnessing the health benefit. In this study, seven varieties of the coastal fish were analysed for the content of EPA/DHA. The one locally known as lotte, (Harpadon nehereus) though has low content of total lipids, was found to have high EPA/DHA in its oil. The phospholipids rich fraction was extracted from the total fish oil. The EPA/DHA enriched PUFA was isolated to investigate the potential use for health benefits. EPA/DHA is found to act as protective agent against mercury poisoning studied in cell culture as well as in animal mode. It is found to be highly preventive in diabetes. The lotte is available in the coastal area of Bay of Bengal adjoining West Bengal, India in large scale and it is the first report showing EPA/DHA enriched PUFA in these fish oil that can be availed to harness in important health benefits.

Regulation of tyrosine kinase activity during capacitation in goat sperm.

Protein tyrosine phosphorylation is a key event accompanying sperm capacitation. Although this signaling cascade generates an array of tyrosine-phosphorylated polypeptides, their molecular characterization is still limited. It is necessary to differentiate the localization of the tyrosine-phosphorylated proteins in spermatozoa to understand the link between the different phosphorylated proteins and the corresponding regulated sperm function. cAMP plays a pivotal role in the regulation of tyrosine phosphorylation. The intracellular cAMP levels were raised in goat spermatozoa by the addition of the phosphodiesterase inhibitor, IBMX in conjugation with caffeine. Tyrosine phosphorylation was significantly up-regulated following treatment with these two reagents. Treatment of caudal spermatozoa with IBMX and caffeine, time dependent up-regulated phosphorylation of the protein of molecular weights 50 and 200 kDa was observed. Increased phosphorylation was observed with a combination of IBMX and caffeine treatment. Tyrosine phosphorylation in caput spermatozoa was not affected significantly under these conditions. The expression level of tyrosine kinase in sperm was examined with specific inhibitors and with anti-phosphotyrosine antibody. The indirect immunofluorescence staining was carried out on ethanol permeabilized sperm using anti-phosphotyrosine antibody. Western blot analysis was done using two separate PKA antibodies: anti-PKA catalytic and anti-PKA RIalpha. Almost no difference was found in the intracellular presence of the PKA RIalpha and RIIalpha subunits in caput and caudal epididymal spermatozoa. However, the catalytic subunit seemed to be present in higher amount in caudal spermatozoa. The results show that caprine sperm displays an enhancement of phosphorylation in the tyrosine residues of specific proteins under in vitro capacitation conditions.

Characterization of a low-molecular-mass stimulator protein of Mg2+-independent Ca2+-ATPase: effect on phosphorylation/dephosphorylation, calcium transport and sperm-cell motility.

A 14 kDa cytosolic protein purified from bovine brain homogenate has been recently reported as a stimulator of goat spermatozoa Mg2+-independent Ca2+-ATPase. In the present study, we demonstrate the formation of the [gamma-32P]ATP-labelled phosphoenzyme as the 110 kDa phosphoprotein and its rapid decomposition in presence of the stimulator protein. Together with the cross-reactivity of this 110 kDa protein with an anti-SERCA (sarcoplasmic/endoplasmic reticulum Ca2+-ATPase) 2a antibody, the ATPase can now be conclusively said to belong to the SERCA family, which is activated by the stimulator. The ability of the stimulator to enhance the Ca2+ transport has been elucidated from 45Ca2+ uptake studies and was found to be sensitive to Ca2+ channel blockers. CD revealed an alpha-helical structure of the stimulator. The amino acid analysis suggests that it is composed primarily of hydrophobic and some acidic amino acid residues. The pI of 5.1 has been re-confirmed from two-dimensional electrophoresis. Immuno-cross-reactivity studies indicate that the stimulator or similar proteins are present in cytosolic fractions of liver, kidney or testes in different species, but brain is the richest source. Proteomic analyses of its trypsinized fragments suggest its similarity with bovine THRP (thyroid hormone-responsive protein). The physiological significance of the stimulator has been suggested from its ability to activate sperm-cell motility.

Structural and functional characterization and physiological significance of a stimulator protein of Mg2+-independent Ca2-ATPase isolated from goat spermatozoa.

Recently a low-molecular-mass protein purified from goat testes cytosol has been reported from our laboratory which is found to stimulate Mg2+ -independent Ca2+-ATPase without any significant effect on Mg2+-dependent Ca2+-ATPase. In the present study, detailed structural and functional characterization, as well as the physiological significance of the protein has been described. The stimulatory effect is found to be inhibited by known inhibitors of P-type ATPases, vanadate and lanthanum chloride. Monitoring of the phosphoenzyme intermediate by autoradiography has shown that the stimulation of the ATPase is due to the enhancement in the rate of dephosphorylation of the overall reaction step. Along with the stimulation of the enzyme activity, the protein is found to enhance the calcium uptake. Amino acid analysis data show that the stimulator contains about 26% non-polar amino acid facilitating easy penetration to the hydrophobic core of the membrane bound ATPase. Circular dichroism analysis of the protein suggested the presence of all secondary structural elements. The Western-blotting experiment shows its expression level is the highest in goat testes. Peptide fragments obtained in MALDI-MS analysis when subjected to MSDB database search by MASCOT search engine reveals that the proteins of close similarity with the protein under study are actin related protein 2/3 complex subunit, peptidyl-prolyl cis-trans isomerase and gastrin releasing peptide precursor. Besides, the protein under study is also shown to decrease the forward motility of goat sperm without having any significant effect on the total motility indicating its possible role in fertility regulation.

Purification of protein from a crude mixture through SDS-PAGE transfer method.

SDS-polyacrylamide gel electrophoresis (SDS-PAGE) transfer method was used for purification and enrichment of the protein from crude sample. Coomassie bluc/ZnSO4 stained protein band(s) containing intact polyacrylamide gel were loaded on to another polyacrylamide gel either alone or as pooled gel bands. Two/three bands were combined together and arranged tightly over one another, sealed with stacking gel and ran in another gel, which was quite useful for enrichment and purification of a particular protein from a complex mixture. Recovery of protein by gel transfer method was found to be 70% in case of ZnSO4 staining, whereas around 30% recovery was possible, following Coomassie blue staining. The method described here for purification of protein(s) from a complex mixture, following gel transfer procedure could be useful for further characterization of the desired protein.

Stimulation of Mg2+-independent form of Ca2+-ATPase by a low molecular mass protein purified from goat testes cytosol.

A low molecular mass protein purified from goat (Capra hircus) testes cytosol following gel filtration and anion exchange chromatographic separation stimulates Mg(2+)-independent Ca(2+)-ATPase activity without any significant effect on Mg(2+)-dependent Ca(2+)-ATPase. Stimulation of the ATPase is due to an increase in the rate of dephosphorylation of the overall reaction step of the enzyme. Binding of the stimulator increases the affinity of Ca(2+)-ATPase for Ca(2+). An analysis of enzyme kinetics reveals a reversible type of binding of the stimulator to the ATPase and non-competitive type of stimulation with respect to the substrate. Stimulation seems due to binding of the protein at a single site following Michaelis-Menten model. The protein can also counter the effect of calcium antagonists exerted on the ATPase. The pI of the protein is 6.2 and its molecular mass has been determined to be 13, 961 by Q-TOF-MS.

Regulation of Mg2+-independent Ca2+-ATPase by a low molecular mass protein purified from bovine brain.

The goat sperm microsomal membranes have been found to contain an Mg2+-independent Ca2+-ATPase, a low affinity but highly active enzyme sharing similarities with the SERCA family of ATPases. The present study reports the identification and characterization of a 14 kilodalton cytosolic protein from bovine brain which can act as an endogenous stimulator of the enzyme with an S50 (concentration producing 50% stimulation) of 0.8 mu molar. Kinetic analysis suggests that the stimulation is noncompetitive with respect to the substrate, and the binding site(s) of the stimulator and substrate are distinct. Binding of the stimulator to the enzyme is reversible. The stimulator increases the affinity of the enzyme for calcium as evident from a decrease in K0.5 of the enzyme for calcium in presence of the stimulator. Radioactive labeling of the enzyme with [gamma-32P]-ATP suggests that the stimulator enhances the rate of dephosphorylation of the phosphoenzyme intermediate without altering the phosphorylation reaction step. The stimulatory effect of the protein has been observed only for the Mg2+-independent form of the enzyme, the Mg2+-dependent form being unaffected.