PubMed · 14817033
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G CSERNYEI. 1950. [Experiments with pulp phosphatase].. https://pubmed.ncbi.nlm.nih.gov/14817033/
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Calcium pump-catalyzed (18)O exchange between inorganic phosphate and water was studied to test the hypothesis that all P-type pumps bind Mg(2+) before P(i) and validate utilization of the rate equation for ordered binding to interpret differences between site-directed mutants and wild-type enzyme. The results were remarkably similar to those obtained earlier with sodium pump (Kasho, V. N., Stengelin, M., Smirnova, I. N., and Faller, L. D. (1997) Biochemistry 36, 8045- 8052). The equation for ordered binding of Mg(2+) before P(i) fit the data best with only a slight chance (0.6%) of P(i) binding to apoenzyme. Therefore, P(i) is the substrate, and Mg(2+) is an obligatory cofactor. The intrinsic Mg(2+) dissociation constant from metalloenzyme (K(M) = 3.5 +/- 0.3 mm) was experimentally indistinguishable from the sodium pump value. However, the half-maximal concentration for P(i) binding to metalloenzyme ((K(p)(')=6.3+/-0.6 mM)) was significantly higher ( approximately 6-fold), and the probability of calcium pump forming phosphoenzyme from bound P(i) (P(c) = 0.04 +/- 0.03) was significantly lower ( approximately 6-fold) than for the sodium pump. From estimates of the rate constants for phosphorylation and dephosphorylation, the calcium pump appears to catalyze phosphoryl group transfer less efficiently than the sodium pump. Ordered binding of Mg(2+) before P(i) implies that both calcium pump and sodium pump form a ternary enzyme.metal.phosphate complex, consistent with molecular structures of other haloacid dehalogenase superfamily members that were crystallized with Mg(2+) and phosphate, or a phosphate analogue, bound.
In a previous study, we showed that protein kinase C betaII (PKC betaII) translocated to a novel juxtanuclear compartment as observed in several cell types (Becker, K. P., and Hannun, Y. A. (2003) J. Biol. Chem. 278, 52747-52754). In this study, we noted the absence of this translocation in MCF-7 breast cancer cells, and we examined the mechanisms underlying this selectivity of response. We show that sustained stimulation of PKC betaII with 4beta-phorbol 12-myristate 13-acetate (PMA) resulted in accumulation of ceramide in MCF-7 cells but not in those cells that showed juxtanuclear translocation of PKC betaII. Addition of exogenous ceramides or formation of endogenous ceramide by the action of bacterial sphingomyelinase prevented PMA-induced translocation of PKC betaII in HEK 293 cells. On the other hand, inhibition of ceramide accumulation with fumonisin B1 restored the ability of PMA to induce translocation of PKC betaII in MCF-7 cells. Taken together, the results showed that endogenous ceramide is both necessary and sufficient for preventing juxtanuclear translocation of PKC betaII in response to PMA. Investigation of the mechanisms of ceramide generation in response to PMA revealed that PMA activated the salvage pathway of ceramide formation and not the de novo pathway. This conclusion was based on the following: 1) the ability of fumonisin B1 but not myriocin to inhibit ceramide formation, 2) the ability of PMA to induce increases in palmitate-labeled ceramide only under chase labeling but not acute pulse labeling, 3) the induction of the levels of sphingosine but not dihydrosphingosine in response to PMA, and 4) induction of sphingomyelin hydrolysis in response to PMA. Together, these results define a novel pathway of regulated formation of ceramide, the salvage pathway, and they define a role for this pathway in regulating juxtanuclear translocation of PKC betaII.
The effect of palmitic acid (PA) and oleic acid (OA) on electrical parameters of planar membranes was studied. We found a substantial difference between the effects of PA and OA on proton transfer. PA induced a small increase in conductance, requiring a new technique for estimating proton-mediated currents across low-conductance planar bilayers in which an electrometer is used to measure the transmembrane current under virtual short circuit (SCC). Open-circuit voltage and SCC were used to determine proton and leak conductances. OA caused a marked increase in membrane conductance, allowing the use of a voltage-clamp technique. From SCC data, we were able to estimate the flip-flop rate constants for palmitate (1 x 10(-6) s(-1)) and oleate (49 x 10(-6) s(-1)) anions. Cholesterol, included in the membrane-forming solution, decreased importantly the leak conductance both in membranes unmodified by FA and in membranes modified by PA added to the bath.