Biomedical subjects

S Engelender

Publications and source records attributed to S Engelender.

22 records · Page 2Linked to original sources

PubMed1996

Pharmacological differentiation between intracellular calcium pump isoforms.

We studied the Ca(2+)-ATPase isoforms of sarco/endoplasmic reticulum (SERCA) derived from cerebellum, cardiac muscle, and skeletal muscle. The Mg2+ dependence varied among the three enzyme preparations. The Ca2+ transport in skeletal muscle vesicles, but not in cerebellar or cardiac vesicles, was activated by free Mg2+ concentrations varying from 0.1 to 0.3 mM. Concentrations of Mg2+ of > 1 mM inhibited Ca2+ transport in all three vesicle preparations but with more pronounced effect in cerebellar and cardiac vesicles. At 10-80 microM, trifluoperazine activated Ca2+ uptake in cerebellar and cardiac vesicles but not in skeletal muscle vesicles. The activation was due to an increase in the coupling ratio between Ca2+ transport and ATP hydrolysis and was observed only in the presence of ATP concentrations of > 100 microM. The Ca2+ transport in all three vesicle preparations was inhibited by trifluoperazine concentrations of > 100 microM. The inhibition promoted by trifluoperazine was prevented by the addition of dimethylsulfoxide (10% v/v) to the medium. The Ca2+ efflux from loaded vesicles was increased by arsenate and even more by trifluoperazine. In skeletal muscle vesicles, the efflux promoted by arsenate was several-fold faster than that promoted in vesicles derived from cerebellum or cardiac muscle. In skeletal muscle, the enhancement of Ca2+ efflux promoted by both arsenate and trifluoperazine was antagonized by thapsigargin, Ca2+, Mg2+, and K+. These agents partly antagonized the enhancement of Ca2+ efflux promoted by trifluoperazine in cardiac vesicles but had little or no effect in the cerebellar vesicles. Finally, Mg.Pi and Mg.ATP, the two substrates that phosphorylate the Ca(2+)-ATPase, antagonized the effect of trifluoperazine in all of the preparations tested. The concentration of ATP needed was in the same range as that of the second K(m) value for ATP (50-300 microM) of the SERCA isoforms. The results indicate that the effect of the drugs on the cytosolic Ca2+ homeostasis may vary depending on the target tissue.

Animals↗

PubMed1995

The Ca(2+)-ATPase isoforms of platelets are located in distinct functional Ca2+ pools and are uncoupled by a mechanism different from that of skeletal muscle Ca(2+)-ATPase.

Vesicles derived from the dense tubular system of platelets possess a Ca(2+)-ATPase that can use either ATP or acetyl phosphate as a substrate. In the presence of phosphate as a precipitating anion, the maximum amount of Ca2+ accumulated by the vesicles with the use of acetyl phosphate was only one-third of that accumulated with the use of ATP. Vesicles derived from the sarcoplasmic reticulum of skeletal muscle accumulated equal amounts of Ca2+ regardless of the substrate used. When acetyl phosphate was used in platelet vesicles, the transport of Ca2+ was inhibited by Na+, Li+, and K+; in sarcoplasmic reticulum vesicles, only Na+ caused inhibition. When ATP was used as substrate, the different monovalent cation had no effect on either sarcoplasmic reticulum or platelet vesicles. The catalytic cycle of the Ca(2+)-ATPase is reversed when a Ca2+ gradient is formed across the vesicle membrane. The stoichiometry between active Ca2+ efflux and ATP synthesis was one in platelet vesicles and two in sarcoplasmic reticulum vesicles. The coupling between ATP synthesis and Ca2+ efflux in sarcoplasmic reticulum vesicles was abolished by arsenate regardless of whether the vesicles were loaded with Ca2+ using acetyl phosphate or ATP. In platelets, uncoupling was observed only when the vesicles were loaded using acetyl phosphate. In both sarcoplasmic reticulum and platelet vesicles, the effect of arsenate was antagonized by thapsigargin (2 microM), micromolar Ca2+ concentrations, P(i) (5-20 mM), and MgATP (10-100 microM). Trifluoperazine also uncoupled the platelet Ca2+ pump but, different from arsenate, this drug was effective in vesicles that were loaded using either ATP or acetyl phosphate. Trifluoperazine enhanced Ca2+ efflux from both sarcoplasmic reticulum and platelet vesicles; thapsigargin, Ca2+, Mg2+, or K+ antagonized this effect in sarcoplasmic reticulum but not in platelet vesicles. The data indicate that the Ca(2+)-transport isoforms found in sarcoplasmic reticulum and in platelets have different kinetic properties.

Arsenates↗

PubMed1993

Protein interactions with a gender-specific gene of Schistosoma mansoni: characterization by DNase I footprinting, band shift and UV cross-linking.

A schistosome gender specific gene (F-10), was used as a probe to characterize DNA binding proteins from adult male and female Schistoma mansoni. Using the band-shift and DNase I footprinting methods, it was found that proteins from male and female worms bound to the intact F-10 gene and to restriction fragments corresponding to different domains of the gene, generating relatively long protected sites. Clear differences between male and female proteins were only observed when nuclear proteins were tested. Thus, gender-specific binding was detected in fragments corresponding to the 5' and 3' ends. UV-induced cross-linking between schistosome proteins and a synthetic oligonucleotide bearing a steroid response element present in the 3' untranslated end of the F-10 gene, revealed a major DNA binding protein with a molecular mass of 30 kDa, in both male and female worms. These results suggested that the activation of transcription of the F-10 gene may depend essentially on nuclear proteins.

Animals↗

PubMed1992

Protein-DNA associations in a gender-specific gene of Schistosoma mansoni: characterization by UV cross-linking, DNase I footprinting and band shift assays.

Protein extracts obtained from male and female schistosomes were incubated with a gender-specific gene, F-10, transcribed only in adult females and encoding a major egg-shell protein. The protein/DNA interaction was measured using the band shift, DNase-I-footprinting and UV cross-linking techniques. The results showed a clear band shift when a 302 bp restriction fragment containing the 3' end of the gene was incubated with either female or male proteins. This fragment also contained a putative steroid hormone regulatory element (HRE). In contrast, only the male proteins produced a shift with the 495 bp fragment corresponding to the middle region of the gene. DNase I footprinting showed that proteins from males and females interacted with the F-10 gene by binding to multiple adjacent sites along the DNA, thus generating relatively long protected fragments of approximately 100 bp. This result suggested that the adjacent binding of several moles of protein occurred at the 5' end of the gene. UV cross-linking between schistosome proteins and a 21 bp synthetic oligonucleotide containing the F-10 HRE, evidenced proteins having MWS of 30, 45 and 65 kDa. These proteins are presumably involved in the regulation of transcription of the F-10 gene.

Animals↗