Recycling of platelet phosphorylation and cytoskeletal assembly

The shape change and aggregation of washed platelets induced by 10 microM arachidonic acid (AA) can be reversed by 20 ng/ml prostacyclin (PGI2), but these platelets can be reactivated by treatment with 30 microM epinephrine and subsequent addition of 10 microM AA mixture. These events may be modulated by cAMP since 2 mM dibutyryl cAMP also reversed activation without reactivation by epinephrine and AA. We examined protein phosphorylation and formation of cytoskeletal cores resistant to 1% Triton X-100 extraction of these platelets and correlated these processes with aggregation, fibrinogen binding, and changes in ultrastructure. Unactivated platelet cores contained less than 15% of the total actin and no detectable myosin or actin-binding protein. AA-induced cytoskeletal cores, which contained 60-80% of the total actin, myosin, and actin-binding protein as the major components, were disassembled back to unactivated levels by PGI2 and then fully reassembled by epinephrine and AA. Phosphorylation of myosin light chain and a 40,000-dalton protein triggered by AA (two- to fivefold) was reversed to basal levels by PGI2 but was completely restored to peak levels upon addition of the epinephrine and AA mixture. The reversibility of actin-binding protein phosphorylation could not be established clearly because both PGI2 and dibutyryl cAMP caused its phosphorylation independent of activation. With this possible exception, cytoskeletal assembly with associated protein phosphorylation, aggregation, fibrinogen binding, and changes in ultrastructure triggered by activation are readily and concertedly recyclable.     

Exchange assay for androgen receptors in the presence of molybdate

Several reports have shown that sodium molybdate stabilizes steroid hormone receptors. We have utilized these observations to develop an exchange assay for the androgen receptor at elevated temperatures. Exchange was found to be complete after 30 min at 30 degrees C. Receptor degradation was negligible during this treatment. Scatchard analysis indicated that the dissociation constant of the androgen receptor was similar both in the absence (Kd = 3.9 nM) and presence (Kd = 2.9 nM) of molybdate. Steroid specificity of the androgen receptor was unaltered by this treatment. The exchange procedure was reproducible, with an interassay variation of 2.45% and intraassay variation less than 10.0%. Using this assay, highest concentrations of androgen binding were measured in androgen target tissues of the rat (Dunning R3327 tumor, prostate and seminal vesicle; 23.37, 20.20 and 19.84 fmol/mg protein respectively). Lower concentrations were observed in other tissues (lung, brain, heart, spleen, liver and kidney; 9.06, 5.63, 3.50, 2.42, 2.33 and 1.36 fmol/mg protein respectively). These results demonstrate that molybdate stabilization of the androgen receptor allows efficient steroid exchange without significant alteration of the receptor's steroid binding properties. Furthermore, this exchange assay can be used to obtain a reasonable measurement of receptor concentrations in different androgen target tissues.     

The submicrosomal distribution of dolichyl phosphate and dolichyl phosphate phosphatase in rat liver

Rat liver microsomes were isolated and fractionated into Golgi, smooth endoplasmic reticulum (SER), and rough endoplasmic reticulum (RER), and the purity of these preparations was determined. The dolichyl phosphate (Dol-P) content of whole microsomes and of each of the submicrosomal fractions was estimated using high pressure liquid chromatography. Dol-P accounts for 4 and 40% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in whole liver and in purified microsomes, respectively. Concentrations equal to 58, 77, and 108 ng of Dol-P/mg of protein were found in Golgi, SER, and RER, respectively. These values represent 3, 36, and 54% of the sum of the alcohol, the fatty acyl esters of dolichol, and monophosphate forms present in each of these same fractions, respectively. Increases in the Dol-P content of rat liver were observed as early as 12 h after turpentine-induced inflammation and increased 2-fold over 36 h. In this system, Dol-P accounts for no more than 50% of the sum of all phosphorylated and pyrophosphorylated dolichol intermediates present. The specific activity for dolichyl phosphate phosphatase was highest by more than a factor of 2 in Golgi membrane. Specific activities obtained for SER and RER were 42 and 11% of those present in Golgi. The major requirement for Dol-P is thought to be for the saccharide and oligosaccharide transferase reactions which are presumed to take place in RER. The discovery of significant quantities of Dol-P in Golgi and SER is consistent with a possible role of Dol-P in the transport of sugars required for glycoprotein synthesis and processing from a cytosolic to luminal orientation.     

Cholesteryl hemisuccinate alters membrane fluidity, angiotensin receptors, and responses in adrenal glomerulosa cells

We examined the effects of cholesteryl hemisuccinate on membrane fluidity and angiotensin II (AII) actions in bovine adrenal glomerulosa cells. Incubating cells with cholesteryl hemisuccinate decreased membrane fluidity and markedly inhibited AII binding. The effect on binding was characterized by a decrease in AII receptor number. The effects of AII on phosphatidyl inositol turnover and calcium fluxes, proposed intermediaries of AII actions on aldosterone secretion, were less impaired than AII binding by cholesteryl hemisccinate. AII stimulation of aldosterone secretion was preserved despite the decrease in AII binding after cholesteryl hemisuccinate treatment. These results indicate that AII binding can be dissociated from its effects on aldosteronogenesis by a reagent that alters membrane fluidity.     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

Thymus cell differentiation and in vivo T-cell migration. I. Migration of lectin-selected thymocytes

The in vivo quantitative distribution and tissue positioning of mouse thymocytes selected in vitro by Lyt phenotype and lectin binding properties were examined. Lyt 1+2- thymocytes were selected for by cytotoxic elimination; peanut agglutinin (PNA) and soybean agglutinin (SBA) binding and nonbinding thymocyte fractions were separated by an agglutinin technique. Selected cell suspensions were labelled in vitro with 51chromium (51Cr) or [3H]adenosine. Labeled washed cells were injected intravenously into syngeneic recipients which were killed at 1, 24 or 48 hr. In recipients of 51Cr-labeled cells, tissues were collected for gamma counting, and the overall percentage recovery of injected radiolabel from the various tissues was assessed. Tissues collected from recipients of [3H]adenosine-labeled cells were fixed, sectioned, and processed for autoradiography; the positioning of labeled cells within the tissues was determined. Selected Lyt 1+2-, PNA-, and SBA- sets all showed significantly enhanced entry into lymph nodes and intestinal lymphoid tissues. Entry of SBA+ cells into these tissues was comparable to that of peripheral T cells. PNA- and SBA- selected sets, but not Lyt 1+2- selected cells, also showed increased localization to the spleen and lungs, and decreased localization to the liver. By autoradiography, PNA- cells entered lymphoid tissues much more than PNA+ cells, and at 1 hr fewer PNA+ cells in spleen were associated with lymphoid follicles. At 24 and 48 hr almost all labeled cells in lymphoid tissues were positioned in T-dependent areas. These results suggest that enrichment for thymocyte subpopulations described as "mature" also enriches for cells with the ability to enter lymphoid tissue. They also suggest that interactions at other tissue sites are important in the determination of in vivo migration, and that surface carbohydrate composition is an important factor in this determination.     

Separation and properties of human brain hexosaminidase C

Hexosaminidase C was separated from human brain supernatant by immunoadsorption of the A and B forms on to a column of immobilized antibody followed by preparative starch-block electrophoresis. There were some differences in the properties of hexosaminidase C preparations after each of these stages, shown by comparison of their heat-inactivation characteristics and filtration through Bio-Gel P-200. The C form prepared by both separation steps had properties which differed markedly from those of the A and B isoenzymes; its molecular weight was much larger, greater than 200000, it had optimum activity between pH6 and 7 and could not be successfully eluted from DEAE-cellulose, even with high salt concentrations, or from Sephadex G-200. These results seem to support the proposal that the C form is under a separate genetic control from the others.