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1.
Tyrosine protein kinase activities have been demonstrated in transformed and normal cell systems. So far, few data on the quantity of protein-bound phosphotyrosine in intact cells have been published. A knowledge of the stoichiometric increase in phosphotyrosine in cells after hormonal induction could be of interest when evaluating the importance of the tyrosine protein kinase activities found. By the addition of a known amount of unlabeled phosphotyrosine to the precipitated protein of 32P-phosphate-labeled cells it was possible after alkaline hydrolysis to spectrophotometrically follow the phosphotyrosine during consecutive chromatographies of the material. From the specific radioactivity of the purified phosphotyrosine the initial concentration of [32P]phosphotyrosine could be calculated. The method proved to be useful for the determination of [32P]phosphotyrosine is small amounts of cells. The minimum detectable amount of [32P]phosphotyrosine was about 1 pmol, and as an example, only 2.5 X 10(6) fibroblasts were needed. By this method it was shown that platelet-derived growth factor increased protein-bound [32P]phosphotyrosine from 600 to 3,200 pmol/g of fibroblasts, while insulin only increased the [32P]phosphotyrosine from 110 to 120 pmol/g of hepatocytes. 相似文献
2.
The perivascular neuropeptide Y (NPY) innervation and its relation to adrenergic nerves of uterine arteries from non-pregnant and pregnant guinea pigs was analyzed immunocytochemically. The NPY content of the uterine artery was, in addition, measured radioimmunologically (RIA). Vasomotor effects of NPY per se and in combination with other vasoconstrictors were examined using a sensitive in vitro method. Pregnancy did not visibly affect density and distribution of NPY-immunoreactive fibres. The NPY fibres contained in addition immunoreactivity to dopamine-beta-hydroxylase (marker for noradrenergic neurons). RIA revealed a slight decrease of NPY content during pregnancy, probably due to the increased smooth muscle volume of uterine arteries. The contractile effect of NPY on uterine arteries was weak, while vasoconstriction induced by various agonists was potentiated by NPY, particularly during pregnancy. It is concluded that perivascular NPY-containing nerve fibres may be involved in the dramatic blood flow alterations that occur in the uterine circulation in connection with pregnancy and partus. 相似文献
3.
Studies on the substrate specificity of cAMP-dependent protein kinase using diastereomeric peptides.
M Eller A Sepp R Toomik P Ekman J J?rv U Ragnarsson L Engstr?m 《Biochemistry international》1991,25(3):453-460
A set of six different diastereomeric hexapeptides RRASVA, each with a D-amino acid residue successively in the six positions, was synthesized and tested as substrates of protein kinase A. It was found that the peptide with D-Ser was neither a substrate, nor an inhibitor of the enzyme. The other five peptides were active as substrates with slightly lower kcat values than that of the all-L amino acid peptide. However, the apparent Km values increased by one to two orders of magnitude, especially when the second arginine or the alanine residue preceding the serine was substituted. The results are discussed. 相似文献
4.
O Nilsson B W?ngberg A Wigander K Lundmark A Dahlstr?m H Ahlman A Bjartell R Ekman 《Peptides》1991,12(5):1077-1083
Delta sleep-inducing peptide (DSIP)-like immunoreactive (LI) material has been detected in nine different human pheochromocytoma tumors by immunocytochemistry. In primary tumors subjected to indirect immunofluorescence a variable number of tumor cells (25-75%) showed positive cytoplasmic labeling after incubation with DSIP antiserum. Tumor cells grown in culture were strongly labeled by the DSIP antiserum with DSIP-LI concentrated to cell bodies. Electron microscopic immunocytochemistry (immunogold labeling) of pheochromocytoma cells demonstrated DSIP-LI over the dense core of secretory granules. The presence of DSIP-LI in several HPLC fractions from conditioned culture media indicates secretion of DSIP-LI from cultured pheochromocytoma cells. The observations suggest that DSIP-LI is synthesized and stored in secretory granules before release. The different HPLC profiles from each of the tumors may reflect differences in processing or turnover of DSIP-LI in pheochromocytoma cells. 相似文献
5.
Cerebrospinal fluid (CSF) from patients without neurological disorder was analyzed after Sep-Pak extraction for beta-endorphin (beta-EP)-immunoreactive components by combined reversed-phase high-performance liquid chromatography (HPLC) and radioimmunoassay. A C-terminal directed antibody detected one major immunoreactive component, probably identical with beta-EP1-31. An N-terminal directed antibody detected several immunoreactive components. One co-eluted with beta-EP1-31 but the others are probably C-terminal truncated or otherwise modified forms of beta-EP1-31. However, they eluted differently from beta-EP1-16 (alpha-endorphin), beta-EP1-26, 1-27 and alpha,N-acetyl-beta-EP1-31. Alternatively, some of the fragments may represent C-terminal extended forms of pro-enkephalin A-derived Met-enkephalin. A Met-enkephalin antiserum detected several immunoreactive components probably representing N-terminal extended forms; neither of them were identical with the beta-EP-immunoreactive components. The results illustrate the heterogeneity of the beta-EP-immunoreactive components in CSF and the need to characterize the beta-EP radioimmunoassay before its application to biological extracts. 相似文献
6.
7.
Absorption of iron has been followed after the intramuscular injection of 12 piglets with iron-dextrin and iron-sorbitol, labelled with Fe59. Disappearance from the site of injection was followed by a method developed for external radioactivity measurements. Blood levels of Fe59 were measured at intervals during a period of 51 days. The tissue distribution pattern of Fe59 was examined in six of the piglets after 30 days. Both iron-dextrin and iron-sorbitol were rapidly absorbed, iron-sorbitol more rapidly. After 20 minutes, 50 per cent of the iron-sorbitol had been absorbed and less than one per cent remained at the site of injection after 48 hours. The amount of Fe59 remaining at the site of injection of iron-dextrin had decreased by half by 60 minutes but some ten per cent had still not been absorbed after ten days. When given as iron-dextrin (94 mg Fe per piglet), the level of Fe59 in the blood remained fairly constant for 14 days. On piglets given iron-sorbitol, (100 mg Fe each) the blood level declined steadily. The amount of Fe59 in the liver, spleen and bone marrow was much greater 30 days after the injection of iron-dextrin than after iron-sorbitol. The relation was reversed in the kidneys, presumably because of the greater excretion of iron-sorbitol in the urine. 相似文献
8.
The failure of cartilage mineralization in osteochondrotic cartilage may be due to an impaired proteoglycan production. Thein vitro production of proteoglycans was therefore studied in the joint cartilage of growing pigs, aged 9–18 weeks, after incubation of cartilage samples with35S-sulfate. Cartilage was obtained from different areas of the femoral condyles and samples from these areas were further divided into three layers, where the superficial layer contains articular cartilage and the basal layers consist of growth cartilage. There was no significant difference in the overall amount of35S-proteoglycans synthesized in different areas of the condyles. However, the total production of35S-proteoglycans per mg tissue was highest in the basal layer in all areas. This was not due to a larger number of cells; the superficial layer contained more DNA per mg tissue than the basal layer. Gel chromatography on Sepharose CL-2B of the cartilage extracts, which resulted in the separation of large proteoglycans (K
av 0.4) from proteoglycans of small hydrodynamic size (K
av 0.8), showed that the relative amount of large proteoglycans increased with the distance from the articular surface. Again, no difference in the relative amounts of large and small proteoglycans were found when cartilage from different areas were compared. Osteochondrotic cartilage was detected in the pigs aged 12–18 weeks. In areas where osteochondrotic cartilage were present, the total production of35S-proteoglycans was lowered and the relative amount of large proteoglycans was less than that found in the adjoining areas devoid of osteochondrotic lesions. The data available indicate that the higher relative amount of small proteoglycans in the osteochondrotic cartilage was partly caused by degradation of the large proteoglycans (aggrecan). 相似文献
9.
Rat liver fructose-1,6-bisphosphatase was partially phosphorylated in vitro and separated into unphosphorylated and fully phosphorylated enzyme. The effects of fructose 2,6-bisphosphate and AMP on these two enzyme forms were examined. Unphosphorylated fructose-1,6-bisphosphatase was more easily inhibited by both effectors. Fructose 2,6-bisphosphate affected both K0.5 and Vmax, while the main effect of AMP was to lower Vmax. Fructose 2,6-bisphosphate and AMP together acted synergistically to decrease the activity of fructose-1,6-bisphosphatase, and since unphosphorylated and phosphorylated enzyme forms are affected differently, this might be a way to amplify the effect of phosphorylation. 相似文献
10.
Age-dependent content of polymerized lipids in Sphagnum fuscum 总被引:1,自引:0,他引:1
The polymerized lipids of Sphagnum fuscum cell wall fragments were found to be composed of long chain hydroxy acids, long chain dicarboxylic acids, fatty alcohols and fatty acids. Their content, on a dry weight basis, was low in the topmost 3 cm of the shoot and increased with shoot age (and depth). A pronounced increase (16-fold) occurred in the contents of hydroxy acids which comprised 76% of the totals at the depth of 40–43 cm. The increase at the depth of 40-43 cm is considered to be at least partly associated with the frequently found destruction of the most suscptible part, the thin-walled stem center. The results suggest that aliphatic lipid polymers are present and acumulated in cell walls resistant to breakdown. 相似文献