Phosphorylation of vitronectin by a protein kinase in human plasma. Identification of a unique phosphorylation site in the heparin-binding domain

Incubation of human plasma with 27 nM [gamma-32P]ATP in the presence of 20 mM MnCl2 results in the phosphorylation of several proteins detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. About 60% of the incorporated radioactivity is found in a 75-kDa protein containing [32P] phosphoserine. The amino-terminal amino acid sequence of the purified 75-kDa [32P]phosphoprotein is identical to that of vitronectin (also termed serum spreading factor or complement S protein). Rabbit antiserum against vitronectin precipitates greater than 90% of the 75-kDa [32P]phosphoprotein from plasma. Reverse phase chromatography of [32P]vitronectin degraded sequentially with CNBr and chymotrypsin yields one major labeled peptide. The sequence of the peptide, Ser-Arg-Arg-Pro-[32PO4]Ser-Arg-Ala-Thr, corresponds to residues 374-381 which are located in the heparin-binding fragment of vitronectin identified by Suzuki et al. [1984) J. Biol. Chem. 259, 15307-15314). Vitronectin could potentially be phosphorylated in vivo with ATP released from injured cells or secreted by platelets activated during hemostasis.     

57Fe and 1H electron-nuclear double resonance of three doubly reduced states Escherichia coli sulfite reductase

We have employed electron-nuclear double resonance (ENDOR) spectroscopy to study the 57Fe hyperfine interactions in the bridged-siroheme [4Fe-4S] cluster that forms the catalytically active center of the two-electron-reduced hemoprotein subunit of Escherichia coli NADPH-sulfite reductase (SiR2-). Previous electron paramagnetic resonance (EPR) and M?ssbauer studies have shown that this enzyme oxidation state can exist in three distinct spectroscopic forms: (1) a "g = 2.29" EPR species that predominates in unligated SiR2-, in which the siroheme Fe2+ is believed to be in an S = 1 state; (2) a "g = 4.88" type of EPR species that predominates in SiR2- in the presence of small amounts of guanidinium sulfate, in which the siroheme Fe2+ is in an S = 2 state; and (3) a classical "g = 1.94" type of EPR species that is seen in SiR2- ligated with CO, in which the siroheme Fe2+ is in an S = 0 state. In all three species, the cluster is in the [4Fe-4S]1+ state, and two distinct types of Fe site are seen in M?ssbauer spectroscopy. ENDOR studies confirm the M?ssbauer assignments for the cluster 57Fe in the g = 1.94 state, with A values of 37, 37, and 32 MHz for site I and ca. 19 MHz for site II. The hyperfine interactions are not too different on the g = 2.29 state, with site I Fe showing more anisotropic A values of 32, 24, and 20 MHz (site II was not detected).(ABSTRACT TRUNCATED AT 250 WORDS)     

Quantification and partial characterization of the hamster sperm proacrosin-acrosin system

The proacrosin-acrosin proteinase system was measured and partially characterized in unpurified extracts of washed hamster epididymal sperm. Autoactivation experiments demonstrated that proacrosin accounted for greater than 98% of the acrosin activity in the sperm extracts from individual animals. Several bands of proteinase activity were observed on gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoretic (gelatin-SDS-PAGE) zymography. The major proteinase activities in the nonactivated extracts corresponded to relative molecular masses (Mr) of 51,000 to 56,000, while less distinct digestion occurred with relative molecular masses of 37,000 to 49,000. It was demonstrated that after a serial dilution of the sperm extract, the proteinase activity in as few as 6,000 sperm could readily be detected by the gelatin-SDS-PAGE methods. Time-course activation studies showed that the zymogen was completely converted to active proteinase in 45-60 min at pH 8.0 and 25 degrees C. This autoconversion process was markedly inhibited by calcium, sodium, and heparin. However, each of these compounds stimulated the proteolytic activity of acrosin. These studies demonstrate that the proacrosin-acrosin system can be investigated in extracts of nonpurified hamster epididymal sperm.     

Inverted micellar intermediates and the transitions between lamellar, cubic, and inverted hexagonal amphiphile phases. III. Isotropic and inverted cubic state formation via intermediates in transitions between L alpha and HII phases

Inverted cubic and isotropic phases have been observed in phospholipid and glycolipid systems. These phases exhibit characteristic morphologies in freeze-fracture electron micrographs, isotropic 31P-NMR resonances and (in some cases) cubic X-ray diffraction patterns. It is proposed here that these phases may form from the same intermediates that are involved in lamellar/inverted hexagonal (L alpha/HII) phase transitions, and that it is possible that these cubic and isotropic phases are metastable. According to a kinetic theory of L alpha/HII phase transitions, intermediates in such transitions can form structures known as interlamellar attachments (ILAs). It is shown that ILAs should form in large numbers during L alpha/HII transitions in systems like those reported to form inverted cubic or isotropic structures. ILAs cannot readily assemble into either the HII phase or well-ordered arrays of L alpha phase bilayers, and represent a kinetic trap for intermediates in L alpha/HII transitions (although it is possible that they are marginally more stable in a thermodynamic sense than the L alpha phase in a small temperature range below TH). It is also shown that arrays of ILAs should form metastable arrays with the same morphology and isotropic 31P-NMR resonances that are observed in isotropic and inverted cubic states. In particular, under some circumstances ILAs will assemble into a structure identical to the bicontinuous inverted cubic phase previously described in monoglycerides and very similar in morphology to structures observed in phospholipid systems. Finally, since isotropic and cubic states form from ILAs, which also can mediate fusion of unilamellar vesicles, unilamellar vesicles should fuse to at least some extent under the same conditions in which multilamellar samples of the same lipid form isotropic or inverted cubic states. This correlation has been observed.     

Immunoreactivity and ouabain-dependent phosphorylation of (Na+ + K+)-adenosinetriphosphatase catalytic subunit doublets

Sodium dodecyl sulfate-polyacrylamide gel electrophoresis with 6% polyacrylamide was used to resolve the 100-kDa catalytic (alpha subunit) polypeptide of (Na+ + K+)-adenosinetriphosphatase from various tissues. The catalytic subunit was identified on immunoblots with antisera against mouse brain catalytic subunit and lamb kidney holoenzyme. Immunoblots and Coomassie Blue-stained companion gels showed double species of the 100-kDa subunit in sucrose gradient fractions of mouse brain and kidney, bovine grey and white matter, purified lamb kidney and duck salt gland holoenzyme, electroplax microsomes, and NaI-extracted microsomes of goldfish and rat brain. The apparent molecular mass differences between the two species in each tissue all ranged between 5 and 8 kDa. Both forms in rat brain and lamb kidney enzyme contain common epitopes reactive with antibodies immunoaffinity-purified on either species from mouse brain. In addition, ouabain-dependent acid-stable inorganic phosphate incorporation was tested with mouse brain, lamb kidney, and electroplax enzyme. Ouabain-dependent phosphorylation was demonstrated in both species in lamb kidney and electroplax and in the larger of the two forms in mouse brain. These results suggest that double species of the phosphorylatable subunit are present generally in epithelial as well as excitable tissues and in fish and avian as well as mammalian species. Work is needed to elucidate their qualitative and quantitative characteristics in different tissues.     

Insect Feeding Deterrents in Endophyte-Infected Tall Fescue

The presence of an endophytic fungus, Acremonium coenophialum, in tall fescue (Festuca arundinacea) deterred aphid feeding by Rhopalosiphum padi and Schizaphis graminum. Both species of aphid were unable to survive when confined to endophyte-infected tall fescue plants. Feeding deterrents and toxic factors to R. padi and Oncopeltus fasciatus, large milkweed bug, were primarily associated with a methanol extract obtained when endophyte-infected tall fescue seed was serially extracted with hexane, ethyl acetate, and methanol. The concentrations of pyrrolizidine alkaloids were determined to be 30 to 100 times greater in the methanol extract than in the hexane and ethyl acetate extracts.     

Partial purification and characterization of an actin-bundling protein, band 4.9, from human erythrocytes

Band 4.9 (a 48,000-mol-wt polypeptide) has been partially purified from human erythrocyte membranes. In solution, band 4.9 polypeptides exist as trimers with an apparent molecular weight of 145,000 and a Stokes radius of 50 A. Electron microscopy shows that the protein is a three-lobed structure with a radius slightly greater than 50 A. When gel-filtered rabbit muscle actin is polymerized in the presence of band 4.9, actin bundles are generated that are similar in appearance to those induced by "vinculin" or fimbrin. The bundles appear brittle and when they are centrifuged small pieces of filaments break off and remain in the supernatant. At low band 4.9 to actin molar ratios (1:30), band 4.9 lowers the apparent steady-state low-shear falling ball viscosity by sequestering filaments into thin bundles; at higher ratios, the bundles become thicker and obstruct the ball's movement leading to an apparent increase in steady-state viscosity. Band 4.9 increases the length of the lag phase and decreases the rate of elongation during actin polymerization as measured by high-shear Ostwald viscometry or by the increase in the fluorescence of pyrene-labeled actin. Band 4.9 does not alter the critical actin monomer concentration. We hypothesize that band 4.9, together with actin, erythrocyte tropomyosin, and spectrin, forms structures in erythroid precursor cells analogous to those formed by fimbrin, actin, tropomyosin, and TW 260/240 in epithelial brush borders. During erythroid development and enucleation, the actin filaments may depolymerize up to the membrane, leaving a membrane skeleton with short stubs of actin bundled by band 4.9 and cross-linked by spectrin.     

Formation of prostaglandins by ovarian carcinomas

Tissue contents of prostaglandins (PG) PGE2, PGE2a and 6-keto-PGF1a (degradation product of PGI2) were determined in specimens of advanced human ovarian cancer (n = 11). The PG levels (ng/mg tissue protein) varied widley: PGE2 17-515; PGF2a 2-43 and 6-keto-PGF1a 5-105. Tumors of patients without response to chemotherapy contained more PGE2, PGF2a and 6-keto-PGF1a than did tumors responding to chemotherapy. PG production was investigated in two ovarian carcinoma-derived cell lines. The ability of these cells to synthesize PG varied depending on the cell density. An increase of cell number was associated with a decrease of PG yield. PG formation was inhibited by indomethacin in a concentration-dependent manner. The present study suggests that ovarian carcinoma cells form PG in vivo and vitro.     

Effect of the ovulatory cycle on oviductal sperm storage in the domestic fowl

Anovulatory domestic hens (pregnant mare serum-treated) and normally cyclic domestic hens were artificially inseminated with 0.034 ml of pooled semen. A subsequent microscopic assessment of the uterovaginal sperm storage glands on days 1, 3, 6, 9 and 12 post-insemination indicated that the sperm glands emptied over time in the normally cyclic hens, but not in the anovulatory hens. The data suggest that events associated with ovulation and/or oviposition are important to the sperm gland emptying process.