Free amino acids from pollens

Pollens from Pinus canariensis, P. nigra, P. pinaster, P. pinea, Castanea sativa, Magnolia grandiflora, Olea sativa cv frantoio, cv itrana, cv pisciottana were examined for their free amino acid composition. A large amount of proline was found in all species; pollens of Olea also contain a large amount of serine.     

Characteristics of the effect of S-100 proteins on the assembly-disassembly of brain microtubule proteins at alkaline pH in vitro

The ability of S-100 proteins to inhibit the assembly of brain microtubule proteins (MTPs) in the presence of microM levels of Ca2+ increases as a function of pH. This seems to be due to an increasingly larger inhibitory effect of S-100 on the nucleation and, probably, on the elongation of microtubules (MTs) as the pH raises. In the presence of microM Ca2+ levels, the ability of S-100 to disassemble MTs also increases linearly with the pH, suggesting that the larger inhibitory effect of S-100 on MTP assembly at alkaline than at acidic pH may depend on both a decrease in the assembly rate and an increase in the disassembly rate. Also, S-100 inhibits the assembly of phosphocellulose-purified tubulin to a larger and larger extent as the pH raises. S-100 brings about its effect on MT assembly-disassembly probably by sequestering soluble tubulin, though additional mechanisms cannot be excluded. The present data are briefly discussed in relation to the role attributed to changes in intracellular pH in the regulation of the state of assembly of cytoplasmic MTs.     

Rapid immunofluorescent determination of cells in the S phase in pea root meristems: An alternative to autoradiography

Photosystem II (PS II) activity and the localization of ribulose-l,5-bisphosphate (RuBP) carboxylase (EC 4.1.1.39) were studied in primary leaves of young maize plants ( Zea mays L. cv. Fronica) by tetra-nitro-blue-tetrazoliumchloride reduction and immunolocalization, respectively. In tissue of 3-day-old plants all chloroplasts were structurally identical. From day 4 they developed into their typical appearance of mesophyll and bundle sheath chloroplasts. First PS II-activity was present in both types of chloroplasts. From day 4 it disappeared in bundle sheath chloroplasts concomitant with the loss of grana. RuBP carboxylase on the other hand was only present in bundle sheath chloroplasts at all stages of development. Thus, the control of the development of the photosystems and the Calvin cycle enzymes seem to differ.     

Properties of carboxymethylated cross-linked hemoglobin A

The selective carboxymethylation of the N-terminal amino groups of hemoglobin A with glyoxylic acid and sodium cyanoborohydride has been studied as a function of the state of ligation of hemoglobin. The N-terminal residues have been established as the primary sites of reaction by peptide mapping of the tryptic digest of each chain and subsequent amino acid analysis of the modified peptides. With oxyhemoglobin, the desired derivatives with a carboxymethyl group at the N-terminal of either or both chains amounted to 55% [Di Donato, A., Fantl, W. J., Acharya, A. S., & Manning, J. M. (1983) J. Biol. Chem. 258, 11890-11895]. In the present study it is shown that with deoxyhemoglobin the amount of the desired derivative is increased to 75%. The oxygen equilibrium curve of hemoglobin A carboxymethylated on its four N-terminal residues [0.5 mM as tetramer in 50 mM [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (Bis-Tris), pH 7.5, 37 degrees C] had a P50 value of 30 mmHg (Hill coefficient n = 2.8, alkaline Bohr value = 0.4) compared to a P50 of 9 mmHg for unmodified hemoglobin under the same conditions (n = 2.5, alkaline Bohr value = 0.5). In carboxymethylated oxyhemoglobin A, cross-linked with the mild agent glycolaldehyde for 3.5 h, there was 85% of Mr 64,000 species and 15% of Mr 128,000 or higher species. For the former, the extent of cross-linking between two subunits was 19%. For the latter, there was 29% of two cross-linked subunits and 13% of three cross-linked subunits. Termination of cross-linking, which may be desirable in some circumstances, can be successfully achieved with isonicotinic acid hydrazide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of S-100 proteins and S-100-binding proteins in a detergent-resistant EDTA/KCl-extractable fraction from bovine brain membranes

The Triton X-100-resistant residue of brain membranes contains appreciable amounts of S-100 proteins. This fraction of S-100 can be solubilized by high concentrations of EDTA plus or minus high concentrations of KCl. Whereas KCl (0.6 M) extracts the detergent-resistant S-100, NaCl (1 M) does not. Endogenous Ca2+ is required and is sufficient for S-100 to remain associated with the detergent-resistant residue. However, 0.6 M KCl extracts a further fraction of Triton X-100-resistant S-100. In contrast, the Triton X-100-extractable fraction of S-100 resists the action of EDTA. These data suggest that Ca2+ regulates the extent of association of S-100 with Triton X-100-resistant components in brain membranes, whereas the association of S-100 with the lipid bilayer of brain membranes and/or with some intrinsic membrane proteins is less Ca2+-regulated. Several S-100-binding proteins are identified in the detergent-resistant residue of brain membranes by an overlay procedure.     

Interaction of S-100b protein with cardiolipin vesicles as monitored by electron spin resonance, pyrene fluorescence and circular dichroism

The interaction of S-100b protein with cardiolipin (CL) vesicles has been studied by electron spin resonance, pyrene fluorescence, and circular dichroism. Electron spin resonance and pyrene fluorescence data indicate that S-100b binds to the polar surface of vesicles Ca2+-independently. In the presence of Ca2+, S-100b potentiates the Ca2+-induced clustering of the polar headgroups of CL molecules and causes a further reduction in the Ca2+-dependent decrease in the lateral mobility of the pyrene inserted into the lipid bilayer, which points to an effect of the protein on the hydrophobic core of the lipid bilayer through a larger perturbation of its polar surface. Circular dichroism analyses indicate that CL vesicles cause a decrease in the alpha-helical content of S-100b, analogous to that produced by Ca2+ and that the effects of CL vesicles and of Ca2+ on the secondary structure of the protein are supra-additive. By this technique, we found that the affinity of Ca2+ for S-100b increases substantially in the presence of CL vesicles, even in the presence of physiologic concentrations of KCl, suggesting that once S-100b had interacted with CL vesicles it assumes a new conformation in which its Ca2+-binding properties are greatly enhanced. These results are discussed in relation to binding of S-100b proteins to natural membranes, and to a possible involvement of S-100b in the regulation of membrane structural organization.     

Synthesis of the marine epoxy sterol 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol

The synthesis of 9 alpha,11 alpha-epoxy-5 alpha-cholest-7-ene-3 beta,5,6 beta-triol (1), a highly oxygenated marine sterol containing a 9,11-epoxide moiety in the nucleus, is described. Epoxy sterol 1 was synthesized from cholesta-5,7-dien-3 beta-ol. Oxidation of this sterol with m-chloroperbenzoic acid followed by hydrolysis and acetylation furnished 5 alpha-cholest-7-ene-3 beta,5,6 alpha-triol 3,6-diacetate (2). Mercuric acetate dehydrogenation of diacetate 2, followed by oxidation with manganese dioxide and epoxidation with m-chloroper-benzoic acid, afforded 9 alpha,11 alpha-epoxy-3 beta,5-dihydroxy-5 alpha-cholest-7-en-6-one (5). Reduction of 5 with lithium aluminum hydride gave the desired compound 1. The structures of all synthetic intermediates were confirmed by 1H and 13C nuclear magnetic resonance (NMR) spectroscopy. A reassignment of resonances for carbons 1, 8, and 15 in the 13C NMR spectrum of 1, based on 2D-NMR correlation spectroscopy, has been accomplished.     

Inhibition of adenylate cyclase by transglutaminase-catalyzed reactions in pigeon erythrocyte ghosts

We report the occurrence in pigeon erythrocytes of a soluble Ca2+-dependent transglutaminase (TGase) activity. The effect of the erythrocyte ghost protein modifications, determined by TGase-catalyzed reactions, on adenylate cyclase, phospholipid methyltransferase I and II activities and on the lipidic matrix fluidity of the membrane was investigated by using a purified guinea pig liver TGase preparation. The results showed a significant inhibitory effect of such modifications both on the basal and on the variously stimulated (by NaF, Gpp(NH)p alone or in the presence of 1-isoproterenol) adenylate cyclase activity. By contrast, both the phospholipid methylation and the fluidity of the lipidic matrix of the membrane were unaffected by TGase-mediated reactions. These data suggest a new possible inhibitory mechanism of the cyclic AMP synthesis which might be triggered by the enhancement of the cytosolic Ca2+ concentration.