A major secretory protein from rat seminal vesicle binds ejaculated spermatozoa

The rat seminal vesicle produces large amounts of a protein-rich fluid that greatly contributes to semen volume. RSV IV, a protein abundantly secreted from this gland, binds in vitro to rat epididymal spermatozoa. However, there is no evidence that this protein may have an in vivo role as a sperm-coating antigen. We report in this paper that high-molecular-weight RSV IV immunologically related proteins can be detected on ejaculated spermatozoa, but not on epididymal spermatozoa. After incubation of purified RSV IV with ejaculated spermatozoa in freshly recovered semen or with epididymal spermatozoa in a medium containing the coagulating gland secretion, sperm-bound proteins with analogous properties were detected. These results support the hypothesis that RSV IV is modified at ejaculation to an high-molecular-weight, sperm-coating antigen.     

Microtubule-associated protein 2 (MAP2)-immunoreactive neurons in the retina of Bufo marinus: colocalisation with tyrosine hydroxylase and serotonin in amacrine cells

Summary Neuron populations in the retina of the toad, Bufo marinus, were labelled with a monoclonal antibody raised against microtubule-associated protein 2 (MAP2). A subpopulation of cones, probably corresponding to the blue-sensitive small single cones, large diameter amacrine cells in the most proximal row of the inner nuclear layer and some large ganglion cells in the ganglion cell layer were labelled. Double labelling experiments were carried out to establish the colocalisation of MAP2 with known putative transmitter substances of the anuran amacrine cells. MAP2 was colocalised in a subpopulation of serotonin-immunoreactive and in all tyrosine hydroxylase-immunoreactive amacrine cells. The results indicate, that the MAP2 content in the neurons of the anuran retina can be correlated with other well-defined neurochemical and/or physiological properties.On leave from Department of Zoology, Attlia József University, Szeged, Hungary     

Analysis of Con A-binding glycoproteins in synaptosomal membranes

Concanavalin A (Con A)-binding proteins obtained from solubilized synaptosomal membranes of bovine brain were analyzed by two-dimensional electrophoresis (2DE), and were identified by peroxidase conjugated Con A (Con A-peroxidase staining), after transfer from 2DE gel to nitrocellulose paper. The Con A-binding proteins were resolved up to 40 spots, ranging in isoelectric points (pI) from 4.5 to 8.0 and molecular weight (MW) from 10 kDa to 120 kDa. Most of the Con A-binding proteins were streaked across a pH gradient and/or exhibited as multiple spots, indicating broad charge and molecular weight heterogeneity. The presence of protein groups that showed high affinities for Con A were revealed. Most interesting group (named GP51), which consisted of seven spots separated horizontally in charge heterogeneity (pI5.85-7.5) with MW 51kDa, was characterized by its binding to an immobilized protein A gel. This implies that GP51 is related to immunoglobulins and/or GP51 may be a new member of the immunoglobulin supergene family.     

Antigenic heterogeneity of strains of Saccharomyces cerevisiae and Candida albicans recognised by serum antibodies from patients with Crohn''s disease

Abstract Vitronectin, a serum and extracellular matrix protein involved in immunological reactions, interacts with Helicobacter pylori strains. Of the 20 H. pylori strains tested three strains bound more than 50% of the vitronectin added, five strains bound 25–40%, nine strains bound 10–20% and three strains bound 5–8% vitronectin. Two strains, one with high- and one with low-binding properties, were selected for further characterization of ¹²⁵I-vitronectin binding. Binding to the urea-activated ¹²⁵I-labelled vitronectin was fast, saturable and reversible when an excess of unlabelled vitronectin was added to the bacteria with bound ¹²⁵I-vitronectin. The binding was heat- and protease-sensitive, suggesting that the binding was mediated by bacterial cell-surface proteins. Since components such as fetuin and orosomucoid but not asialofetuin inhibited the binding, sialic-acid specific proteins, related to H. pylori sialic-acid specific haemagglutinins, were probably involved.     

Molecular modeling of the amphipathic helices of the plasma apolipoproteins.

In this paper we propose a classification of the amphipathic helical repeats occurring in the plasma apolipoprotein sequences. It is based upon the calculation of the molecular hydrophobicity potential around the helical segments. The repeats were identified using a new autocorrelation matrix, based upon similarities of hydrophobic and hydrophilic properties of the amino acid residues within the apolipoprotein sequences. The helices were constructed by molecular modeling, the molecular hydrophobicity potential was calculated, and isopotential contour lines drawn around the helices yielded a three-dimensional visualization of the hydrophobicity potential. Two classes of apolipoproteins could be differentiated by comparing the hydrophobic angles obtained by projection of the isopotential contour lines on a plane perpendicular to the long axis of the helix. The isopotential contour lines around apo AI, AIV, and E are more hydrophilic than hydrophobic, whereas they are of similar intensity for apo AII, CI, and CIII. In both cases discoidal lipid-protein complexes are generated, with the amphipathic helices around the edge of the lipid core. The long axis of the helices is oriented parallel to the phospholipid acyl chains and the hydrophilic side of the helix toward the aqueous phase. As a result of the differences in hydrophobicity potential, the contact between the hydrophobic side of the helices and the phospholipid acyl chains is larger for apo AII, CI, and CIII than for the other apolipoproteins. This might account for the greater stability of the discoidal complexes generated between phospholipids and these apoproteins.     

Crystal structure of human immunoglobulin fragment Fab New refined at 2.0 A resolution.

The three-dimensional structure of the human immunoglobulin fragment Fab New (IgG1, lambda) has been refined to a crystallographic R-factor of 16.9% to 2 A resolution. Rms deviations of the final model from ideal geometry are 0.014 A for bond distances and 3.03 degrees for bond angles. Refinement was based on a new X-ray data set including 28,301 reflections with F > 2.5 sigma(F) from 6.0 to 2.0 A resolution. The starting model for the refinement procedure reported here is from the Brookhaven Protein Data Bank entry 3FAB (rev. 1981). Differences between the initial and final models include modified polypeptide-chain folding in the third complementarity-determining region (CDR3) and the third framework region (FR3) of VH and in some exposed loops of CL and CH1. Amino acid sequence changes were determined at a number of positions by inspection of difference electron density maps. The incorporation of amino acid sequence changes results in an improved VH framework model for the "humanization" of monoclonal antibodies.     

Detection of native-like models for amino acid sequences of unknown three-dimensional structure in a data base of known protein conformations.

We present an approach which can be used to identify native-like folds in a data base of protein conformations in the absence of any sequence homology to proteins in the data base. The method is based on a knowledge-based force field derived from a set of known protein conformations. A given sequence is mounted on all conformations in the data base and the associated energies are calculated. Using several conformations and sequences from the globin family we show that the native conformation is identified correctly. In fact the resolution of the force field is high enough to discriminate between a native fold and several closely related conformations. We then apply the procedure to several globins of known sequence but unknown three dimensional structure. The homology of these sequences to globins of known structures in the data base ranges from 49 to 17%. With one exception we find that for all globin sequences one of the known globin folds is identified as the most favorable conformation. These results are obtained using a force field derived from a data base devoid of globins of known structure. We briefly discuss useful applications in protein structural research and future development of our approach.     

Ribosomal protein S14 is not responsible for the Minute phenotype associated with the M(1)7C locus in Drosophila melanogaster.

Summary A locus associated with a severe Minute effect has been mapped at 7C on the X chromosome of Drosophila melanogaster. Previous work has suggested that this Minute encodes ribosomal proteins S14A and S141B. We have made a chromosomal deficiency that removes the S14 ribosomal protein genes, yet does not display the Minute phenotype. These data suggest that the S14 genes do not actually correspond to the Minute locus.     

Immunological identification of yeast SCO1 protein as a component of the inner mitochondrial membrane

Summary The SCO1 gene of Saccharomyces cerevisiae encodes a 30 kDa protein which is specifically required for a post-translational step in the accumulation of subunits 1 and 2 of cytochrome c oxidase (COXI and COXII). Antibodies directed against a -Gal::SCO1 fusion protein detect SCO1 in the mitochondrial fraction of yeast cells. The SCO1 protein is an integral membrane protein as shown by its resistance to alkaline extraction and by its solubilization properties upon treatment with detergents. Based on the results obtained by isopycnic sucrose gradient centrifugation and by digitonin treatment of mitochondria, SCO1 is a component of the inner mitochondrial membrane. Membrane localization is mediated by a stretch of 17 hydrophobic amino acids in the amino-terminal region of the protein. A truncated SCO1 derivative lacking this segment, is no longer bound to the membrane and simultaneously loses its biological function. The observation that membrane localization of SCO1 is affected in mitochondria of a rho ⁰ strain, hints at the possible involvement of mitochondrially coded components in ensuring proper membrane insertion.     

Identification and genetic analysis of semidwarfism-related proteins in rice (Oryza sativa L.)

Summary By transferring a semidwarf gene (sd-1) from Taichung Native 1 into a tall Japanese cultivar, Norin 29, through seven backcrosses, a semidwarf near-isogenic line SC-TN1 was obtained. The proteins of the embryo in Norin 29 and SC-TN1 were separated by two-dimensional electrophoresis. Most of the proteins showed the same electrophoretic pattern. However, it was found that there was a difference in the appearance of two basic glycoproteins designated as SRP-1 and SRP-2. These proteins exhibited the same molecular mass, but different isoelectric points. Hybridization results indicated that a single locus controls SRP-1 and SRP-2 with codominant alleles. The gene symbol Srp was given to this locus, with alleles Srp-1 and Srp-2 responsible for SRP-1 and SRP-2, respectively. Srp-2 was found in all of the semidwarf cultivars and lines having sd-1, except a tall cultivar Tsaiyuan-chung. This finding suggests that Srp-2 may be closely linked with sd-1. The amounts of these proteins markedly increased after water absorption of the seed, suggesting that these proteins may be related to the early development of the plant.