The proteins of the content of the secretory granules of the rat parotid gland

The proteins of the secretory granules of the rat parotid gland were characterized by sodium dodecylsulfate gel electrophoresis, by chromatography of [³H]prolinelabeled proteins on DEAE-cellulose and by amino acid analysis.Sodium dodecylsulfate gel electrophoresis of the secretory granule content showed five principal proteins and a limited number of minor components. Only two of the principal bands could be identified as known secretory enzymes of the parotid gland. One was identified as the α-amylase and one as deoxyribonuclease. Peroxidase and ribonuclease form minor portions of the secretory proteins.The other three major proteins constitute, together, about 60% by weight, of the secretory granule content proteins. Of these, one which represents more than 30% of the total granule protein was found to contain uniquely high amounts of leucine residues (21 mole%). Another one of these principal proteins was relatively rich in cysteine residues (7 mole%).The fifth principal protein was found to contain high amounts of proline (28 mole%) glutamic acid (17 mole%) and glycine (18 mole%) residues. Its amino acid composition was very similar to that of the proline-rich proteins that were previously shown to be present in the membrane isolated from these granules. This protein, however, differed from the “membranous” proline-rich proteins by several criteria.Two minor glycoproteins of the secretory granule content were also found to be rich in proline residues (37 mole%). As with the other proline-rich proteins of the granule, they contained no sulphur-containing amino acids, stained faintly pink with Coomassie Blue and were underestimated by the Lowry method. They differ however, from all the other proline-rich proteins of the granule by having a significantly higher content of threonine, less glycine (9 mole%) and much less glutamic acid (3 mole%).Of the principal proteins, only the deoxyribonuclease and the half-cystine-rich proteins were positively stained by periodic acid Schiff staining.The possible functions of the leucine-rich, the half cystine-rich and the various proline-rich proteins are discussed.     

Basic protein changes during the final stages of sperm maturation in the house cricket

Polyacrylamide gel electrophoresis has been used to analyse basic protein changes during the final stages of spermiogenesis in the house cricket. Mature sperm were obtained from the spermathecae of inseminated females. Their basic protein is electrophoretically heterogeneous, with two major and two minor components, all of unusually high mobilities, as expected ofprotamine. No histones are present. Testis also contains basic protein components of high mobilities, although in small amount relative to the histones present. Testis preparations were centrifuged on a density gradient of colloidal silica to separate nuclei of different stages of spermiogenesis from each other, and it was found that very late spermatids contain relatively large amounts of protamine. At least seven different protamine-like components, each with a different mobility, occur during the final maturation stages. The particular components present, and their abundancies, vary during development. The complement first found in spermatids is different from that of a later spermatid; still another complement is found in sperm from the seminal vesicle; and still another in mature sperm. Components which are abundant in spermatids are progressively eliminated, while components which are barely detectable in them gradually increase in abundance to become the major components of the basic protein complement at maturity.     

The impacts of cytoplasmic incompatibility factor (cifA and cifB) genetic variation on phenotypes

Wolbachia are maternally transmitted, intracellular bacteria that can often selfishly spread through arthropod populations via cytoplasmic incompatibility (CI). CI manifests as embryonic death when males expressing prophage WO genes cifA and cifB mate with uninfected females or females harboring an incompatible Wolbachia strain. Females with a compatible cifA-expressing strain rescue CI. Thus, cif-mediated CI confers a relative fitness advantage to females transmitting Wolbachia. However, whether cif sequence variation underpins incompatibilities between Wolbachia strains and variation in CI penetrance remains unknown. Here, we engineer Drosophila melanogaster to transgenically express cognate and non-cognate cif homologs and assess their CI and rescue capability. Cognate expression revealed that cifA;B native to D. melanogaster causes strong CI, and cognate cifA;B homologs from two other Drosophila-associated Wolbachia cause weak transgenic CI, including the first demonstration of phylogenetic type 2 cifA;B CI. Intriguingly, non-cognate expression of cifA and cifB alleles from different strains revealed that cifA homologs generally contribute to strong transgenic CI and interchangeable rescue despite their evolutionary divergence, and cifB genetic divergence contributes to weak or no transgenic CI. Finally, we find that a type 1 cifA can rescue CI caused by a genetically divergent type 2 cifA;B in a manner consistent with unidirectional incompatibility. By genetically dissecting individual CI functions for type 1 and 2 cifA and cifB, this work illuminates new relationships between cif genotype and CI phenotype. We discuss the relevance of these findings to CI’s genetic basis, phenotypic variation patterns, and mechanism.     

A spin label study of erythrocyte membranes during simulation of freezing

Summary Human erythrocytes were labeled with stearic acid spin labels, and no change was detected in membrane fluidity under hyperosmotic stress, going from isotonicity to about 3000 mOsm. Intact erythrocytes labeled with an androstane spin label and submitted to simulation of freezing show the onset of irreversible structural breakdown occurring in a saline solution at 2,000 mOsm. Ghosts labeled with maleimide spin label (4-maleimide-2,2,6,6-tetramethylpiperidinooxyl) when submitted to solutions of increasing osmolalities (pH 7.4), exhibit protein conformational changes that are irreversible after a simulated freeze-thaw cycle. After sonication of maleimide spin-labeled ghosts, membrane buried sulfhydryl groups become exposed. Such preparations showed behavior similar to the unsonicated when in saline hyperosmolal medium (pH 7.4). Such results suggest the ionic strength of the medium as the determining factor of the detected conformational changes. Maleimide spin-labeled ghosts in 300 mOsm saline solution (pH 7.4) were treated with ascorbic acid (spin destruction of nitroxides), and the kinetic analysis indicates that 65% of the labeled sites are located at the external interface of the membrane or in hydrophilic channels. Deformation and rearrangements of membrane components in solutions of increasing osmolalities apparently are related to protein conformational changes, on the outside surface of erythrocyte membranes, with a significant amount being structurally dissociated of lipids.