Tyrosine hydroxylase in secretory granules from bovine adrenal medulla. Evidence for an integral membrane form

Intact secretory granules isolated from bovine adrenal medulla express tyrosine hydroxylase (TH) activity. Granule-associated TH sediments on continuous sucrose gradients with dopamine beta-hydroxylase, a marker for granule membranes, indicating that TH is associated with chromaffin granules. Membranes prepared from lysed granules retain TH, whereas granule contents are free of the enzyme. TH immunoreactivity was detected in granule membranes by immunoblot analysis using a polyclonal antiserum against TH. TH immunoreactivity cannot be removed from membranes by washes in high ionic strength buffers and is only partially removed from membranes by treatment with either urea or Na2CO3. TH can be removed from granule membranes by the detergents Nonidet P-40, Triton X-100, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate. Treatment of membranes with a phosphatidylinositol-specific phospholipase C did not remove TH, ruling out the possibility of a glycosyl phosphatidyl anchor. Fractionation of granule membranes by temperature-induced phase separation in Triton X-114 revealed that TH is recovered in phases in which integral (detergent phase) and hydrophobic (phospholipid phase) membrane proteins are typically found. By contrast, TH from adrenal cytosol fractionated exclusively into the aqueous phase along with other soluble proteins. Digestion of granules with various protease enzymes revealed that TH is resistant to degradation, suggesting that the enzyme is embedded within membranes. TH becomes phosphorylated when intact granules are exposed to the catalytic subunit of the cAMP-dependent protein kinase, indicating that at least the N-terminal region of TH is exposed on the cytoplasmic surface of granules. These results establish that a fraction of TH is an integral component of bovine granule membranes. The association of TH with granule membranes may play a role in coordinating TH activity and catecholamine release.     

Interactions among coexisting larval Odonata: an in situ experiment using small enclosures

Field experiments using small replicated enclosures focused on interactions between larval populations of Epitheca cynosura and Ladona deplanata (Odonata: Anisoptera) — two species that emerge in early spring. The presence of Epitheca reduced the total biomass of Ladona, but Ladona had no significant effect on Epitheca. These early-emerging species reduced the biomass of small instars of late-emerging Anisoptera which colonized enclosures during the experiments; and the late-emerging Anisoptera seem to have inhibited colonization by Zygoptera larvae. Results are consistent with the importance of predatory (cannibalism or mutual predation) interactions in this community.     

Processing and amino acid sequence analysis of the mouse mammary tumor virus env gene product.

The envelope proteins of mouse mammary tumor virus (MMTV) are synthesized from a subgenomic 24S mRNA as a 75,000-dalton glycosylated precursor polyprotein which is eventually processed to the mature glycoproteins gp52 and gp36. In vivo synthesis of this env precursor in the presence of the core glycosylation inhibitor tunicamycin yielded a precursor of approximately 61,000 daltons (P61env). However, a 67,000-dalton protein (P67env) was obtained from cell-free translation with the MMTV 24S mRNA as the template. To determine whether the portion of the protein cleaved from P67env to give P61env was removed from the NH2-terminal end of P67env and as such would represent a leader sequence, the NH2-terminal amino acid sequence of the terminal peptide gp52 was determined. Glutamic acid, and not methionine, was found to be the amino-terminal residue of gp52, indicating that the cleaved portion was derived from the NH2-terminal end of P67env. The NH2-terminal amino acid sequences of gp52's from endogenous and exogenous C3H MMTVs were determined though 46 residues and found to be identical. However, amino acid composition and type-specific gp52 radioimmunoassays from MMTVs grown in heterologous cells indicated primary structure differences between gp52's of the two viruses. The nucleic acid sequence of cloned MMTV DNA fragments (J. Majors and H. E. Varmus, personal communication) in conjunction with the NH2-terminal sequence of gp52 allowed localization of the env gene in the MMTV genome. Nucleotides coding for the NH2 terminus of gp52 begin approximately 0.8 kilobase to the 3' side of the single EcoRI cleavage site. Localization of the env gene at that point agrees with the proposed gene order -gag-pol-env- and also allows sufficient coding potential for the glycoprotein precursor without extending into the long terminal repeat.     

Retention of human skin fibroblast fatty acid modifications during maintenance culture

Summary The fatty acid composition of cultured human skin fibroblasts was modified by adding either oleic or linoleic acid to the growth medium. After the cultures became confluent, they were washed and transferred to different maintenance media in order to determine the stability of the various fatty acyl modifications. Some changes in fatty acid composition occurred under all conditions. When the maintenance medium was supplemented with fatty acid, the cellular neutral lipid and phospholipid fatty acyl composition were altered markedly within 16 to 24 hr. If no supplemental fatty acid was available during the maintenance period, however, the modified fatty acyl compositions were sufficiently retained so that appreciable differences between the cells enriched with oleate and linoleate persisted for at least 48 to 72 hr. This considerable degree of stability occurred when either 10% delipidized fetal bovine serum or 10% fetal bovine serum containing its inherent lipids were present in the maintenance medium. Although the triglyceride content of the fatty acid-modified cells was quite labile, neither the cholesterol nor phospholipid content changed appreciably during culture in any of the maintenance media. Since the fatty acid compositional differences persisted during several days of maintenance under certain conditions, these modified cultures appear to be a useful experimental system for assessing the effect of lipid structure on fairly long-term cellular functions. This work was supported by Arteriosclerosis Specialized Center of Research Grant HL14230 from the National Heart, Lung and Blood Institute, National Institutes of Health.     

A general method for permeabilizing monolayer and suspension cultured animal cells

A wide variety of animal cells have been successfully permeabilized to non-penetrating molecules, using lysolecithin. The sizes of molecules that can enter the cells can be controlled by varying the concentration of lysolecithin. The cells become permeable to small molecules and maintain viability following treatment with low lysolecithin concentrations. At higher concentrations the cells become permeable to proteins but do not retain viability. Lysolecithin permeabilization should permit many studies of the effects of non-penetrating compounds on cellular processes.     

Synthesis of glycosyl halides and glycosides via 1-O-sulfonyl derivatives

The reaction of an aldose derivative containing a free anomeric hydroxyl group with trifluoromethanesulfonic anhydride or methanesulfonic anhydride, in the presence of halide ion and s-collidine, furnishes a glycosyl halide; if an alcohol is then introduced, glycoside synthesis is effected in an overall, “one-pot” reaction. Several α-d-glucopyranosides, including disaccharides, have been prepared in high yield by using 2,3,4,6-tetra-O-benzyl-d-glucose as the aldose, and generating the corresponding glycosyl bromide(s) in situ. As a halide-exchange step is incorporated in the reaction sequence, orthoacetate formation was favored in reactions of 2,3,4,6-tetra-O-acetyl-d-glucose, such as occurs with per-O-acetylglycosyl halides. Methanesulfonic anhydride promotes glycosidation or orthoester formation in the absence of halide ion, as well as in its presence, whereas formation of an intermediate glycosyl halide appears to be necessary in order to moderate the more vigorous reactions of the trifluoro derivative. The analogous reaction of methanesulfonyl chloride with an aldose provides a ready route to glycosyl chlorides. Under the conditions employed for these various syntheses, acid-sensitive protecting groups may be used, including cyclic and acyclic acetals and O-trityl substituents.     

Inverse correlation between species lifespan and capacity of cultured fibroblasts to convert benzo(a)pyrene to water-soluble metabolites

Diploid fibroblasts from lung and skin of eight mammalian species were cultured and the capacity of these cell strains to convert benzo(a)pyrene (BP) to water-soluble metabolites was determined. The rate of conversion is dependent upon culture density and varies widely among different species. There is a very good inverse correlation between species lifespan and the capacity of cultured fibroblasts from these species to metabolize BP to water-soluble forms. Since these cell systems have also shown a good inverse correlation between species lifespan and biological activity of 7,12-dimethylbenz(a)anthracene (DMBA), these data suggest that the capacity to metabolize polycyclic hydrocarbon carcinogens may be closely related to lifespan in mammalian species.