Effect of inserted oxysterols on phospholipid packing in normal and sickle red blood cell membranes

Fourier transform infrared (FTIR) spectroscopy was used to examine the effect of oxysterol insertion into normal and sickle RBC membranes and the total lipid extracts of the membranes. Examination of the FTIR C-H stretch and fingerprint regions reveal that the insertion of 7 alpha- and 7 beta-hydroxycholesterol has the greatest effect on the fluidity of RBC membranes and lipid extracts. The results confirm the observation that sterol molecules are oriented in the membrane so that the 7 position is located in the phospholipid head group region at the lipid/water interface. The substitution of a keto for a hydroxy group at the number seven position decreases the effect of the sterol on membrane packing.     

Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes.

In contrast to endothelin-1 (ET-1) and several of its analogues, sarafotoxin S6c (S6c) was a much more potent inhibitor of [125I]-ET-1 binding in rat hippocampus and cerebellum (Ki approximately 20 pM) than in rat atria and aorta (Ki approximately 4500 nM), suggesting the existence of ET-1 receptor subtypes (aorta/atria, ETA; hippocampus/cerebellum, ETB). S6c was a potent activator of PI turnover in hippocampus (EC50 approximately 10 nM) but not atria (EC50 greater than 1 microM), unlike ET-1 which was active in both tissues. S6c, therefore, is a highly selective ETB agonist. Furthermore, S6c was a potent pressor agent in the pithed rat (ED25 mm Hg approximately 0.1 nmoles/kg, i.v.), suggesting that the ETB receptor subtype may be important in cardiovascular function.     

Physical map of two D. melanogaster DNA segments containing sequences coding for the 70,000 dalton heat shock protein.

The isolation of the two hybrid plasmids 56H8 and 132E3, which contain D. melanogaster (Dm) DNA sequences complementary to the mRNA coding for the 70,000 dalton heat shock protein, has been reported (Schedl et al., 1978). Here we compare the sequence arrangement in the two cloned Dm DNA segments by restriction, cross-hybridization and heteroduplex analysis. The results show that the two cloned DNA segments derive from nonoverlapping regions of the Dm genome; that they contain homologous regions present once in 56H8 and twice in 132E3; and that each homologous region is composed of three distinct contiguous sequence elements, x, y and z, which together define a 3 kb common unit. While the 2.5 kb z elements show a high degree of sequence homology in all three common units, the three x and y elements display an intriguing relationship. The localization of the mRNA coding sequences within each of these common units is presented in the accompanying paper (Artavanis-Tsakonas et al., 1979).     

Regional distribution of membrane-bound γ-glutamyl transpeptidase activity in mouse brain

Activity of membrane-bound -glutamyl transpeptidase (-GTP) was examined in various regions of mouse brain, in capillaries of the cerebral cortex and in telencephalic choroid plexuses. The level of activity in the capillaries was double and that of the choroid plexus nine times that of the -GTP activity found in the brain, septum, hippocampus, hypothalamus, thalamus, cerebellum, frontal cortex, pons, medulla oblongata, and amygdala. Histochemically the -GTP activity was demonstrated in the surface membranes of choroidal cells and in the endothelium of small capillaries.The activities of -GTP of cerebral cortex, choroid plexus, and capillaries from rabbit were 5–17 times greater than those from corresponding areas of mouse brain. While 30 mM methionine stimulated (in vitro) the enzyme from mouse brain, no such effect was observed with the enzyme activity from rabbit brain. The -GTP activity from the capillaries of cerebral cortex of both mouse and rabbit was not effected by the presence of methionine.These findings suggest existence of differences in the specificity of -GTP activity in these two species.     

Biosynthesis of a ubiouitin-related peptide in rat brain and in human and mouse pituitary tumors

A biosynthetic labeled peptide structurally related to the thymic peptide ubiquitin was first identified fortuitously in bovine pars intermedia cells in regard to its partial NH₂ terminal amino acid sequence (Met 1, Leu 8, 15 and Lys 6, 11, 27, 29, 33) after a protein segment data bank search. A peptide with the same behavior on carboxymethylcellulose chromatography and polyacrylamide gel electrophoresis has been purified after labeling experiments in two areas of rat brain, hypothalamus and striatum, and in a mouse and a human ACTH-secreting pituitary tumors. It represents about 1 to 10% of the total labeled proteins in the various experiments. Its identity with the above mentioned bovine pituitary peptide was confirmed by microsequence analysis with respect to Met 1, Lys 6, 11 in hypothalmus, Met 1 in striatum, and Lys 6, 11, 27, 29, 33 in the two pituitary tumors. The availability of standard purified ubiquitin allowed us to show that labeled and cold peptides have the same electrophoretic mobility and elution volume on Sephadex G-50 chromatography this further confirms their identity. Possible interests of such a biosynthetic characterization of a ubiquitin-related peptide are discussed, particularly in view of the structural relationship of ubiquitin to the non histone component of nuclear protein A-24, and as a test of tissue viability and biosynthetic efficiency in our in vitro biosynthetic systems.     

The influence of sugars on nitrate reductase induction by exogenous nitrate or nitrite in excised pisum sativum roots

Nitrate reductase (NR) induction is enhanced by exogenously supplied sucrose in excised pea roots exposed to both exogenous nitrate and exogenous nitrite. NR synthesis is preferentially supported by sugars transported to the cells at the moment, however NR induction can take place for some time without exogenous sugar influx if roots are saturated with sugars during precultivation. Steady high NR levels are dependent on steady sugar and nitrate influxes. NR induction is low in roots precultivated for 20 h without sucrose although sugar content is still high in them. This suggests that compartmentation of sugars in the cells is of major importance during NR induction. Total nitrate content in roots exposed to nitrate is not influenced by sucrose supplied together with nitrate. Some nitrite is oxidized to nitrate in roots exposed to exogenous nitrite ; we assume that this nitrate is responsible for NR induction. Our results indicate that sugars, besides many indirect effects on NR induction, may also directly influence NR synthesis either as coinducers or as derepressors of NR synthesis. Our results further show that NR is not a product-inducible enzyme.     

Laser Raman spectroscopy of lipid-protein systems Differences in the effect of intrinsic and extrinsic proteins on the phosphatidylcholine Raman spectrum

Laser Raman spectroscopy is used to examine the interactions of intrinsic and extrinsic proteins with the lipid layer structure. The interactions of cytochrome c and cytochrome c oxidase with lipids have been well established by others using a variety of techniques. Cytochrome c is thought to act as an extrinsic membrane protein while cytochrome c oxidase is thought to act as an intrinsic membrane protein. The lipid-cytochrome c and lipid cytochrome c oxidase systems are used to assist in interpreting the spectral changes due to extrinsic and intrinsic protein interactions. The two types of proteins examined produced differential changes in the lipid hydrocarbon CH stretch Raman modes for both dimyristoyl and dipalmitoyl phosphatidylcholine. The plasma proteins albumin and fibrinogen were also found to differentially affect the lipid hydrocarbon CH stretch Raman modes. These proteins appear to interact with lipids in an extrinsic manner different from that of cytochrome c.     

On the importance of the binding of lectins to cell surface receptors at low lectin concentrations

The binding of soybean agglutinin to human and rabbit erythrocytes, before and after treatment with trypsin, was reinvestigated with special emphasis on measurements at very low lectin concentrations. This communication presents two features of the binding that are observed only at the low concentrations used. (1) The trypsinized erythrocytes bind more lectin molecules than untreated cells at low concentrations (0.1–1.0 μg/ml), even though the total number of binding sites appears to be the same for both treated and untreated cells. It is suggested that this difference could explain, at least in part, the much higher susceptibility of the trypsin-treated cells to agglutination by soybean agglutinin. (2) At low site occupancy the binding of soybean agglutinin exhibits positive cooperativity, indicating a conformational change in the membrane. Trypsin-treated cells exhibit this effect at much lower lectin concentrations than untreated cells.