Deletion analysis of the internal signal-anchor domain of the human asialoglycoprotein receptor H1.

The human asialoglycoprotein receptor H1 is a single-spanning membrane protein with the amino terminus facing the cytoplasm and the carboxy terminus exposed on the exoplasmic side of the plasma membrane. It has been shown earlier that the transmembrane segment, residues 38-65, functions as an internal signal directing protein synthesis to the endoplasmic reticulum and initiating membrane insertion. This process is co-translational and mediated by signal recognition particle (SRP). To identify subsegments within this region containing the signal information, we prepared deletion mutants at the level of the cDNA and analysed them in a wheat germ in vitro translation system with microsomes as the target membrane. Insertion and membrane anchoring were judged by the glycosylation of the protein, its resistance to exogenous protease and the extent to which it can be extracted from the microsomes by alkaline treatment. It was found that very small deletions already reduce the stability of membrane anchoring. However, nearly half of the transmembrane domain can be deleted, both from the amino-terminal and from the carboxy-terminal side, without completely abolishing membrane insertion. Several mutants, although not inserted, still interact with SRP. The results support the notion that the main feature of a signal sequence is a hydrophobic stretch of sufficient length (10-12 residues in our sequence), and indicate that recognition by SRP is not sufficient for membrane insertion.     

Organization of the murine immunoglobulin VH complex in the inbred strains.

Deletion mapping analyses have been employed to order the heavy chain variable region (VH) gene families in three inbred murine strains. These nine VH gene families have been positioned with respect to the J558 and 3660 VH families in A/J (Ighe) as follows: 3609-J558-(J606,VGAM3-8,S107)-3660-(X24,Q52,7183 )-DH. Maps generated with respect to J558 in the BALB/c (Igha) and C57BL/6 (Ighb) strains are consistent with these results. The organization of the VH complex produced by deletion mapping is quite different from the accepted map generated by other methods, particularly in that J558 is more DH distal and 3660 is more DH proximal than previously thought. The order presented here is compatible with VH rearrangement frequencies suggesting preferential utilization of DH-proximal VH gene segments. Our data also indicate that interspersion of some VH family members may be a common feature of the murine VH complex since the 3609 VH family is interdigitated in the three strains and a Q52 VH gene segment is interspersed in C57BL/6.     

Stimulation by D-glucose of mitochondrial oxidative events in islet cells.

Antibodies were elicited to FAD by using the hapten N-6-(6-aminohexyl)-FAD conjugated to the immunogenic carrier protein bovine serum albumin. Cross-reactivity was determined by Ouchterlony double-diffusion analysis with N-6-(6-aminohexyl)-FAD coupled to rabbit serum albumin. Anti-FAD IgG was partially purified by (NH4)2SO4 precipitation followed by DEAE-cellulose/CM-cellulose and bovine serum albumin-agarose chromatography. The partially purified anti-FAD IgG fraction failed to inhibit the catalytic activities of the flavin-containing enzymes nitrate reductase, xanthine oxidase and succinate dehydrogenase, whereas enzyme activity could be inhibited by addition of antibodies elicited against the native proteins. However, the partially purified anti-FAD IgG fraction could be used as a highly sensitive and specific probe to detect proteins containing only covalently bound flavin, such as succinate dehydrogenase, p-cresol methylhydroxylase and monoamine oxidase, by immuno-blotting techniques. Detection limits were estimated to be of the order of femtomolar concentrations of FAD with increased sensitivity for the 8 alpha-N(3)-histidyl linkage compared with 8 alpha-O-tyrosyl substitution.     

Cellular free cholesterol in Hep G2 cells is only partially available for down-regulation of low-density-lipoprotein receptor activity.

We have previously shown that in Hep G2 cells and human hepatocytes, as compared with fibroblasts, the low-density lipoprotein (LDL) receptor activity is only weakly down-regulated after incubation of the cells with LDL, whereas incubation with high-density lipoproteins (HDL) of density 1.16-1.20 g/ml (heavy HDL) strongly increased the LDL-receptor activity. To elucidate this difference between hepatocytes and fibroblasts, we studied the cellular cholesterol homoeostasis in relation to the LDL-receptor activity in Hep G2 cells. (1) Interrupting the cholesteryl ester cycle by inhibiting acyl-CoA: cholesterol acyltransferase (ACAT) activity with compound 58-035 (Sandoz) resulted in an enhanced LDL-mediated down-regulation of the receptor activity. (2) The stimulation of the receptor activity by incubation of the cells with cholesterol acceptors such as heavy HDL was not affected by ACAT inhibition. (3) Incubation of the Hep G2 cells with LDL, heavy HDL or a combination of both grossly affected LDL-receptor activity, but did not significantly change the intracellular content of free cholesterol, suggesting that in Hep G2 cells the regulatory free cholesterol pool is small as compared with the total free cholesterol mass. (4) We used changes in ACAT activity as a sensitive (indirect) measure for changes in the regulatory free cholesterol pool. (5) Incubation of the cells with compactin (2 microM) without lipoproteins resulted in a 4-fold decrease in ACAT activity, indicating that endogenously synthesized cholesterol is directed to the ACAT-substrate pool. (6) Incubation of the cells with LDL or a combination of LDL and heavy HDL stimulated ACAT activity 3-5 fold, whereas incubation with heavy HDL alone decreased ACAT activity more than 20-fold. Our results suggest that in Hep G2 cells exogenously delivered (LDL)-cholesterol and endogenously synthesized cholesterol are primarily directed to the cholesteryl ester (ACAT-substrate) pool or, if present, to extracellular cholesterol acceptors (heavy HDL) rather than to the free cholesterol pool involved in LDL-receptor regulation.     

Neutrophil association and degradation of normal and acute-phase high-density lipoprotein 3.

The interaction of normal and acute-phase high-density lipoproteins of the subclass 3 (N-HDL3 and AP-HDL3) with human neutrophils and the accompanying degradation of HDL3 apolipoproteins have been studied in vitro. The chemical composition of normal and acute-phase HDL3 was similar except that serum amyloid A protein (apo-SAA) was a major apolipoprotein in AP-HDL3 (approx. 30% of total apolipoproteins). 125I-labelled AP-HDL3 was degraded 5-10 times faster than 125I-labelled N-HDL3 during incubation with neutrophils or neutrophil-conditioned medium. Apo-SAA, like apolipoprotein A-II (apo-A-II), was more susceptible than apolipoprotein A-I (apo-A-I) to the action of proteases released from the cells. The amounts of cell-associated AP-HDL3 apolipoproteins at saturation were up to 2.8 times greater than N-HDL3 apolipoproteins; while apo-A-I was the major cell-associated apolipoprotein when N-HDL3 was bound, apo-SAA constituted 80% of the apolipoproteins bound in the case of AP-HDL3. The associated intact apo-SAA was mostly surface-bound as it was accessible to the action of exogenous trypsin. alpha 1-Antitrypsin-resistant (alpha 1-AT-resistant) cellular degradation of AP-HDL3 apolipoproteins also occurred; experiments in which pulse-chase labelling was performed or lysosomotropic agents were used indicated that insignificant intracellular degradation occurred which points to the involvement of cell-surface proteases in this degradation.     

A rapid assay for measuring the activity and the Mg2+ and Ca2+ requirements of phosphatidate phosphohydrolase in cytosolic and microsomal fractions of rat liver.

1. A rapid extraction and purification scheme was designed for the recovery of [3H]diacylglycerol formed during the assay of phosphatidate phosphohydrolase. 2. The importance of removing polyvalent cations, particularly Ca2+, from the phosphatidate and other reagents used in the assay of the phosphohydrolase activity was demonstrated. This was achieved mainly by treating the phosphatidate with a chelating resin and by adding 1 mM-EGTA and 1 mM-EDTA to the assays. 3. The activity of the phosphohydrolase in dialysed samples of the soluble and microsomal fractions of rat liver was very low. 4. Addition of optimum concentrations of MgCl2 resulted in a 110-167-fold stimulation in activity. 5. CaCl2 was also able to stimulate phosphohydrolase activity, but to a much smaller extent than MgCl2. 6. Chlorpromazine, an amphiphilic cation, inhibited the reaction when it was measured in these experiments by using a mixed emulsion of phosphatidylcholine and phosphatidate at pH 7.4. 7. Microsomal fractions that were preincubated with albumin contained very low activities of the Mg2+-dependent phosphohydrolase. When these were then incubated with the soluble fraction in the presence of oleate, the soluble phosphohydrolase attached to the microsomal membranes, and it retained its high dependency on Mg2+.     

Effects of lactation and removal of pups on the rate of triacyglycerol/fatty acid substrate cycling in white adipose tissue of the rat.

The rate of the triacylglycerol/fatty acid substrate cycle was measured in vivo in adipose tissue of virgin and lactating rats with pups removed. The rate decreased by 70% in adipose tissue of lactating rats and increased 9-fold on removal of the pups. Similar differences in cycling rate were seen in adipose tissue incubated in vitro in the presence of isoprenaline.     

The synthesis of fluorescent chlorotriazinylaminofluorescein-concanavalin A and its use as a glycoprotein stain on sodium dodecyl sulphate/polyacrylamide gels.

Concanavalin A, a specific glycoprotein probe, was optimally labelled to a maximum stoichiometry of 0.4 mol of chlorotriazinylaminofluorescein (CTAF)/mol of concanavalin A monomer under mild reaction conditions (pH 8.0, 6 h), and under these conditions the CTAF concanavalin A preparation retains its carbohydrate-binding ability and is able to penetrate SDS/7.5-15%-polyacrylamide gradient gels. CTAF-concanavalin A gives fluorescent bands for the glycoproteins transferrin, fetuin and deoxyribonuclease and shows no fluorescent response for the non-glycoproteins bovine serum albumin and soya-bean trypsin inhibitor. The detection limit of sensitivity for CTAF-concanavalin A, which is similar to that of fluorescein isothiocyanate-concanavalin A, is in the range 5-25 micrograms of glycoprotein. CTAF-concanavalin A is a suitable probe for the detection of glycoproteins in higher-percentage (greater than or equal to 10%) SDS/polyacrylamide gels, and will probably have other applications in, for example, fluorescent energy transfer and other structure-function studies.     

The influx of Ca2+ induced by the administration of glucagon and Ca2+-mobilizing agents to the perfused rat liver could involve at least two separate pathways.

The Ca2+-mobilizing actions of ADP, ATP and epidermal growth factor (EGF) and their interaction with glucagon were studied in a perfused liver system incorporating a Ca2+-selective electrode. ADP (1-100 microM), ATP (1-100 microM) and EGF (10-50 nM) all induced a net efflux, followed by a net uptake of Ca2+ in the intact liver. The co-administration of glucagon (or of cyclic AMP) with these agents resulted in a synergistic potentiation of the Ca2+ uptake response in a way which resembles the synergism observed when glucagon is administered with phenylephrine, vasopressin or angiotensin [Altin & Bygrave (1986) Biochem J. 238, 653-661]. The inability of diltiazem, verapamil and nifedipine to inhibit the Ca2+-influx response suggests that the stimulation of Ca2+ influx does not occur through voltage-sensitive Ca2+ channels. By contrast, the synergistic effects of glucagon in the stimulation of Ca2+ influx are inhibited by 10 mM-neomycin, and a lowering of the extracellular pH to 6.8. Simultaneous measurements of perfusate Ca2+ and pH changes suggest that the Ca2+ influx response is not mediated by a Ca2+/H+ exchange. The inability of neomycin and low extracellular pH to inhibit the refilling of the hormone-sensitive pool of Ca2+, after the administration of Ca2+-mobilizing agents alone, provides evidence for the existence in liver of at least two Ca2+-influx pathways, or mechanisms for regulating Ca2+ influx.