Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Sulfate-chloride exchange transport in a glioma cell line

Transport of SO4(2-) was studied in the glioma cell line LRM55 to determine whether it is mediated by the Cl-/HCO3- exchanger or the K+/Cl- cotransporter previously described in these cells (Wolpaw, E.W. and Martin, D.L. (1984) Brain Res. 297, 317-327). 35SO4(2-) influx was saturable with SO4(2-). External SO4(2-) stimulated 35SO4(2-) efflux, indicating an exchange mechanism. External Cl- was a competitive inhibitor of 35SO4(2-) influx. Internal Cl- stimulated 35SO4(2-) influx and external Cl- stimulated 35SO4(2-) efflux, indicating that Cl- is an exchange substrate for the SO4(2-) carrier. Also, SO4(2-) flux was sensitive to SITS, DIDS and furosemide. However, saturating external SO4(2-) did not inhibit 36Cl- influx and did not inhibit 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 36Cl- efflux via the Cl-/HCO3- exchanger. Moreover, K+ did not stimulate 35SO4(2-) influx as it does Cl- influx. These findings indicate that SO4(2-) transport into these cells is mediated by an exchange carrier distinct from both the Cl-/HCO3- exchanger and the K+/Cl- cotransporter. While Cl- is an alternative substrate for the SO4(2-) porter, this carrier is responsible for only a minor fraction of total Cl- flux in these cells.     

Effect of phosphatidylcholine and free fatty acids on the activity of pancreatic lipase-colipase

Mixed micelles of bile salt and phospholipids inhibit the lipase-colipase-catalysed hydrolysis of triacylglycerols. Free fatty acids can reverse this inhibition and reactivate lipase-colipase. This reactivation is either due to the formation of a high-affinity complex between lipase and colipase induced by free fatty acids and/or to a change of the quality of the interface. Lauric acid, oleic acid and linoleic acid are the most potent reactivators, while short-chain free fatty acids have no effect and long-chain, saturated free fatty acids inhibit the lipase-colipase activity further. The physiological relevance of these results is evident as the glyceride emulsion reaching the duodenum already contains free fatty acids due to the activity of lingual lipase in the stomach.     

Changes in host cell membrane activities in response to adhesion of Neisseria gonorrhoeae

Physiological changes in host cell model membranes (intact human erythrocytes and ghosts) as a consequence of bacterial adhesion were studied with special reference to Neisseria gonorrhoeae. Membrane activities examined were transport of K+, Cl- ions, pyruvate kinase, Na-K-dependent ATPase, and cAMP. We found that K+ and Cl- transport were affected, more so in membranes with attached pilated (P+) organisms than in those with apilated (P-) isogenic strains. In N. gonorrhoeae and in several other species of gram-negative bacteria studied, hemagglutination titres were directly correlated with effects on anion transport, suggesting that perturbations in anion transport are an immediate result of adhesion. Of three P+ gonococcus strains tested, two depressed Na-K-ATPase activity in the membrane, indicating a possible effect on the Na-K pump. Pyruvate kinase activity associated with the membrane appeared to be stimulated by attached gonococci, again by P+ strains to higher levels than P- organisms. Clearly, some enzyme properties of host membranes are intrinsically affected by bacterial adhesion. Human polymorphonuclear neutrophils were also investigated, and with some exceptions, changes observed in leukocyte enzyme activities tended to parallel those in erythrocytes. Since hypochlorous acid production is considered to be an important microbicidal mechanism in neutrophils, interference with Cl- transport could jeopardize their role in host defense.     

Prokaryotic features of a nucleus-encoded enzyme. cDNA sequences for chloroplast and cytosolic glyceraldehyde-3-phosphate dehydrogenases from mustard (Sinapis alba)

Two cDNA clones, encoding cytosolic and chloroplast glyceraldehyde-3-phosphate dehydrogenases (GAPDH) from mustard (Sinapis alba), have been identified and sequenced. Comparison of the deduced amino acid sequences with one another and with the GAPDH sequences from animals, yeast and bacteria demonstrates that nucleus-encoded subunit A of chloroplast GAPDH is distinct from its cytosolic counterpart and the other eukaryotic sequences and relatively similar to the GAPDHs of thermophilic bacteria. These results are compatible with the hypothesis that the nuclear gene for subunit A of chloroplast GAPDH is of prokaryotic origin. They are in puzzling contrast with a previous publication demonstrating that Escherichia coli GAPDH is relatively similar to the eukaryotic enzymes [Eur. J. Biochem. 150, 61-66 (1985)].     

Codon reading patterns in Saccharomyces cerevisiae mitochondria based on sequences of mitochondrial tRNAs

The sequences of Saccharomyces cerevisiae mitochondrial tRNA Arg1, tRNA Arg2, tRNA Gly, tRNA Lys2, tRNA Leu amd tRNA Pro are reported. Special structural features were found in tRNA Pro, which has A8, C21, A48 instead of the constant residues U8, A21 and pyrimidine 48, and in tRNA Lys2, which has a U excluded from base-paring and bulging out from the TpsiC stem. The tRNA Arg1, tRBA Lys2 and tRNA Leu, which belong to two-codon families ending in a purine, have a modified uridine in the wobble position, which prevents misreading of C and U. It is likely to be 5-carboxymethylaminomethyluridine. tRNA Gly and tRNA Pro have an unmodified uridine in the wobble position allowing the reading of all four codons of a four-codon family. However, tRNA Arg2, which is a minor species and belongs to the CGN four-codon family, has an unmodified A in the wobble position. This unusual feature raises the problem of the mechanism by which the codons CGA, CGG and CGC are recognized.     

Two novel matrix proteins isolated from articular cartilage show wide distributions among connective tissues

Two proteins of Mr = 58,000 and 59,000, respectively, were purified from 4 M guanidinium chloride extracts of articular cartilage by dissociative CsCl-density gradient centrifugation followed by gel chromatography on Sephadex G-200 and ion exchange chromatography on DEAE-cellulose. The two proteins differ in ionic properties and only the one with Mr = 59,000 bound to the ion exchanger. Although the two proteins showed dissimilar peptide patterns after proteolysis, their amino acid composition was similar, with very high contents of leucine and aspartic acid/asparagine. The two proteins showed no cross-reactivity in radioimmunoassays. By use of these assays, the proteins were demonstrated in extracts of most connective tissues, with high contents of about 0.1% of tissue wet weight determined in several types of cartilage. Among the non-cartilage connective tissues, tendon and sclera had the highest contents of the proteins, i.e. about 0.1% of the tissue wet weight. Bone extracts, on the other hand, contained insignificant amounts of the proteins. Only the Mr = 59,000 protein was detected in serum, its concentration being about 33 micrograms/l. Both proteins were shown to be localized in the extracellular matrix of cartilage, predominantly in the territorial matrix, by using indirect immunofluorescence.     

Apolipoprotein B mediates the capacity of low density lipoprotein to suppress neutrophil stimulation by particulates

Low density lipoprotein (LDL) inhibits phagocytosis of certain negatively charged particulates and also inhibits subsequent cellular secretory and oxidative responses to these particulates. In the present work, we have defined the structural features of LDL involved in this activity. Starch-heptane extraction depleted greater than 95% of neutral lipids but had little effect on the capacity of LDL to inhibit monosodium urate crystal- or polystyrene latex bead-induced neutrophil chemiluminescence (CL). Liposomes containing gamma-palmitoyl-beta-oleoylphosphatidylcholine (PC) with unesterified cholesterol (PC:cholesterol = 2:1), PC and sphingomyelin (PC:sphingomyelin = 2.3:1), or PC alone lacked the capacity to inhibit urate-induced CL. However, incorporation of apoB-100 into liposomes via cholate dialysis rendered them nearly as inhibitory for urate-induced neutrophil CL as LDL on a protein weight basis. Moreover, delipidated apoB-100, containing less than 3% residual phospholipid, inhibited neutrophil responses to urate crystals or latex beads (degranulation and superoxide anion release) in a stimulus-specific manner. Modifications of the lysine residues of apoB (e.g. acetylation) reduced both the capacity of LDL to inhibit urate crystal-induced CL and to bind to urate crystals. The effects of apoB lysine residue modification were reversible, proportional to the extent of modification, and were not attributable to alteration of the net charge of apoB. Thus, the apoB-100 of LDL both mediates and shares the capacity of native LDL to inhibit certain neutrophil responses to particulates.     

Islet activating protein inhibits physiological responses evoked by cardiac muscarinic acetylcholine receptors. Role of guanosine triphosphate binding proteins in regulation of potassium permeability

The involvement of GTP binding proteins in muscarinic acetylcholine receptor (mAChR) mediated responses of cultured chick embryonic cardiac muscle cells was studied by using islet activating protein (IAP) from Bordetella pertussis. Incubation of cells for 24 h with IAP resulted in inhibition of subsequent IAP-catalyzed incorporation of [alpha-32P]ADP-ribose into membrane proteins of Mr 39 000 (No alpha) and 41 000 (Ni alpha); treatment of cultures with 5 ng/mL IAP was sufficient to ADP-ribosylate all available No alpha and Ni alpha. Inhibition of forskolin-stimulated cAMP accumulation by the muscarinic agonist carbachol was abolished in cultures pretreated with IAP. The affinity of carbachol for the mAChR in membranes from IAP-treated cells was considerably decreased compared to control membranes and was not further decreased by addition of guanyl-5'-yl imidodiphosphate. In contrast, the affinity of carbachol for the mAChR on intact cells was not affected by pretreatment with IAP. To investigate the involvement of No and/or Ni in mAChR-mediated increases in K+ permeability, the effect of IAP treatment on mAChR stimulation of 86Rb+ efflux was determined. Treatment of cultures with 5 ng/mL IAP for 24 h completely blocked the stimulation of 86Rb+ efflux evoked by carbachol. Because previous work has shown that mAChR regulation of K+ permeability is independent of changes in cAMP levels, these results suggest a role for No and/or Ni in coupling the mAChR directly to K+ channels in the heart.