The sequential transfer of internalized, cell surface sialoglycoconjugates through the lysosomes and Golgi complex in HeLa cells

Surface sialoglycoproteins of HeLa cells were labeled by NaB[3H]4 reduction after oxidation with NaIO4, yielding seven major radioactive bands as visualized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. When labeled cells are reincubated in growth medium, all of these major classes of glycoproteins are internalized and all but one (105 kDa) are recycled, i.e. subsequently reappear on the surface. The surface-labeling patterns over time remain qualitatively similar, but changes in relative specific activity of the bands suggest some preferential degradation of individual glycoproteins. Analytical fractionation at various time points after labeling suggests that the surface molecules pass through the lysosomal compartment and subsequently accumulate in the Golgi and Golgi-related compartments before returning to the surface. Inhibition of lysosomal function with chloroquine or NH4Cl prevents the accumulation and subsequent recycling. The pathway is confirmed with preparative fractionation into surface membrane, prelysosomal, lysosomal, Golgi, and Golgi-related compartments. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrates a degree of preferential handling of the glycoproteins on this pathway, e.g. the 180-kDa band is relatively reduced at the endocytic/prelysosomal stage and the 105-kDa band appears to be degraded in its first passage through the lysosomes. The other bands recycle 10-20 times before being degraded.     

The binding of [3H]mepyramine to histamine H1 receptors in monkey brain

Several laboratories have reported ligand binding studies using radioactive histamine H1 antagonists to label the H1 receptors in mammalian brain. We have extended these studies to a detailed examination of the binding of [3H]mepyramine to monkey brain and have shown that the distribution is similar to that in man, with specific binding sites being concentrated in the frontal cortex with relatively low binding to the pons and basal ganglia. The binding shows a single saturable component with a KD of about 1 nM and a Hill plot slope close to unity. These observations are the same for all structures tested. Comparison with data from other laboratories suggests that in this species, the histamine receptor is the same as that in peripheral tissues. From Ki values for various ligands and comparison of KD estimates in other species, the receptor seems to be essentially identical to the H1 receptor in central and peripheral tissues of the guinea pig and also to that in human brain. The rat and possibly the dog have minor differences from the monkey in terms of KD values for [3H]mepyramine binding.     

Inhibition of glycine N-methyltransferase activity by folate derivatives: implications for regulation of methyl group metabolism

Glycine N-methyltransferase, an enzyme that uses S-adenosylmethionine to methylate glycine with the production of sarcosine, was recently shown to be identical with a major folate binding protein of rat liver (Cook, R.J. and Wagner, C. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 3631-3634). We now present evidence that 5-methyltetrahydropteroylpentaglutamate (5-CH3-H4PteGlu5) is bound with high specificity, and is a powerful inhibitor of the enzyme. It is proposed that this information may be used to modify the "methyl trap" hypothesis which describes how the availability of one-carbon units is regulated by folate, vitamin B12 and methionine.     

Clustering of glycine and NG,NG-dimethylarginine in nucleolar protein C23

Protein C23 (Mr 110 000, pI = 5.5), a major phosphoprotein in the nucleolus of mammalian cells, has been shown to contain 1.3 mol% of NG,NG-dimethylarginine (DMA) [Lischwe, M.A., Roberts, K.D., Yeoman, L.C., & Busch, H. (1982) J. Biol. Chem. 257, 14600-14602]. A tryptic peptide from protein C23 that contains DMA has been isolated and sequenced. Its sequence is Gly-Glu-Gly-Gly-Phe-Gly-Gly-DMA-Gly-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA- Gly-Gly- Gly-DMA-Gly-Gly-DMA-Gly-Gly-Phe-Gly-Gly-DMA-Gly-DMA-Gly-Gly-Phe-Gly-Gly- DMA-Gly-Gly-Phe-DMA-Gly-Gly-DMA-Gly-Gly-Gly-Gly-Asp-Phe-Lys. This peptide contains 34 glycine, 10 DMA, and 6 phenylalanine residues and has clusters of glycine and NG,NG-dimethylarginine interspersed with phenylalanine residues. A similar domain has been found at the amino terminus of a nucleolar protein of Mr 34,000, pI = 8.5. This sequence array may represent a conserved domain characteristic of a certain class of nuclear proteins. All of the methylated arginine residues in protein C23, the 34-kilodalton protein, and myelin basic protein [Carnegie, P.R. (1971) Biochem. J. 123, 57-67] have at least one adjacent glycine. Access of certain arginine methylases to arginine residues may be sterically possible because of the lack of a side chain on the adjacent glycine residue(s).     

Purification of gamma-glutamyltransferase by phenyl boronate affinity chromatography. Studies on the acceptor specificity of transpeptidation by rat kidney gamma-glutamyltransferase

gamma-Glutamyltransferase has been purified from rat kidney by a novel procedure using phenyl boronate affinity chromatography. The highly purified enzyme has been studied with respect to acceptor specificity for a number of amino acids, amino acid analogues, dipeptides and tripeptides. The acceptor activity is specific for L-amino acids. The amino acids and the majority of the essential amino acids are poor acceptors while the sulphur-containing amino acids are the best acceptors. The acceptor activity is modulated by the substitution of the amino acid side chain. Substitution of the side chain at the delta, gamma or beta positions results in a proportionally decreasing ability to act as acceptor. The carbonyl moiety of the gamma-carboxy group of the acceptor appears to be essential for acceptor activity, absence of an alpha-carboxy carbonyl group increases the Kappm of the acceptor approximately 100-fold.     

Solubilization and purification of hepatic microsomal trans-2-enoyl-CoA reductase: evidence for the existence of a second long-chain enoyl-CoA reductase

The present study describes the solubilization and purification of a NADPH-specific trans-2-enoyl-CoA reductase from rat liver microsomes. The final preparation was purified to near homogeneity and had a minimal molecular weight of 51,000 +/- 2,000, as judged by sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis. This enzyme specifically used NADPH, as cofactor, and was chromatographically (2',5'-ADP-agarose) separated from another trans-2-enoyl-CoA reductase which utilized either NADH or NADPH as cofactor. The NADPH-specific trans-2-enoyl-CoA reductase catalyzed the reduction of trans-2-enoyl-CoAs from 4 to 16 carbon units. The Km values for crotonyl-CoA, trans-2-hexenoyl-CoA, and trans-2-hexadecenoyl-CoA were 20, 0.5, and 1.0 microM, while the Km value for NADPH was 10 microM. Although N-ethylmaleimide, heat treatment, and limited proteolysis with trypsin affected the reduction of short-chain (C4) and long-chain (C16) substrates equally, and in spite of the fact that a single protein band was observed on SDS-gels, at the present time one cannot state unequivocally that the purified preparation contained only one reductase. trans-2-Hexenoyl-CoA, for example, did not inhibit the reduction of trans-2-hexadecenoyl-CoA to palmitoyl-CoA and trans-2-decenoyl-CoA to decanoyl-CoA whereas it strongly inhibited the conversion of crotonyl-CoA to butyryl-CoA. The potential implications of this finding are discussed. Finally, the reductase preparation was shown not to contain either heme, nonheme iron, or a flavin prosthetic group.     

Light-induced conformational change in rhodopsin detected by modification of G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation

CNBr treatment of rod outer segments was performed in dark and in light conditions. With the subsequent modified rhodopsin and opsin the cGMP phosphodiesterase activation system was reconstituted. The recombination systems exhibited greatly reduced G-protein binding, GTP gamma S binding and cGMP phosphodiesterase activation. The reduction in activity of these three steps of the PDE activation cascade is most significant with modified opsin and is shown to be due to its inability to bind the G alpha subunit. The correlation between the localization of CNBr cleavage in dark and light conditions and these results is strongly indicative that a light-induced conformational change occurs in two extradiscal regions of rhodopsin.     

Linear relationship of phlorizin-binding capacity and hexose uptake during differentiation in a clone of LLC-PK1 cells

With a clone of (Cl 4) of LLC-PK cells, which develop a high capacity for Na+-dependent hexose uptake over time (days) in culture, we show that increasing uptake capacity is paralleled by an increase in the number of phlorizin-binding sites in the population. The linear relationship between binding and hexose transport is the same whether the cells differentiate spontaneously or are induced by either methylisobutylxanthine or hexamethylene bisacetamide. The constancy of the relationship suggests that the primary factor in transport development is the number of transporters in the cells rather than other possible factors like a change in membrane potential or decreased efflux. The Kd for phlorizin binding is .08 +/- .04 microM, and corresponds to Ki of 0.10 microM for transport inhibition. The turnover number of the transporter is estimated to be 170 +/- 40 molecules per second of alpha-methyl glucoside.     

Transepithelial transport of nonelectrolytes in the rabbit mandibular salivary gland

Summary The characteristics of nonelectrolyte secretion by the rabbit mandibular salivary gland have been investigated in anin vitro perfused preparation. The concentrations of¹⁴C-labeled nonelectrolytes were measured in saliva samples collected over a range of flow rates during the secretory response of the gland to continuous acetylcholine infusion. Of the nine nonelectrolytes studied, the two particularly lipid-soluble molecules, ethanol and antipyrine, appeared in the saliva at approximately the same concentration as in the perfusate, regardless of the secretory flow rate. The more polar molecules (urea, ethanediol, thiourea, glycerol, erythritol, mannitol and sucrose) appeared at saliva/perfusate concentration ratios () which showed a strong dependence on flow. With the exception of thiourea, this could be attributed to the combined contributions of diffusion and solvent drag.For the polar nonelectrolytes, estimates have been obtained of both the permeability coefficients of the gland (P) and the solvent-drag filtration coefficients (1–). The relation between 1– and molecular radius suggests that small polar nonelectrolytes and the bulk of the secreted water cross the epithelium via aqueous channels that are approximately 0.8 nm in width. The location of the channels remains uncertain because tissue space measurements indicate that the nonelectrolytes most affected by solvent drag have access to both transcellular and paracellular pathways.