Regulation of the human organic cation transporter hOCT1

The human organic cation transporter type 1 (hOCT1) is an important transport system for small organic cations in the liver. Organic cation transporters are regulated by different signaling pathways, but the regulation of hOCT1 has not yet been studied. In this work, we have for the first time investigated the regulation of hOCT1. hOCT1 was expressed in Chinese hamster ovary cells (CHO-hOCT1) and in human embryonic kidney cells (HEK293-hOCT1). Its activity was monitored using microfluorimetry with the fluorescent organic cation 4-(4-(dimethylamino)styryl)-N-methylpyridinium (ASP(+)) as substrate. hOCT1 expressed in CHO-cells was inhibited by protein kinase A (PKA) activation (1 microM forskolin, -58 +/- 6%, n = 12), calmodulin inhibition (0.1 microM calmidazolium, -68 +/- 3%, n = 6; 10 microM ophiobolin A, -48 +/- 10%, n = 7), calmodulin-dependent kinase II inhibition (1 microM KN62, -78 +/- 4%, n = 12), and inhibition of p56(lck) tyrosine kinase (10 microM aminogenistein, -35 +/- 7%, n = 12). The apparent affinities for TEA(+) were lower in CHO-hOCT1 than in HEK293-hOCT1, while those for TPA(+) and quinine were almost identical; the rank order of EC(50) values (TPA(+) > quinine > TEA(+)) was independent of the expression system. EC(50) values for TEA(+) in CHO-hOCT1 or HEK293-hOCT1 were increased under calmidazolium incubation (6.3 and 1.4 mM, respectively). hOCT1 was inhibited by PKA and endogenously activated by calmodulin, calmodulin-dependent kinase II, and p56(lck) tyrosine kinase. Regulation pathways were the same in the two expression systems. Since apparent substrate affinities depend on activity of regulatory pathways, the expression system plays a role in determining the substrate affinities.     

Genomic affinities in Arachis section Arachis (Fabaceae): molecular and cytogenetic evidence

Section Arachis is the largest of nine sections in the genus Arachis and includes domesticated peanut, A. hypogaea L. Most species are diploids (x=10) with two tetraploids and a few aneuploids. Three genome types have been recognized in this section (A, B and D), but the genomes are not well characterized and relationships of several newly described species are uncertain. To clarify genomic relationships in section Arachis, cytogenetic information and molecular data from amplified fragment length polymorphism (AFLP) and the trnT-F plastid region were used to provide an additional insight into genome composition and species relationships. Cytogenetic information supports earlier observations on genome types of A. cruziana, A. herzogii, A. kempff-mercadoi and A. kuhlmannii but was inconclusive about the genome composition of A. benensis, A. hoehnei, A. ipaensis, A. palustris, A. praecox and A. williamsii. An AFLP dendrogram resolved species into four major clusters and showed A. hypogaea grouping closely with A. ipaensis and A. williamsii. Sequence data of the trnT-F region provided genome-specific information and showed for the first time that the B and D genomes are more closely related to each other than to the A genome. Integration of information from cytogenetics and biparentally and maternally inherited genomic regions show promise in understanding genome types and relationships in Arachis.     

Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding

The immunochemical reaction of monoclonal antibodies directed against native membrane proteins was investigated after their separation in sodium dodecyl sulfate polyacrylamide gels and electrotransfer to nitrocellulose. Nonspecific binding of antibodies to membrane proteins, which was increased by beta-mercaptoethanol treatment or heat denaturation of the antibodies, could be significantly reduced if 1 M D-glucose plus 10% (v/v) glycerol was added during the incubation with the antibodies. It was found that specific antibody binding was drastically reduced by SDS treatment of the membrane proteins. During the electrotransfer to nitrocellulose and the simultaneous removal of SDS, some increase in antibody binding was observed. Considerable renaturation of antigenic sites in the blotted proteins could be induced if the nitrocellulose blots were incubated for 16 h at 37 degrees C in phosphate-buffered saline. With the introduction of both modifications, the renaturation step, and the addition of D-glucose and glycerol to reduce nonspecific antibody binding, the immunoblot technique may be successfully applied to detect conformational antibodies against membrane proteins.     

Electron microscopic immunohistochemical localization of components of Na+-cotransporters along the rat nephron

The localization of Na+-cotransport proteins in cortex and outer medulla of rat kidney was investigated with five monoclonal antibodies. Recently, it was found that these antibodies altered Na+-D-glucose cotransport and/or Na+-dependent high affinity phlorizin binding in pig kidney cortex and that three of these antibodies interacted also with Na+-cotransporters for lactate, L-alanine and/or L-glutamate (Koepsell, H., K. Korn, A. Raszeja-Specht, S. Bernotat-Danielowski, D. Ollig, J. Biol. Chem. 263, 18,419-18,429 (1988]. In pig and rat the monoclonal antibodies bind to two brush-border membrane polypeptides with identical molecular weights and isoelectric points of 75,000 and pI 5.5, and 47,000 and pI 5.4. These polypeptides have been previously identified as components of the porcine renal Na+-D-glucose cotransporter (Neeb, M., U. Kunz, H. Koepsell, J. Biol. Chem. 262, 10,718-10,727 (1987] and may also be part of other Na+-cotransporters. The electron microscopic localization of antibody binding was demonstrated by protein A-gold labeling on ultrathin plastic sections. Three antibodies bound to brush-border membranes of proximal convoluted and straight tubules. In the proximal convoluted tubules all antibodies reacted with apical endocytic vacuoles, apical dense tubules and lysosomes. Since dense tubules are supposed to originate from endocytic vacuoles and to fuse with brush-border membranes the data suggest recycling of Na+-cotransporters in the proximal convoluted tubule. In the outer medulla two antibodies bound to apical membranes of descending thin limbs (DTL) of short loops of Henle and to apical and basal membranes of DTL of long loops of Henle. Three antibodies bound to apical membranes of collecting ducts. These data indicate that Na+-cotransporters or homologous proteins exist beyond the proximal tubule.     

Identification of an Mr 75000 component of the H+/D-glucose cotransporter from Zea mays with monoclonal antibodies directed against the mammalian Na+/D-glucose cotransporter

Monoclonal antibodies which interact with the mammalian Na+/D-glucose cotransporter and bind to Mr 75,000 and Mr 47,000 polypeptide components of this transporter have been described (Koepsell, H., Korn, K., Raszeja-Specht, A., Bernotat-Danielowski, S. and Ollig, D. (1988) J. Biol. Chem., 263, 18419-18429). The interaction of these antibodies with plasma membranes from Zea mays L. coleoptiles containing an H+/D-glucose cotransporter was studied. Four monoclonal antibodies cross-reacted with Mr 75,000 and Mr 33,000 polypeptides. One of these antibodies, which inhibits Na+/D-glucose cotransport in the kidney and stimulates Na+/D-glucose cotransport in intestine, stimulates electrogenic uptake of 3-O-methyl-D-[14C]glucose in plant membrane vesicles. The data indicate common epitopes in the mammalian Na+/D-glucose cotransporter and the H+/D-glucose cotransporter of plants and suggest that both transporters contain an Mr 75000 polypeptide component.     

Effects of Low Root Temperature on Ion Uptake and Ion Translocation in Wheat

Roots of wheat seedlings (Triticum aestivum L. cv. Weibulls Starke) were cooled (+1°C) for 24 h while the shoots were kept at 25°C. The treatment induced an increased water deficit in the leaves. Fresh weight, dry weight, and the uptake and distribution of potassium and calcium were measured before and after cooling. Growth, measured both as fresh weight and dry weight increase, was reduced during the cold treatment. Afterwards (at 20°C), growth recovered to nearly pre-stress rates. Analysis of the potassium fluxes in and out of the roots by ⁸⁶Rb techniques showed that influx, and to a lesser extent efflux, were inhibited at low temperature. The result was a net potassium uptake rate of one-third that of unstressed plants. After the cooling period the potassium influx increased to the rate of control plants. The potassium efflux increased to one and one-half times the rate of unstressed wheat so that net uptake was negative. The increase in potassium efflux was explained by a higher permeability of the root cell membranes after cooling. The net uptake of calcium was reduced to one-third by root cooling. Contrary to potassium uptake, calcium uptake increased under post-stress conditions, partly due to a low efflux rate. During root cooling there was a redistribution of dry matter from the leaves down towards the lower part of the shoot. Afterwards the original distribution of dry matter was reestablished. The net flow of potassium and calcium followed a similar pattern as dry matter, suggesting a growth-regulated flow.     

Different classes of nucleotide binding sites in the (Na+ + K+)-ATPase studied by affinity labeling and nucleotide-dependent SH-group modifications

The ATP analog 6-[(3-carboxy-4-nitrophenyl)thiol]-9-beta-D-ribofuranosylpurine 5'-triphosphate (Nbs6ITP) is slowly hydrolyzed at pH 7.4 by the (Na+ + K+)-ATPase, whereas it binds covalently at pH 8.5 and inhibits the enzyme irreversibly. Time courses of irreversible inhibition could only be fitted to a model in which the enzyme can exist in two slowly interchangeable states, one of which is enzymatically active and binds Nbs6ITP first reversibly and then covalently. Arguments that the covalent binding occurs at a low affinity nucleotide binding site are: (a) similarity of the Ki Nbs6ITP for the reversible and the irreversible inhibition and of K0.5 for ATP protection; (b) stoichiometry of covalent Nbs6ITP binding per alpha subunit of 0.8; and (c) change of complex substrate dependence of the enzyme to a Michaelis-Menten type after Nbs6ITP modification. This change in kinetics and the finding that the Nbs6ITP inactivation at a low affinity nucleotide binding site is increased by micromolar ADP concentrations indicates that the (Na+ + K+)-ATPase contains two different nucleotide binding sites. Since studies of nucleotide effects on enzyme inactivation by 5,5'-dithiobis(2-nitrobenzoic acid) did not confirm the hypothesis of an SH-group in a nucleotide binding site, Nbs6ITP may bind to another functional group, e.g. to an OH-group of tyrosine.     

Degeneration of Streptomyces niveus with Repeated Transfers

Novobiocin production by Streptomyces niveus decreased drastically as the culture was transferred at regular intervals under both sporulating and nonsporulating conditions. Addition of degenerated live mycelium as second inoculum to shake flask fermentations already inoculated with a high-producing strain resulted in sharply depressed novobiocin formation. Fractionated medium of low-producing strain containing either no cells or dead cells had no adverse effect on the antibiotic yield of the high-producing mycelium. It appears that the low-producing mycelium was outgrowing the high-producing mycelium. A study of the growth rates of the two types of mycelium in a clear broth medium indicated no differences in generation time. However, the low-producing strain proved to have a higher efficiency of carbohydrate utilization, thus overgrowing the high-producing strain. It was speculated that culture instability of S. niveus is due to heterocaryosis.