Asymmetry of the Na+-succinate cotransporter in rabbit renal brush-border membranes

We have investigated the symmetry of Na⁺-succinate cotransport in rabbit renal brush-border membrane vesicles. Succinate influx and efflux kinetics were measured under voltage-clamped conditions using [¹⁴C]succinate and a rapid filtration procedure. Both influx and efflux were Na⁺-dependent, saturable, temperature-sensitive, and influenced by the trans Na⁺ and succinate concentrations. The system was judged to be asymmetric, since the maximal velocity for influx was 3-fold higher than that for efflux, and trans Na⁺ inhibited influx more than efflux. This may be due to the asymmetrical insertion of the transporter in the brush-border membrane, which leads to differences in either the forward and backward translocation rates of the fully loaded carrier or the Na⁺ and succinate binding constants at the inner and outer faces of the membrane.     

Detection of DC electric dipoles in background fields by the nurse shark

Summary Two nurse sharks (Ginglymostoma cirratum) were trained to respond to the galvanic dipole fields of steel spheres with and without a background electric field. Detection ranges were increased by a factor of 1.6 with background-field-on over background-field-off for dc fields in the range of 0.01 V/cm to 0.04 V/cm independent of background field strength. No increase in detection range was found for a background field strength of 0.005 V/cm. Similar results were obtained for an ac background field of 0.005 V/cm zero-to-peak at frequencies from 0.2 Hz to 1.6 Hz. These results indicate that threshold sensitivity was increased by a factor of 4 with the background field on. Further observations made on one shark, electric dipoles, and a dc background field of 0.02 V/ cm, are consistent with the factor of four increase in threshold sensitivity with the background field on found using the steel balls. The reason for the observed increase in sensitivity is unknown.     

Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma

A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of ? 65,000 (HA-BR₆₅). N-terminal amino acids in the complex were selectively ^l4C-carbamylated. The resulting derivatized HA-BR₆₅ was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a M_r by ? 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR₆₅ to a polypeptide of ? 55,000 (HA-BR₅₅) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with M_r ? 109,000 (HA-BR₁₀₉) as well as HA-BR₆₅. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at M_r ? 120,000 (HA-BR₁₂₀) and ? 140,000 (HA-BR₁₄₀). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.     

Changes in the metabolism of the proteoglycans from sheep articular cartilage in response to mechanical stress

The effect of altered mechanical stress on the metabolism of sheep articular cartilage has been investigated. A simple experimental model involving the immobilisation of a single sheep foreleg was used to study the effect of increased or decreased functional demand on the chemical composition of, and the incorporation of labelled acetate into, the proteoglycans of sheep articular cartilage. By immobilisation of one of the sheep forelegs, mechanical stress is removed from that particular joint, while increased stress is placed on the other foreleg. The load distribution about the two hind legs remains essentially the same. After a 4-week immobilisation period there was a significant increase in the hexuronic acid content of the cartilage from the loadbearing ankle joint, and a corresponding decrease in the hexuronic acid content of the non-loadbearing joint cartilage. Hexosamine analyses of the cartilage from each joint showed that the major chemical occurred in the chondroitin sulphate fraction. From analyses of the extracted and isolated proteoglycans from each experimental joint it was evident that there was a significant decrease in the molecular weight of the proteoglycan from the non-loadbearing joint. In vitro studies showed increased incorporation of labelled acetate into the chondroitin sulphate fraction from the loadbearing joint but a corresponding decreased incorporation into the non-loadbearing immobilised joint cartilage. These results suggest that the changes observed in the chemical composition of the cartilage from the loadbearing and non-loadbearing joints may be accounted for in part by changes in the biosynthesis of the cartilage proteoglycan in response to altered functional demand.     

An Artifact Of Liquid Emulsion Autoradiography

The localization of ³H-opiatcs in the myenteric plexus of the guinea pig ileum is subject to systematic artifact when stretch preparations of the myenteric plexus are dipped into liquid Kodak NTB-3 emulsion for autoradiography. The cause of the artifact was determined to be a discontinuous distribution, or retraction, of emulsion over plexuses. The apposition of frozen freeze-dried ilial sections to dried photographic emulsion avoids this source of error.     

Ouabain-insensitive salt and water movements in duck red cells. II. Norepinephrine stimulation of sodium plus potassium cotransport

Catecholamines induce net salt and water movements in duck red cells incubated in isotonic solutions. The rate of this response is approximately three times greater than a comparable effect observed in 400 mosmol hypertonic solutions in the absence of hormone (W.F. Schmidt and T. J. McManus. 1977 a.J. Gen. Physiol. 70:59-79. Otherwise, these two systems share a great many similarities. In both cases, net water and salt movements have a marked dependence on external cation concentrations, are sensitive to furosemide and insensitive to ouabain, and allow the substitution of rubidium for external potassium. In the presence of ouabain, but the absence of external potassium (or rubidium), a furosemide-sensitive net extrusion of sodium against a large electrochemical gradient can be demonstrated. When norepinephrine-treated cells are incubated with ouabain and sufficient external sodium, the furosemide-sensitive, unidirectional influxes of both sodium and rubidium are half- maximally saturated at similar rubidium concentrations; with saturating external rubidium, the same fluxes are half-maximal at comparable levels of external sodium. In the absence of sodium, a catecholamine-stimulated, furosemide-sensitive influx of rubidium persists. In the absence of rubidium, a similar but smaller component of sodium influx can be seen. We interpret these results in terms of a cotransport model for sodium plus potassium which is activated by hypertonicity or norepinephrine. When either ion is absent from the incubation medium, the system promotes an exchange-diffusion type of movement of the co-ion into the cells. In the absence of external potassium, net movement of potassium out of the cell leads to a coupled extrusion of sodium against its electrochemical gradient.     

Mechanism of suppression in Drosophila. V. Localization of the purple mutant of Drosophila melanogaster in the pteridine biosynthetic pathway

The suppressible eye color mutant purple (pr) of Drosophila melanogaster is known to be unable to synthesize a wild-type complement of pteridine eye pigments. This study measures the reduced levels of drosopterins, sepiapterin, and an unidentified presumed pteridine in pr and pr ^bw. Pteridine analyses in double mutants combining pr with one of three other eye color mutants sepia, Henna-recessive³, and prune², suggest that the metabolic block in pr occurs prior to sepiapterin biosynthesis. Measurements of GTP and GTP cyclohydrolase in pr showed wild-type levels and indicate the metabolic block in pr to be at one of the steps converting dihydroneopterin triphosphate to sepiapterin. Quantitation of pteridines in suppressed purple [su(s) ²; pr and pr; su(pr) ^e3] shows restoration of pteridines to wild-type or nearly wild-type levels.T. G. W. is a predoctoral trainee supported by Grant GM 1974 from the National Institute of General Medical Sciences, National Institutes of Health.The Oak Ridge National Laboratory is operated by Union Carbide Corporation for the U.S. Energy Research and Development Administration.     

COLONY DEVELOPMENT IN EUDORINA ELEGANS (CHLOROPHYTA,VOLVOCALES)1

A detailed ultrastructure study was made of cell division and colony development in Eudorina elegans Ehrenberg. At the onset of cell division and prior to nuclear division the nucleus moved from the cell center to the cell surface. During nuclear division the nuclear membrane remained intact, except for openings occurring at the nuclear poles. The spindle microtubules appeared to arise from a MTOC-like (microtubule organizing centers) structure, while centrioles were absent from the nuclear poles. Following telophase, daughter nuclei formed which were separated by several distinct bands of endoplasmic reticulum. Cytokinesis occurred with formation of a cleavage furrow, associated with a typical phycoplast band of microtubules. However, cytokinesis was incomplete, resulting in formation of cytoplasmic bridges between the plakeal cells. Upon completion of up to five successive cell divisions, the plakea underwent inversion, which appeared to involve the production of colonial envelope material and rearrangement of cytoplasmic bridges. A new hypothesis concerning inversion is postulated based on these observations.