Na+-dependent transport of glycine in renal brush border membrane vesicles: Evidence for a single specific transport system

The uptake of glycine in rabbit renal brush border membrane vesicles was shown to consist of glycine transport into an intravesicular space. An Na⁺ electrochemical gradient (extravesicular>intravesicular) stimulated the initial rate of glycine uptake and effected a transient accumulation of intravesicular glycine above the steady-state value. This stimulation could not be induced by the imposition of a K⁺, Li⁺ or choline⁺ gradient and was enhanced as extravesicular Na⁺ was increased from 10 mM to 100 mM. Dissipation of the Na⁺ gradient by the ionophore gramicidin D resulted in diminished Na⁺-stimulated glycine uptake. Na⁺-stimulated uptake of glycine was electrogenic. Substrate-velocity analysis of Na⁺-dependent glycine uptake over the range of amino acid concentrations from 25 μM to 10 mM demonstrated a single saturable transport system with apparent K_m = 996 μM and V_max = 348 pmol glycine/mg protein per min. Inhibition observed when the Na⁺-dependent uptake of 25 μM glycine was inhibited by 5 mM extravesicular test amino acid segregated dibasic amino acids, which did not inhibit glycine uptake, from all other amino acid groups. The amino acids d-alanine, d-glutamic acid, and d-proline inhibited similarly to their l counterparts. Accelerative exchange of extravesicular [³H]glycine was demonstrated when brush border vesicles were preloaded with glycine, but not when they were preloaded with l-alanine, l-glutamic acid, or with l-proline. It is concluded that a single transport system exists at the level of the rabbit renal brush border membrane that functions to reabsorb glycine independently from other groups of amino acids.     

Functional membrane vesicles from the nervous system of insects: I. Sodium- and chloride-dependent γ-aminobutyric acid transport

(1) A synaptosomal fraction obtained from locust nervous tissue has been shown to possess an active γ-aminobutyric acid transport mechanism. This activity is preserved and even enriched by the membrane vesicles derived from osmotically shocked synaptosomes. (2) Electron-microscopy examination indicates that the above membrane vesicles are derived predominantly from the neuronal plasma membrane and are devoid of any internal cellular organelles and components. Active transport of γ-aminobutyric acid into these vesicles has been demonstrated with artificially imposed ion gradients as the sole energy source. (3) γ-Aminobutyric acid transport can be driven by an Na⁺ gradient (out>in) and /or by a gradient of Cl^? (out>in). This process is absolutely dependent on the simultaneous presence of both types of ion in the external medium. The stimulation of the process by valinomycin indicates that γ-aminobutyric acid transport is an electrogenic process which is stimulated by a membrane potential (interior negative).     

Use of tritiated 3-O-methyl-d-glucose for studies of membrane transport caveat

Tritiated 3-O-methyl-d-glucose has many useful attributes as a model substance for studies of the transport of glucose across cell membranes. However, preparations of high specific radioactivity can decompose within a few months, producing radioactive impurities that can cause a several-fold increase in the apparent rate of sugar transport. In our investigation radioactive contaminants entered frog skeletal muscle cells by free diffusion rather than by facilitated transport. Much of the contaminating radioactive material could be removed by evaporating the solvent and redissolving the sugar. Tritiated sugar samples that had a specific activity below 0.1 Ci/mmol remained stable and suitable for transport measurements after several years of storage at -20°C. In order to evaluate the suitability of a given tritiated preparation of sugar for transport measurements, it is recommended that its behavior be compared with that of a stable reference standard of low specific activity.     

Effect of N-ethylmaleimide on leucine transport in chang liver cells. I. Stimulatory effect on Na+-independent transport at low concentrations of leucine

Pretreatment of Chang liver cells with $\text{N-}\text{ethylmaleimide}$ (0.5 or 1 mM) stimulated Na⁺-independent uptake of leucine at low concentrations (?1 mM). The stimulatory effect of $\text{N-}\text{ethylmaleimide}$ on the uptake of leucine measured in Na⁺-replete medium was completely blocked by the addition of $\text{b-2-}\text{aminobicyclo}\text{[2,2,1]}\text{heptane}\text{-2-}\text{carboxylate}$ (5 mM), which shows that the L system participates in the stimulation. The Na⁺-dependent uptake of glycine was depressed by $\text{N-}\text{ethylmaleimide}$ pretreatment. The stimulation of the Na⁺-independent component of leucine uptake continued for at least 30 min after $\text{N-}\text{ethylmaleimide}$ treatment, while the inhibition of glycine uptake was progressive with time and the Na⁺-dependent uptake of leucine became depressed later, after the treatment. It has been demonstrated that treatment of cells with $\text{N-}\text{ethylmaleimide}$ is capable of increasing the Na⁺-independent influx of leucine and at the same time slightly decreasing the efflux of it. These results suggest that $\text{N-}\text{ethylmaleimide}$ attacks the Na⁺-independent system of amino acid transport at the reactive SH groups(s) of relevant protein(s) in favor of specific activation of that system in this cell.     

Inhibition of topoisomerase I by heparin

DNA topoisomerase I isolated from mouse mammary cacinoma cells was shown to be inhibited by heparin, the dose giving 50% inhibition (IC₅₀) being 0.20 μg/ml. Other chemically related acid mucopolysaccharides including heparan sulfate, dermatan sulfate etc. were more than 500 times less active than heparin. When the amount of enzyme was doubled relative to the substrate the inhibition was reversed. Addition of heparin to assay mixtures after the initiation of the reaction immediately inhibited the enzyme reaction.     

Binding properties of 23S,25-dihydroxyvitamin D3: an in vivo metabolite of vitamin D3

23$\text{S}$,25-Dihydroxyvitamin D₃ was isolated from the plasma of vitamin D₃-toxic pigs. An ultraviolet absorbance spectrum confirmed its purity. The configuration of the 23-hydroxyl group was determined to be S by comparison of the natural product with synthetic 23$\text{R}$,25- and 23$\text{S}$,25-dihydroxyvitamin D₃ by high-pressure liquid chromatography. The affinity of both 23$\text{S}$,25- and 23$\text{R}$,25-dihydroxyvitamin D₃ for the plasma vitamin D binding protein was similar to vitamin D₃. Thus, with respect to the plasma vitamin D binding protein, 23$\text{S}$,25-dihydroxyvitamin D₃ is the least potent, naturally-occurring, dihydroxylated vitamin D₃ metabolite known.     

Inhibition of DNA synthesis in permeabilized L cells by novobiocin

Novobiocin was equipotent in inhibiting DNA and RNA synthesis in cultured mouse L cells. It also suppressed in vitro DNA and RNA synthesis in permeabilized L cells and nuclei; 50 percent inhibition of DNA and RNA synthesis was obtained by 1 mM and 20 mM novobiocin, respectively. ATP antagonized the effect of novobiocin. Nalidixic acid had a weak inhibitory effect on in vitro DNA synthesis; 10 mM nalidixic acid showed 60 percent inhibition. ATP did not antagonize nalidixic acid. The inhibitory effect of novobiocin exceeded that of aphidicolin. These findings suggest a participation of a gyrase- and/or type II topoisomerase-like enzyme in the DNA replication machinery in L cells.     

Studies on the adenosine 3′,5′-monophosphate-dependent protein kinase of Blastocladiella emersonii

Using a combination of Chromatographic and sucrose density gradient techniques under carefully controlled conditions of pH and protease inhibitors, we demonstrate that there is only one form of adenosine 3′,5′-monophosphate-dependent protein kinase in the cytosol fraction of the Blastocladiella emersonii zoospore. If any of these conditions are omitted during extract preparation, one obtains what are apparently multiple forms of the enzyme, which are in reality artifacts due to extensive endogenous proteolytic activity. This endogenous protease is stimulated by alkaline pH and inhibited by antipain. The zoospore protein kinase is similar to type II protein kinase from mammalian cells in several aspects including Chromatographic behavior on DEAE-cellulose column, conditions for subunit dissociation and reassociation, as well as the molecular weight value of the regulatory subunit.     

Partial purification and property of pyridoxine (pyridoxamine)-5′-phosphate oxidase isozymes from wheat seedlings

Isozymes of pyridoxine (pyridoxamine)-5′-phosphate oxidase (EC 1.4.3.5) were isolated from the extract of wheat seedlings by column chromatographies. From DEAE-Sephadex A-50, two fractions having pyridoxine-5′-phosphate oxidase activity were separated by eluting with ~0.075 and ~0.125 m phosphate buffers (pH 8.0). These fractions were further fractionated on a Blue-Sepharose CL-6B column, from which again two activities were eluted by 1.0 m KCl solution. One fraction, designated as E-I, used only pyridoxine 5′-phosphate as substrate, whereas the other, designated as E-II, oxidized not only pyridoxine 5′-phosphate but also pyridoxamine 5′-phosphate with approximately equal rates. The mobility on polyacrylamide disc gel electrophoresis and the substrate specificity of these two fractions were different. Therefore, they were concluded to be isozymes.     

gp110—A major sialoglycoprotein of human cells: Isolation and partial characterization from a malignant melanoma cell line

A major sialoglycoprotein (gp110) was isolated from NP-40 extracts of the human melanoma cell line SK-MEL-37 by concanavalin A-Sepharose and wheat germ agglutinin-Sepharose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A rabbit antiserum was prepared to this concanavalin A- and wheat germ agglutinin-binding glycoprotein and used to study the biochemical properties and distribution of gp110 in human cells. gp110 is highly acidic (pI ~ 3.8–4.0) and located on the cell surface in melanoma cells. It contains sialylated, N-linked complex chains as well as sialylated, O-linked carbohydrate chains. gp110 was detected as a major glycoprotein on all human cell lines tested (except erythrocytes), although its apparent molecular weight varied from cell line to cell line. The pI of gp110 from normal and malignant human kidney epithelial cells was identical, indicating that gp110's from two cell types do not substantially differ in their sialylated carbohydrate moieties.