A new method for the reconstitution of the anion transport system of the human erythrocyte membrane

Summary The anion transport protein of the human erythrocyte membrane, band 3, was solubilized and purified in solutions of the non-ionic detergent Triton X-100. It was incorporated into spherical lipid bilayers by the following procedure: (1) Dry phosphatidylcholine was suspended in the protein solution. Octylglucopyranoside was added until the milky suspension became clear. (2) The sample was dialyzed overnight against detergentfree buffer. (3) Residual Triton X-100 was removed from the opalescent vesicle suspension by sucrose density gradient centrifugation and subsequent dialysis. Sulfate efflux from the vesicles was studied, under exchange conditions, using a filtration method. Three vesicle subpopulations could be distinguished by analyzing the time course of the efflux. One was nearly impermeable to sulfate, and efflux from another was due to leaks. The largest subpopulation, however, showed transport characteristics very similar to those of the anion transport system of the intact erythrocyte membrane: transport numbers (at 30°C) close to 20 sulfate molecules per band 3 and min, an activation energy of approx. 140 kJ/mol, a pH maximum at pH 6.2, saturation of the sulfate flux at sulfate concentrations around 100mm, inhibition of the flux by H₂DIDS and flufenamate (approx.K _l-values at 30°C: 0.1 and 0.7 m, respectively), and right-side-out orientation of the transport protein (as judged from the inhibition of sulfate efflux by up to 98% by externally added H₂DIDS). Thus, the system represents, for the first time, a reconstitution of all the major properties of the sulfate transport across the erythrocyte membrane.     

Does Dimeric Melittin Occur in Aqueous Solutions?

Melittin, a peptide from bee venom, is known to undergo a monomer / tetramer conversion in aqueous solutions. We have studied the possible participation of dimers in the association equilibrium of melittin by sedimentation equilibrium experiments in the analytical ultracentrifuge and subsequent mathematical analysis of the concentration distributions obtained. It was found that the dimeric state is not significantly populated, the contribution of dimer to the total peptide weight probably being below 0.5%.     

In situ hybridization confirms jumping nucleolus organizing regions in Allium

In situ hybridization with a ¹²⁵I-rDNA clone from Vicia faba was performed against Allium cepa and three strains of top onion, which represent hybrids between A. cepa and A. fistulosum. In principle, the labelling patterns correspond to the patterns of the silver-stained nucleolus organizing regions (NORs) in the same species. This strongly supports the inference drawn from the Ag-NOR patterns that NORs can jump between terminal heterochromatin blocks of different Allium chromosomes in the parental species A. cepa as well as in their interspecific hybrids.     

Isolation of a cell-surface receptor for chick neural retina adherons

Embryonic chick neural retina cells release glycoprotein complexes, termed adherons, into their culture medium. When absorbed onto the surface of petri dishes, neural retina adherons increase the initial rate of neural retina cell adhesion. In solution they increase the rate of cell-cell aggregation. Cell-cell and adheron-cell adhesions of cultured retina cells are selectively inhibited by heparan-sulfate glycosaminoglycan, but not by chondroitin sulfate or hyaluronic acid, suggesting that a heparan-sulfate proteoglycan may be involved in the adhesion process. We isolated a heparan-sulfate proteoglycan from the growth-conditioned medium of neural retina cells, and prepared an antiserum against it. Monovalent Fab' fragments of these antibodies completely inhibited cell-adheron adhesion, and partially blocked spontaneous cell-cell aggregation. An antigenically and structurally similar heparan-sulfate proteoglycan was isolated from the cell surface. This proteoglycan bound directly to adherons, and when absorbed to plastic, stimulated cell-substratum adhesion. These data suggest that a heparan-sulfate proteoglycan on the surface of chick neural retina cells acted as a receptor for adhesion-mediating glycoprotein complexes (adherons).     

A new mechanism for altering chromosome number during karyotype evolution

Summary A new mechanism for changing chromosome numbers (preserving the fundamental number of long chromosome arms) during karyotype evolution is suggested. It includes: 1) Occurrence of individuals heterozygous for two interchanges between different arms of three chromosomes (a metacentric and two acrocentric ones). 2) Formation in heterokaryotypes of multivalents during meiosis between the chromosomes involved in the interchanges and their unchanged homologues. 3) Mis-segregation of chromosomes from these multivalents resulting in hypoploid (n-1) and hyperploid (n+1) simultaneously instead of euhaploid gametes. 4) Fusion of n-1 or n+1 gametes which gives rise to (zygotes and) individuals representing homokaryotypes with changed number of chromosomes (2n+2 or 2n-2), but preserves (as compared to the parental karyotypes) the number of long chromosome arms. Under definite conditions, chromosome numbers of the progeny may be changed by this process in both directions (upwards and downwards). The mechanism is free of the difficulties associated with the explanation for such changes by direct Robertsonian interchanges (see Discussion), which are usually considered to be responsible for such alterations in chromosome number. The above-mentioned process has been experimentally documented in Vicia faba and it probably also occurred naturally within the Vicia sativa group.     

Horseradish peroxidase based bienzyme elctrode for isocitrate

Coimmobilized horseradish peroxidase and D-isocitrate dehydrogenase were fixed to an oxygen electrode to assemble a bienzyme electrode for isocitrate determination. The linear measuring range for isocitrate of the sensor is between 0.1 and 2.0 mmol. I^?1, the coefficient of variation (20 measurements) is 3.6%. 8 samples per hour can be assayed. With one sensor preparation 140 measurements can be carried out.     

Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

The nature of the stable noncovalent dimers of band 3 protein from erythrocyte membranes in solutions of Triton X-100

Stable noncovalent dimers of band 3 protein from human erythrocyte membranes, in which state the protein is thought to exist after solubilization by the nonionic detergent Triton X-100, do not occur when purified batches of the detergent are used. Instead, the protein is in a monomer/dimer/tetramer association equilibrium. The stable dimers do appear, however, when the detergent has been 'aged'. They thus seem to be artifacts.     

Influence of a 9-double bond on stereospecific microbial 4,5-reductions

By stereospecific microbial reduction with Rhodosporidium rubrum or Rhodotorula glutinis, 17 alpha-cyano-methyl-4-estren-17 beta-ol-3-one was metabolized to 17 alpha-cyanomethyl-5 alpha-estrane-3 beta,17 beta-diol (50%) and 17 alpha-cyanomethyl-5 alpha-estrane-3 alpha,17 beta-diol (30%). By Clostridium paraputrificum the same substrate was reduced stereospecifically to 17 alpha-cyanomethyl-5 beta-estrane-3 alpha, 17 beta-diol (70%). When the corresponding 9-dehydrogenated compound 17 alpha-cyanomethyl-4,9-estradien-17 beta-ol-3-one (STS 557, a new progestagen) was fermented, yeasts failed in 5 alpha-reducing the 4-double bond. Still Clostridium paraputrificum formed the expected 5 beta-reduced metabolite 17 alpha-cyanomethyl-5 beta-estr-9-ene-3 alpha,17 beta-diol (60%). Structures were elucidated by n.m.r. and mass spectra and partly by circular dichroism. By oxidation of the metabolites, the corresponding 3-oxo compounds 17 alpha-cyanomethyl-5 alpha-estran-17 beta-ol-3-one, 17 alpha-cyanomethyl-5 beta-estran-17 beta-ol-3-one and 17 alpha-cyanomethyl-5 beta-estr-9-en-17 beta-ol-3-one were prepared. The evident influence of the 9-double bond on reduction of the 4-en-3-oxo compound STS 557 preventing 5 alpha-reduction but permitting 5 beta-reduction is discussed in view of the distinctly diminished metabolism of this progestagen in mammals.     

Carbon Dioxide Fixation in Roots and Nodules of Alnus glutinosa: I. Role of Phosphoenolpyruvate Carboxylase and Carbamyl Phosphate Synthetase in Dark CO(2) Fixation, Citrulline Synthesis, and N(2) Fixation

Detached roots and nodules of the N₂-fixing species, Albus glutinosa (European black alder), actively assimilate CO₂. The maximum rates of dark CO₂ fixation observed for detached nodules and roots were 15 and 3 micromoles CO₂ fixed per gram dry weight per hour, respectively. The net incorporation of CO₂ in these tissues was catalyzed by phosphoenolpyruvate carboxylase which produces organic acids, some of which are used in the synthesis of the amino acids, aspartate, glutamate, and citrulline and by carbamyl phosphate synthetase. The latter accounts for approximately 30 to 40% of the CO₂ fixed and provides carbamyl phosphate for the synthesis of citrulline. Results of labeling studies suggest that there are multiple pools of malate present in nodules. The major pool is apparently metabolically inactive and of unknown function while the smaller pool is rapidly utilized in the synthesis of amino acids. Dark CO₂ fixation and N₂ fixation in nodules decreased after treatment of nodulated plants with nitrate while the percentage of the total ¹⁴C incorporated into organic acids increased. Phosphoenolpyruvate carboxylase and carbamyl phosphate synthetase play key roles in the synthesis of amino acids including citrulline and in the metabolism of N₂-fixing nodules and roots of alder.