Limited proteolysis patterns of the B chain of insulin.

The oxidized B chain of insulin was used as a simple model for further consideration of limited proteolysis with low substrate:enzyme ratios. With low B chain:trypsin ratios, the ordinarily slower cleavage rate of the -Lys₂₉-Ala₃₀ bond essentially equaled the cleavage saturation rate of the -Arg₂₂-Gly₂₃ bond. This led to the disappearance of octapeptide which ordinarily forms most rapidly. Heptapeptide and alanine, formed mainly by cleavage of the octapeptide, decreased somewhat at high enzyme relative levels. Trypsin added to B chain formed a single chromatographic peak.     

Two modes of bicarbonate utilization in the marine green macroalga Ulva lactuca

The green marine macroalga Ulva lactuca L. was found to be able to utilize HCO₃^? from sea water in two ways. When grown in flowing natural sea water at 16°C under constant dim irradiance, photosynthesis at pH8.4 was suppressed by acetazolamide but unaffected by 4,4′-diisothiocyanostilbene-2,2′-disulphonate. These responses indicate that photosynthetic HCO₃^? utilization was via extracellular carbonic anhydrase (CA) -mediated dehydration followed by CO₂ uptake. The algae were therefore described as being in a ‘CA state’. If treated for more than 10 h in a sea water flow-through system at pH9.8, these thalli became insensitive to acetazolamide but sensitive to 4,4′-diisothiocyanostilbene-2,2′-disulphonate. This suggests the involvement of an anion exchanger (AE) in the direct uptake of HCO₃^?, and these plants were accordingly described as being in an ‘AE state’. Such thalli showed an approximately 10-fold higher apparent affinity for HCO₃^? (at pH9.4) than those in the ‘CA state’, while thalli of both states showed a very high apparent affinity for CO₂. These results suggest that the two modes of HCO₃^? utilization constitute two ways in which inorganic carbon may enter the Ulva lactuca cells, with the direct entry of HCO₃^?, characterizing the ‘AE state’, being inducible and possibly functioning as a complementary uptake system at high external pH values (e.g. under conditions conducive to high photosynthetic rates). Both mechanisms of entry appear to be connected to concentrating CO₂ inside the cell, probably via a separate mechanism operating intracellularly.     

Substrate particle size affects pit building decision and pit size in the antlion larvae Euroleon nostras (Neuroptera: Myrmeleontidae)

Abstract. The larvae of the antlion Euroleon nostras are pit-builders, constructing pitfall traps in loose sand. The number of pits and the pit diameter are recorded when larvae are kept in substrates with different particle sizes. The most convenient pit-building sand fractions are two fractions with fine sand (≤ 0.23 mm; 0.23–0.54 mm). The largest pits are constructed in sand with a particle size of 0.23–0.54 mm. In this sand fraction, larvae of all three instars most readily build pits. No pits are constructed in sand with a particle size greater than 1.54 mm. First- and second-instar larvae avoid building pits in substrates of particle size 1–1.54 mm, but third-instar larvae construct pits in this sand fraction. It is assumed that the antlion is capable of distinguishing between substrate types and this hypothesis is tested by giving larvae the choice of building a pit in one of four particle-size fractions. Larvae of all three instars prefer to build pits in the fraction with a particle size of 0.23–0.54 mm. Only third-instar larvae build pits in all four fractions, but only occasionally in the coarser fraction.     

Mutations by near-ultraviolet radiation in Escherichia coli strains lacking superoxide dismutase

Om wild-type Escherichia coli, near-ultraviolet radiation (NUV) was only weakly mutagenic. However, in an allelic mutant strain (sodA sodB) that lacks both Mn- and Fe-superoxide dismutase (SOD) and assumed to have excess superoxide anion (O₂⁻), NUV induced a 9-fold increase in mutation above the level that normally occurs in this double mutant. When a sodA sodB double mutant contained a plasmid carrying katG⁺ HP-I catalase), mutation by NUV was reduced to wild-type (sodA⁺ sodB⁺) levels. Also, in the sodA sodB xthA triple mutant, which lacks exonuclease III (exoIII) in addition to SOD, the mutations frequency by NUV was reduced to wild-type levels. This synergistic action of NUV and O₂⁻ suggested that pre-mutational lesions occur, with exoIII converting these lesions to stable mutants. Exposure to H₂O₂ induced a 2.8 fold increase in mutations in sodA sodB double mutants, but was reduced to control levels when a plasmid carrying katG⁺ was introduced. These results suggest that NUV, in addition to its other effects on cells, increases mutations indirectly by increasing the flux of OH^. radicals, possibly by generating excess H₂O₂.     

The nucleotide sequences of barley cytoplasmic glutamate transfer RNAs and structural features essential for formation of δ-aminolevulinic acid

In chloroplasts and a number of prokaryotes, -aminolevulinic acid (ALA), the universal precursor of porphyrins, is synthesized by a multistep enzymatic pathway with glutamyl-tRNA^Glu as an intermediate. The ALA synthesizing system from barley chloroplasts is highly specific in its tRNA requirement for chloroplast tRNA^Glu; a number of other Glu-tRNAs are inactive in ALA formation although they can be glutamylated by chloroplast aminoacyl-tRNA synthetases. In order to obtain more information about the structural features defining the ability of a tRNA to be recognized by the ALA synthesizing enzymes, we purified and sequenced two cytoplasmic tRNA^Glu species from barley embryos which are inactive in ALA synthesis. By using glutamylated tRNAs as a substrate for the overall reaction, we showed that Glu-tRNA reductase is the enzyme responsible for tRNA discrimination.     

The interaction of MgADP with H-ATPase in rat liver mitochondria

The activating anions are found to induce an unexpectedly high (up to 8-fold for sulphite) increase of ATPase activity in intact rat liver mitochondria. This effect is not determined by the observed changes in K_m and K_i (ADP) values. The stimulation seems to be caused by dissociation of the inactive complex of ATPase with Mg·ADP. The quantity of this complex formed in the course of ATP hydrolysis is approx. 90% of the total ATPase content in intact mitochondria. The data on toluene-permeabilized mitochondria suggest that the high content of the complex is a result of the stabilizing effect of some matrix macromolecules.     

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.     

Calcium-dependent anion channel in the water mold,Blastocladiella emersonii

Summary Injection of depolarizing current into vegetative cells of the water moldBlastocladiella emersonii elicits a regenerative response that has the electrical characteristics of an action potential. Once they have been taken past a threshold of about –40 mV, cells abruptly depolarize to +20 mV or above; after an interval ranging from several hundred milliseconds to a few seconds, the cells spontaneously return to their resting potential near –100 mV. When the action potential was analyzed with voltage-clamp recording, it proved to be biphasic. The initial phase reflects an influx of calcium ions through voltage-sensitive channels that also carry Sr²⁺ ions. The delayed, and more extended, phase of inward current results from the efflux of chloride and other anions. The anion channels are broadly selective, passing chloride, nitrate, phosphate, acetate, succinate and even PIPES. The anion channels open in response to the entry of calcium ions, but do not recognize Sr²⁺. Calcium channels, anion channels and calcium-specific receptors that link the two channels appear to form an ensemble whose physiological function is not known. Action potentials rarely occur spontaneously but can be elicited by osmotic downshock, suggesting that the ion channels may be involved in the regulation of turgor.     

Comparison of trout hepatocyte culture on different substrates

Summary Comparisons were made of attachment and viability of rainbow trout (Salmo gairdneri) hepatocytes in short-term (2 days), primary culture on plastic, collagen-coated or extracellular matrix (ECM) coated dishes. Hepatocyte isolation routinely yielded cells with good viability (96%). Cells plated on ECM attached with high efficiency (93%) in contrast to cells cultured on plastic or collagen (∼20%). The cells plated on ECM flattened out and formed monolayers, while the cells on plastic and collagen rounded up and formed multi-cell aggregates in suspension. Viability of cells in all substrates remained high over the 2 day culture period. ECM is the first substrate to support trout-hepatocyte attachment in primary culture. Differentiated liver function was maintained in cells cultured on ECM as evidence by the induction of tyrosine aminotransferase by hydrocortisone (200%). This work was supported in part by research grant R809599010 from the U. S. Environmental Protection Agency. Editor's Statement This paper reports improved methods for culture of trout liver-derived cells that make in vitro investigations of fish metabolism, carcinogenesis and chemical toxicity more feasible than previously applied techniques. Recent interest in fish as models for study and indicators of effects of envionmental and food-related toxins make this work timely, poarticularly since many of the compounds of interest are primarily metabolized by hepatocytes or act on liver as a major target. David W. Barnes     

Characterization of membrane-bound MgATPases, purified by aqueous two-phase partitioning, from young sugar beet roots

Membrane-bound MgATPase activity from roots of young sugar beet ( Beta vulgaris L. cv. Monohill) was investigated in a membrane fraction purified by partition in an aqueous polymer two-phase system. After two steps of "washing" with fresh bottom phase (rich in dextran), the polyethylene glycol rich top phase (U₃) was practically free of mitochondrial membranes (cytochrome oxidase), and the remaining MgATPase activity showed high substrate specificity for ATP. An optimum for the MgATPase activity was found at pH 7. The activation by Na⁺ or K⁺ was strongest on the acid side without any observable shift in pH optimum. Oligomycin had no effect, but vanadate strongly inhibited the U₃ MgATPase and the K⁺ activation was lost. The complex activation pattern achieved by varying the Na⁺/K⁺ ratio at constant total concentration was interpreted as a synergistic (Na⁺+ K⁺)-activation. The U₃ fraction MgATP-ase activity showed a 4-fold increase in the presence of 0.01% Triton X-100 implying that the MgATPase activity is located in vesicles of which 75% or more are sealed with the ATP binding site on the inside. Comparison with the properties of plasma membrane. ATPases from other plants indicated that the U₃ fraction MgATPase was mainly of plasma membrane origin.