Regulation of receptor-mediated calcium influx across the plasma membrane in a human leukemic T-cell line: evidence of its dependence on an initial calcium mobilization from intracellular stores

It has been repeatedly shown that stimulation of a human leukemic T-cell line, JURKAT, by lectins such as phytohaemagglutinin and anti-T3 antibody (OKT3) leads to an elevation in the concentration of cytosolic free Ca2. This Ca2+ transient results from both an intracellular mobilization and an influx of Ca2+ through specific membrane channels. The objective of this study was to investigate the mechanism by which receptor-mediated influx of Ca2+ is regulated in JURKAT cells, which demonstrably lack 'voltage-dependent calcium channels'. It was found that upon increased loading with quin2 or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetate (BAPTA) there was a pronounced decline of both phytohaemagglutinin-stimulated and OKT3-stimulated influx of 45Ca2+. Using 15 microM quin2/AM or 30 microM BAPTA/AM, agonist-stimulated 45Ca2+ influx was almost totally abolished. At these concentrations of both quin2/AM or BAPTA/AM, phytohaemagglutinin and OKT3 could still induce a rise of cytosolic free Ca2+ above 200 nM. In the presence of La3+ (200 microM), which completely inhibited the agonist-induced 45Ca2+ influx, both phytohaemagglutinin and OKT3 were able to raise the concentrations of cytosolic free Ca2+ to well above 200 nM by merely mobilizing Ca2+ from intracellular stores alone. The data suggest that an agonist-induced increase in the concentration of cytosolic free Ca2+, due to mobilization from intracellular stores, could either directly or indirectly, initiate receptor-mediated Ca2+ influx across the plasma membrane in JURKAT cells.     

Termination of gonadal refractoriness in Turkish hamsters, Mesocricetus brandti

The Turkish hamster (Mesocricetus brandti) is a photoperiodic species. In this investigation, we characterized the photoperiodic requirements for termination of gonadal refractoriness, defined as the inability of the animal to respond to short-day treatment with gonadal regression. Paired testes weights were reduced to less than 20% of their original weight by 10 wk of 12L:12D treatment. This was followed by spontaneous testicular recrudescence (completed by Week 25 of 12L:12D treatment), the overt indication of refractoriness to short photoperiods. Next, the period of long-day exposure sufficient for termination of refractoriness was determined. Refractory males were exposed to 16L:8D for 5 to 20 wk. Ten weeks of 16L:8D treatment was enough for the animals to regain the sensitivity to a second challenge of 12L:12D treatment. Fifteen weeks of 20L:4D or 16L:8D terminated refractoriness in female Turkish hamsters; 20L:4D therefore was not interpreted as a short day by refractory hamsters. This was unexpected because in photosensitive animals this photoperiod acts like a short day, causing gonadal regression. These results suggest that Turkish hamsters are similar to Syrian hamsters in that both species require two or more months of long days in summer to recover sensitivity to the short days of the following fall.     

Optimal substrate feeding policy for a fed batch fermentation with substrate and product inhibition kinetics

The optimal substrate feeding policy for the fed batch fermentation which is governed by product and substrate inhibited kinetics is presented. The conjunction point between nonsingular and singular arcs and the feeding policy along the singular arc are derived analytically in terms of the concentrations of substrate and product and the liquid volume. Thus, it is possible to determine the feeding rate by monitoring the state variables (i.e., closed loop control). As a specific example, an optimization study of the fed batch fermentation for ethanol production by Saccharomyces cerevisiae is presented. It is shown that the optimal feeding patterns are heavily dependent upon the initial conditions. The point selectivity provides the guideline for predicting the optimal feeding patterns and explaining the results of rigorous mathematical analysis.     

Studies on naturally occurring proteinous inhibitor for transmethylation reactions

An inhibitor for S-adenosyl-L-methionine (AdoMet)-dependent methyltransferases has been purified from rat liver particulate fraction to apparent homogeneity, as judged by high-performance liquid chromatography, two-dimensional paper electrophoresis and isoelectric focusing chromatography. This inhibitor molecule, which is composed of 27 amino acid residues with an additional fluorescent chromophore, is rich in glycine, contains no basic amino acid, and has an isoelectric point (pI) of 3.70. A single absorption peak was observed at 248 nm in acidic as well as in neutral media, while two peaks were detected in alkaline medium at 206 nm and 248 nm. The former peak was found to be quite labile. The fluorescent spectra with excitation peak at 285 nm and emission peak at 358 nm are greatly influenced by the pH, being the highest in alkaline medium. The purified inhibitor inhibits all the AdoMet-dependent methyltransferases examined.     

The phosphate group of 3-phosphoglycerate accounts for conformational changes occurring on binding to 3-phosphoglycerate kinase. Enzyme inhibition and thiol reactivity studies

Steady-state kinetic study of the inhibition of 3-phosphoglycerate kinase reaction by the substrate analogues D-glycerol 3-phosphate, 2-phosphoglycolate, tartronate and malonate revealed competition with respect to 3-phosphoglycerate. D-Glycerate had no detectable inhibitory effect. The data indicate that (a) the phosphate of 3-phosphoglycerate plays an essential role in the formation of its complex with the enzyme and, taking into account the relatively strong binding of 3-phosphoglycerate, (b) the two charged groups of the substrate might cause a synergic interaction with the protein. The carboxyl-lacking D-glycerol 3-phosphate is a non-competitive inhibitor with respect to MgATP, while all the investigated carboxyl-containing inhibitors compete for MgATP binding. The inhibitory analogues of 3-phosphoglycerate reduce the reactivity of both the two fast-reacting and the five slow-reacting thiol groups of the enzyme molecule. In the case of the fast-reacting thiols the effect is specifically associated with the presence of a ligand's phosphate group. Similarly mainly the phosphate-containing nucleotides and analogues slow down significantly the reaction rate of the fast-reacting thiols, while adenosine is less effective and the competitive inhibitor adenine has no effect at all. MgADP has an especially dramatic effect as compared to MgATP, in line with the known X-ray structural data. The fast-reacting thiols are of particular interest, since their reactivity is possibly controlled by ligand-induced conformational changes. This is shown by the similar ligand protection against alkylation irrespective of the reagent's electrostatic charge (iodoacetamide or iodoacetate) and also by the similar substrate-binding properties of carboxamidomethylated and the unmodified enzyme.     

Spermine as a modulator of membrane fusion: interactions with acidic phospholipids

The interaction of spermine with acidic phospholipids was investigated for its possible relevance to membrane fusion. Equilibrium dialysis was used to measure the binding of spermine and calcium to large unilamellar vesicles (liposomes) of phosphatidate (PA) or phosphatidylserine (PS). Spermine bound to isolated PA and PS liposomes with intrinsic association constants of approximately 2 and 0.2 M-1, respectively. Above the aggregation threshold of the liposomes, the binding of spermine increased dramatically, especially for PA. The increased binding upon aggregation of PA liposomes was interpreted as evidence for the formation of a new binding complex after aggregation. Spermine enhanced calcium binding to PA, while it inhibited calcium binding to PS, under the same conditions. This difference explained the small effect of spermine on the overall rate of calcium-induced fusion of PS liposomes as opposed to the large effect on PA liposomes. The rate increase could be modeled by a spermine-induced increase in the liposome aggregation rate. The preference for binding of spermine to PA over PS suggested a preference for accessible monoesterified phosphate groups by spermine. This preference was confirmed by the large effects of spermine on aggregation and overall fusion rates of liposomes containing phosphatidylinositol 4,5-diphosphate. The large spermine effects on these liposomes compared with phosphatidate- or phosphatidylinositol-containing liposomes suggested that spermine has a strong specific interaction with phosphatidylinositol 4,5-diphosphate. Clearly, phosphorylation of phosphatidylinositol can lead to a large change in the spermine sensitivity of membrane fusion.     

Fusion of liposomes induced by a cationic protein from the acrosome granule of abalone spermatozoa

Lysin, a protein of Mr 16 000 from the acrosome granule of the abalone, is responsible for the dissolution of the egg vitelline layer. The primary structure of this cationic protein projects some hydrophobic domains in the secondary structure. Lysin was found to associate nonselectively with phospholipid bilayers and cause a spontaneous release of encapsulated carboxyfluorescein in liposomes. The association of lysin with phosphatidylcholine liposomes suggests that there is a hydrophobic interaction between lysin and lipid bilayers. Binding of lysin to phospholipid resulted in the aggregation of phosphatidylserine-containing liposomes, but aggregation was not observed in neutral phosphatidylcholine liposomes. Resonance energy transfer and dequenching of fluorescent 1-palmitoyl-2-cis-parinaroylphosphatidylcholine were both used to determine the fusogenic activity of lysin in aggregated liposomes. Results from both assays are consistent. Lysin-induced fusion was observed in all the phosphatidylserine-containing liposomes, and the general trend of fusion susceptibility was phosphatidylserine/phosphatidylcholine (1:2) approximately equal to phosphatidylserine/phosphatidylcholine/phosphatidylethanolamine (1:1:1) greater than phosphatidylserine/phosphatidylethanolamine (1:2). Cholesterol up to 30% did not affect the intrinsic fusion susceptibility. A hydrophobic penetration by protein molecules and the packing of phospholipid bilayers are used to interpret the fusion susceptibility. Lysin-induced liposome aggregation was highly independent of the state of self-association of lysin in ionic medium. However, the fusogenic activity of self-associated lysin was found to be much less than the monodispersed one. Liposomes preincubated with Ca2+ did not fuse initially as readily as those without Ca2+ treatment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a cDNA for human muscle 6-phosphofructokinase

A cDNA for human muscle 6-phosphofructokinase (EC.2.7.1.11) has been isolated from a human fibroblast cDNA library made using the Okayama-Berg procedure. The cDNA isolated as a Bam H1 fragment of the pcD recombinant, pO4, is approximately 2000 bp in length. It represents approximately 1350 bp of the C-terminus coding sequence of the enzyme, approximately 500 bp of the 3'-untranslated region and approximately 150 bp of the vector sequences. The identity of the pO4 cDNA was established by the observation of a high degree of homology (approximately 95%) between the deduced amino acid sequence with the published protein sequence of rabbit muscle 6-phosphofructokinase, and the assignment of the sequence to human chromosome 1 (the known location of PFKM) by using somatic cell hybrids. Based on immunochemical evidence, we had previously predicted not only a remarkable structural conservation of the vertebrate muscle PFK, but also partial structural identity among all three vertebrate PFK isozymes. The pO4 cDNA is, therefore, expected to permit isolation of cDNAs for muscle and non-muscle PFKs from a wide variety of vertebrate species.     

Light-induced potential and current across a large bacteriorhodopsin-asolectin planar membrane stabilized on a polyacrylamide gel surface

A phospholipid bilayer membrane was spread from an organic solvent solution between a polyacrylamide gel surface and an aqueous buffer solution. The membrane was quite similar to the conventional black lipid membrane, but was of a large size and was stable since it was supported on the gel surface. Bacteriorhodopsin, impregnated into the membrane, generated membrane potential and current upon illumination. The induced current was large, and this was attributed to the large area of the present membrane. Remarkable responses of the light-induced potential and current were also observed with a thick layer of organic solvent containing phospholipids. The effects of applied membrane potential, carbonylcyanide-m-chlorophenyl hydrazone (CCCP) and gramicidin were examined on these photoresponses. Steady-state current, which is due to protons flowing through the membrane, was enormously enhanced by applying membrane potential opposite to the photopotential or by adding gramicidin to the membrane-forming solution.