Ruthenium red-sensitive and Ruthenium Red-insensitive release of calcium by mitochondria isolated from rat liver and from rat heart

EGTA (ethanedioxybis(ethylamine)tetra-acetic acid) induced a release of Ca²⁺ from mitochondria isolated from both rat liver and rat heart that was inhibited by Ruthenium Red. The concentration of Ruthenium Red giving half-maximal inhibition was about 350 pmol/mg of protein, a value approximately 7 times greater than that giving half-maximal inhibition of the initial rate of Ca²⁺ transport. The EGTA-induced release of Ca²⁺ was temperature-dependent and was inhibited by the local anaesthetic, nupercaine.Pi, acetate, and tributyltin in the presence of Cl^?, inhibited the Ruthenium Red-sensitive Ca²⁺ release induced by EGTA, whereas these agents enhanced the Ruthenium Red-insensitive release of Ca²⁺ induced by acetoacetate in liver and heart mitochondria and by Na⁺ in heart mitochondria.     

Effect of protein denaturants on the structure of water: Electron spin resonance spin probe study

The effect of the urea-class of protein denaturants on the structure of liquid water was studied. The method chosen was to monitor the microviscosity of the medium by estimating the reorientational correlation time, τ₈ and proton hyperfine linewidth, WH of an inert stable organic free radical (2, 2, 6, 4-tetramethyl-piperid 4-one-l-oxyl) by measuring its electron spin resonance spectra in aqueous denaturant solutions. Urea, thiourea and guanidinium chloride were found to disrupt water structure efficiently and continuously, with the effect significant at low molarities. Dimethyl urea was found to be somewhat less efficient. The relation between the structure-breaking tendency of the denaturants and their ability to weaken hydrophobic interactions is rationalised.     

Calcium ion cycling in rat liver mitochondria

1. Addition of N-ethylmaleimide to rat liver mitochondria respiring with succinate as substrate decreases both the initial rate of Ca(2+) transport and the ability of mitochondria to retain Ca(2+). As a result, Ca(2+) begins to leave the mitochondria soon after it has entered. Half-maximal effects occur at an N-ethylmaleimide concentration of about 100nmol/mg of protein. 2. The efflux of Ca(2+) induced by N-ethylmaleimide is not prevented by Mg(2+) or by Ruthenium Red at concentrations known to prevent Ca(2+) efflux when exogenous phosphate also is present. Swelling of mitochondria does not accompany N-ethylmaleimide-induced Ca(2+) efflux. 3. Addition of Ca(2+) to rat liver mitochondria in the presence of N-ethylmaleimide produces an immediate decrease in DeltaE (membrane potential), which decreases further to only a slight extent over the next 8min. Concomitant with this is an immediate increase and then levelling off of the -59DeltapH (transmembrane pH gradient). 4. Preincubation of rat liver mitochondria with p-chloromercuribenzenesulphonate, which by contrast with N-ethylmaleimide is unable to penetrate the inner mitochondrial membrane, also prevents Ca(2+) retention. The DeltaE and -59DeltapH respond to Ca(2+) addition in a manner similar to that which occurs when N-ethylmaleimide is present. Subsequent addition of mercaptoethanol produces an immediate increase in both DeltaE and -59DeltapH. At the same time Ca(2+) is rapidly accumulated by the organelles. 5. The above data are interpreted as indicating that under the conditions of Ca(2+) efflux seen here, the mitochondria retain their functional integrity. This contrasts with the uncoupling effect of Ca(2+) seen in the presence of P(i), which generally leads to a loss of mitochondrial integrity. We suggest that a unique mechanism of Ca(2+) cycling is able to take place when mitochondria have been treated with N-ethylmaleimide.     

Satellite DNA specific to knob heterochromatin inCucumis metuliferus (Cucurbitaceae)

Buoyant density gradient analysis of nuclear DNA of fourCucumis species showed asymmetric profiles indicating the presence of satellite DNA sequences in the nuclear genome. A highly repeated satellite DNA sequence was isolated from the nuclear genome ofC. metuliferus under neutral CsCl gradients. The satellite DNA constitutes about 4.96% of total nuclear DNA and has 48.06% guanine plus cytosine content. The kinetic complexity of satellite DNA is 150 times smaller than T₄ phage DNA and the base sequence divergence is low.³H-labeled cRNA transcribed from satellite DNA hybridized clearly to six heterochromatic knobs of pachytene chromosomes. The knob heterochromatin can be distinguished by Giemsa C-banding of pachytene chromosomes. Restriction enzyme analysis and Southern blot hybridization indicated that the satellite DNA has a tandem arrangement and predominantly formed two bands of size 210 and 151 base pairs. Absence of knob satellite DNA ofC. metuliferus in the nuclear genomes ofC. melo, C. anguria andC. sativus showed thatC. metuliferus remains isolated within the genusCucumis.     

Dexamethasone increases neutral sphingomyelinase activity and sphingosine levels in 3T3-L1 fibroblasts

The activity of neutral sphingomyelinase (EC 3.1.4.12) in a plasma membrane enriched fraction was found to be increased in dexamethasone treated cells. The elevation of sphingomyelinase activity was blocked by cycloheximide indicating that protein synthesis was required for the steroid action. Ceramidase (EC3.5.1.23) activity was unaffected by the dexamethasone treatment. Levels of sphingosine in 3T3-L1 Cells were also increased after treatment with 10(-7) M dexamethasone for 2 and 4 hours.     

Studies on the stimulation of dolichol-mediated glycosylation by dexamethasone in HeLa cells.

The effect of the addition of 1 microM-dexamethasone on the temporal sequence of the glycosylation process has been studied in HeLa S3G cells. In the presence of delipidized serum, dexamethasone caused an increase in 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase and an accelerated synthesis of dolichols. These events were followed by an increase in the transfer of mannose from GDP-mannose to mannolipid. An increase in the rate of synthesis of lipid-linked oligosaccharides and a stimulation of glycosylation were observed in cells grown in the presence of delipidized serum in the culture medium. The data are consistent with the view that cellular syntheses of lipids and glycoproteins are co-ordinately controlled.     

Primary culture of normal rat adrenocortical cells. I. Culture conditions for optimal growth and function

Rat adrenocortical cells retained their differentiated characteristics over 2 wk in culture without a specific requirement for additives other than inorganic salts, amino acids, vitamins, and fetal bovine serum. The cells were maintained free from fibroblast overgrowth by substitution of D-value in place of L-valine in the medium. Corticotropin (ACTH) inhibited the growth of adrenocortical cells in this medium and the effect was reversible. The adrenocortical cells had a limited capacity for growth as reflected by total cell counts and [3H]thymidine uptake with cells from young animals demonstrated a greater potential for DNA synthesis than cells obtained from mature animals. A very sensitive assay for ACTH using a small number of cells in primary culture also is described.     

N-terminal sequence of porcine big gastrin: Sequence, synthesis, and immunochemical studies

The synthesis of fragments corresponding to the N-terminal region of porcine big gastrin is described. Radioimmunoassay using synthetic peptides supports the revised structure for the hormone.     

Coordinate repression of cholesterol biosynthesis and cytoplasmic 3-hydroxy-3-methylglutaryl coenzyme A synthase by glucocorticoids in HeLa cells.

The stability constants for the calcium and magnesium complexes of rhodanese are >10⁵m^?1 at both high and low substrate concentrations. The stoichiometry of alkaline earth metal ion binding totals close to 1 per 18,500 molecular weight. The usual assay reagents contain sufficient amounts of these metal ions to maintain added enzyme in its metal-complexed form. When reaction mixtures are treated with oxalate to remove calcium ions, inhibition of rhodanese activity is virtually complete under circumstances such that the contribution of magnesium ion is low.Zinc and a number of transition metal ions are inhibitors of rhodanese activity. Studies of the concentration dependence of these effects with zinc, copper, and nickel showed that: 1) Some cyanide complexes of these metals are competitive with the donor substrate, thiosulfate ion. The binding of the copper and zinc complexes is mutually competitive. 2) Another cyanide species of copper appears to combine with the free enzyme to form a functionally active complex. 3) The zinc cyanide species with a net positive charge is an inhibitor competitive with the acceptor substrate, cyanide ion.All of these observations are consistent with a model in which metal ions serve as the electrophilic site of rhodanese.