The phosphorylation potentials generated by respiring Ehrlich ascites tumor mitochondria

The extra- and intramitochondrial phosphorylation potentials (ΔG_p(out) and ΔG_p(in), respectively) generated by respiring Ehrlich ascites tumor mitochondria were determined, using succinate, pyruvate + malate, ascorbate + N,N,N′,N′-tetramethyl-p-phenylenediamine, and ascorbate + ferrocyanide as substrate systems. Values of ΔG_p(out) exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) in post-ADP state 4 respiration were found with succinate as substrate, in agreement with data on normal rat liver mitochondria. ΔG_p(out) values exceeding 15 kcal mol^?1 (62.8 kJ mol^?1) were also observed with ascorbate + TMPD or ascorbate + ferrocyanide as substrates. Slightly lower values of ΔG_p(out) were found with the NAD-linked substrates pyruvate + malate. The intramitochondrial ΔG_p(in) developed by respiring Ehrlich ascites tumor mitochondria respiring on succinate approached 12 kcal mol^?1 (50.2 kJ mol^?1), in agreement with reported values on rat liver mitochondria. The prior accumulation of Ca²⁺ and phosphate by the Ehrlich cell mitochondria did not lower the extramitochondrial ΔG_p(out) developed after a subsequent addition of ADP. Although the rate of oxidative phosphorylation of Ehrlich ascites tumor cells is reduced by intramitochondrial Ca²⁺ and phosphate (Villalobo and Lehninger (1980) J. Biol. Chem., 255, 2457–2464) they are still capable of generating ATP in the suspending medium against a high thermodynamic gradient, as expressed by the [ATP]/[ADP][P_i]mass action ratio.     

Intestinal maturation: The effect of glucocorticoids on in vivo net magnesium and calcium transport in the rat

We investigated with an $\text{in vivo}$ single pass perfusion technique, the effect of glucocorticoids on net magnesium and calcium absorption from the small and large intestine of suckling and adolescent rats. In control rats, rates of net magnesium and calcium absorption were several fold greater in both small and large intestinal segments of suckling rats compared to corresponding rates in segments of adolescent rats. Methylprednisolone 30 mg/kg body weight daily for three days, suppressed significantly net magnesium and calcium absorption from the small and large intestinal segments of suckling rats only. Methylprednisolone had no effect on either net magnesium or calcium absorption in adolescent rats. The mechanism(s) responsible for the observed decrease in net magnesium and calcium absorption in the suckling period by glucocorticoids are discussed.     

Mouse transcobalamin II: Biosynthesis and uptake by L-929 cells

Mouse fibroblast (L-929) cells, in culture, synthesized and secreted into the growth medium a vitamin B₁₂-binding substance which was identical to mouse transcobalamin II (TC II) as judged by the following criteria: (i) gel filtration on Sephadex G-200, (ii) ion-exchange chromatography on DEAE-cellulose and CM-cellulose, and (iii) the ability to facilitate cellular B₁₂ uptake by L-929 cells. The secretion of mouse fibroblast binder was blocked by cycloheximide and puromycin; and in both cases the cells' ability to secrete this binder was partially restored when the inhibitor was removed. Within 30 h after the cells were exposed to [⁵⁷Co]B₁₂ bound to mouse serum TC II (M_r ~ 38,000) the [⁵⁷Co]B₁₂ was bound to a large molecular weight intracellular binder (M_r ~ 120,000) which was not released into the culture medium. During this same incubation period, the cells released free [⁵⁷Co]B₁₂ and [⁵⁷Co]B₁₂ bound to a protein which had the same elution volume as mouse serum TC II on Sephadex G-200.     

The effect of light on four protochlorophyllide-binding polypeptides of barley (Hordeum vulgare)

Protochlorophyllide-binding proteins were investigated and possible changes in the pigment-protein association during light-induced chlorophyll synthesis were analyzed. Three major results were obtained. (1) Four protochlorophyllide-binding polypeptides were separated electrophoretically on polyacrylamide gels and visualized by their fluorescence. The number and size of these protochlorophyllide-binding proteins isolated from darkgrown and illuminated plants were not affected by light. (2) The association of pigment with these proteins was studied using exogenous [³H]protochlorophyllide as substrate in an NADPH-dependent in vitro chlorophyll synthesis assay. It was found that a constant exchange of protein-bound and free pigment occurs and that freshly synthesized chlorophyllide does not accumulate on any of the four pigment-binding proteins in vitro. (3) NADPH does not affect the amount of pigment bound to protein in vitro, even during chlorophyll synthesis which occurred only in the presence of NADPH.     

Parathyroid hormone induced stimulation of calcium uptake by renal microsomes

Cortical and papillary microsomes prepared from feline kidneys perfused with parathyroid hormone (PTH) showed an enhanced ability to accumulate calcium (Ca⁺²). PTH was unable to stimulate Ca⁺² uptake into microsomes prepared from outer medulla. These data suggest that renal microsomes may be a valid model system for studying the action of PTH on Ca⁺² transport in the kidney.     

Rapid efflux of Ca2+ from heart mitochondria in the presence of inorganic pyrophosphate

Inorganic pyrophosphate (PPi) in the intracellular concentration range causes rapid efflux of Ca2+ from rat heart mitochondria oxidizing pyruvate + malate in a low Na+ medium. Half-maximal rates of Ca2+ efflux were given by 20 microM PPi. During and after PPi-stimulated Ca2+ efflux the mitochondria retain their structural integrity and complete respiratory control. Carboxyatractyloside inhibits PPi-stimulated Ca2+ efflux, indicating PPi must enter the matrix in order to promote Ca2+ efflux. Heart mitochondria have a much higher affinity for PPi uptake and PPi-induced Ca2+ efflux than liver mitochondria.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

H+ stoichiometry of sites 1 + 2 of the respiratory chain of normal and tumor mitochondria

The mechanistic stoichiometry for vectorial H+ ejection coupled to electron transport through energy-conserving segments 1 + 2 was determined on cyanide-inhibited mitochondria from rat liver, rat heart, and Ehrlich ascites tumor cells, and on rat liver mitoplasts with ferricyanide or ferricytochrome c as electron acceptors. K+ (+ valinomycin) and Ca2+ were employed as permeant cations. Three different methods were employed. In the first, known pulses of ferricyanide were added, and the total H+ ejected was determined with a glass electrode. Such measurements gave H+/2e-values exceeding 7.0 for both normal and tumor mitochondria with beta-hydroxybutyrate and other NAD-linked substrates; uptake of Ca2+ was also measured and gave the expected q+/2e-ratios. The second type of measurement was initiated by addition of ferricytochrome c to rat liver mitoplasts, with H+ ejection monitored with the glass electrode and ferricytochrome c reduction by dual-wavelength spectrophotometry; the H+/2e-ratios generally exceeded 7.0. In the third type of measurement, mixing and dilution artifacts were eliminated by oxidizing ferrocytochrome c in situ with a small amount of ferricyanide. H+/2e-ratios for rat liver mitoplasts oxidizing beta-hydroxybutyrate consistently approached or exceeded 7.5. Over 150 measurements made under a variety of conditions gave observed H+/2e-ejection ratios significantly exceeding 7.0, which correlated closely with H+/2e-measurements on sites 1 + 2 + 3, sites 2 + 3, and site 2. Factors leading to the deficit of the observed ratios from the integral value 8 for sites 1 + 2 were discussed.     

The effect of A13+ on the physical properties of membrane lipids in Thermoplasma acidophilum.

Using Electron Paramagnetic Resonance Spectroscopy, Al³⁺ was shown to produce a dramatic decrease of membrane lipid fluidity on the microorganism $\text{Thermoplasma}\text{acidophilum}$ at a pH > 2. The ability of Al³⁺ to alter lipid fluidity was enhanced with increasing pH (from 3 to 5). At pH 4, 10^?2 M Al³⁺ increased the lower lipid phase transition by 39°C, and a detectable change was observed with AlCl₃ concentrations as low as 10^?5 M. The ability of Al³⁺ to increase the lower lipid phase transition temperature of $\text{T.}\text{acidophilum}$ is the largest of any cation/lipid interaction yet reported.     

Self-regulation of membrane fluidity The effect of saturated normal and methoxy fatty acid supplementation on Tetrahymena membrane physical properties and lipid composition

Tetrahymena cells elongated and desaturated massive supplements of palmitic or lauric acid at nearly twice the rates employed by unfed cells, thereby maintaining constant the physical properties of their membrane lipids. However, when a mixture of the 9- and 10-monomethoxy derivatives of stearic acid was administered, these compounds were incorporated without further metabolism. The marked fluidizing effect of the phospholipid-bound methoxy-fatty acids elicited an immediate reduction in fatty acid desaturase activity, the pattern of change very similar to that induced by supplements of polyunsaturated fatty acids. The modulation of fatty acid desaturase activity by methoxy-acids clearly seems to be governed by embrane fluidity rather than by some form of end product inhibition of the type which might have been postulated to explain the similar effect caused by polyunsaturated fatty acids.     

A novel effect of 6-azauridine on growth and protein synthesis in wheat embryonic axes

Growth and the rate of protein synthesis in germinating wheat embryonic axes are inhibited by the analog 6-azauridine via a mechanism which is independent of the usual effect of this compound as an inhibitor of de novo synthesis of UTP. The effects on growth and protein synthesis can be separated from that on UTP biosynthesis by analyses of the kinetics by which each effect is maximized following a 1.5-h pulse with 6-azauridine, and by saturation of the responses at different doses of the analog. The inhibitions of growth and protein synthesis are apparently not mediated through the rate of poly A(+) RNA synthesis (reduced as little as 8%), but rather by an effect on translation. Since cordycepin reduces the azauridine inhibitions of growth and protein synthesis, it is suggested that these latter effects of 6-azauridine may depend upon the synthesis of an inhibitory azauridyl-RNA.     

DNA biosynthesis in rat liver mitochondria: Inhibition by sulfhydryl compounds and stimulation by cytoplasmic proteins

The sulfhydryl compounds, 2-mercaptoethanol, dithiothreitol, cysteine. and glutathione inhibit the incorporation of [³H]dTTP or [³H]dATP into mitochondrial DNA by rat liver mitochondria in vitro. The lack of inhibition by non-SH-containing analogs indicates that the SH group is responsible for the inhibition.The inhibition does not result from an effect of the sulfhydryl compounds on precursor permeability, ATP formation, or respiration, or the action of the thiol on the outer mitochondrial membrane. An intact inner membrane is not required for the action of the inhibitor. Furthermore, SH compounds do not appear to exert their effect by activation of a mitochondrial nuclease, chemical breakdown of high molecular-weight mitochondrial DNA or dissociation of membrane-bound DNA from the inner mitochondrial membrane. Incorporation of labeled precursor into DNA by mitochondrial DNA polymerase, when removed from the inner mitochondrial membrane, is not inhibited by SH compounds.Cytoplasmic extracts prepared from rat and mouse tumors and 22-h regenerating rat liver contain a protein(s) not detectable in normal rat liver which can reverse the inhibition by SH compounds of the synthesis of mitochondrial DNA in rat liver mitochondria in vitro.More importantly, when the stimulatory protein(s) is partially purified by affinity chromatography on DNA-cellulose, it is possible to demonstrate that this protein(s) also stimulates the synthesis of mitochondrial DNA by normal rat liver mitochondria in vitro in the absence of the sulfhydryl inhibitor.     

The generation of memory B-cell subpopulations capable of proliferation and expansion of the pool: effect of time and antigen

The ability of antigen to induce proliferation of memory B lymphocytes, thus perpetuating and expanding the memory cell pool, has been examined using an antibody-forming cell (AFC) assay. This method provided confirmation of previous studies in which serum antibody titer was used as a relative measure of pool size and demonstrated directly that the number of antibody-forming cells is increased. Memory cell subpopulations were prepared by lg velocity sedimentation from recently immunized donors (2 weeks) and tested for their ability to proliferate, thus expanding the memory cell pool. Both large and small immature DNP-specific memory cells displayed antigen-dependent and antigen-independent proliferation while mature cells (8 weeks postpriming) were capable only of antigen-dependent proliferation. Chicken γ-globulin (CGG) specific memory cells were also evaluated in this system and were found to differ from DNP-specific cells in several ways. (A) DNP-specific AFC were found to be concentrated in the spleen while CGG-specific AFC were found predominantly in the bone marrow early after transfer and in the spleen upon delayed challenge. (B) The rate of maturation of CGG-specific memory cells capable of antigen-driven proliferation and pool expansion was delayed in comparison to DNP-specific memory cells. The relationship of these functionally defined subsets to previously described memory cell subpopulations is discussed.     

The analysis of the betaine homarine by high pressure liquid chromatography

A procedure is presented which is suitable for the qualitative and quantitative analysis of the betaine homarine in aqueous tissue extracts. After preliminary purification of the extract by gel permeation chromatography on Sephadex G-25, quantitative analysis of the homarine content is performed by high pressure liquid chromatography on a 1-m column of Corasil II.     

Cytochrome P-450 revealed: the effect of the respiratory cytochromes on the spectrum of bacterial cytochrome P-450

Soluble extracts of Bacillus megaterium ATCC 14581 prepared by centrifuging a sonicated cell suspension at 40,000 xg for 30 min apparently contained no cytochrome P-450 unless the culture had been grown in the presence of an inducer: a reduced+CO minus reduced spectrum was used to measure cytochrome P-450 concentration. When the 40,000 xg supernatants from the uninduced cultures were recentrifuged at 105,000 xg the respiratory cytochromes, including one like cytochrome a1, were sedimented, and cytochrome P-450 was observed to be 100 nM or 30 +/- 9 p mol cytochrome P-450/mg protein (n=9). Measurements of cytochrome P-450 in cultures induced with phenobarbital were always higher after ultracentrifugation. There was soluble cytochrome o in all extracts. When cytochrome a1 was present a deep trough at 441 nm developed in the reduced +CO minus reduced difference spectrum of the 40,000 xg supernatant of both the uninduced and the induced cultures. The 40,000 xg supernatant obtained after lysing protoplasts of B. megaterium did not contain cytochrome a1 and always gave a good measure of cytochrome P-450.     

Cumene hydroperoxide and yeast cytochrome P-450: spectral interactions and effect on the genetic activity of promutagens.

Cells of $\text{Saccharomyces}\text{cerevisiae}$, harvested from log phase cultures, contain cytochrome P-450 and are capable of activating promutagens to products that are genetically active in the same cell. The effect of cumene hydroperoxide, a compound known to support cytochrome P-450-mediated reactions, on the activation of a variety of the promutagens was investigated. In all cases the genetic activity of the promutagens was increased. With dimethyl-nitrosamine as the promutagen, the increased rate of gene conversion was linear for at least 1 hr. Yeast cytochrome P-450 was stable in intact cells in the presence of cumene hydroperoxide. However, in microsomal preparations the cytochrome was rapidly destroyed. When cumene hydroperoxide was added to a suspension of intact yeast cells, a spectrum with a Soret maximum at 455 nm — indicative of an interaction with cytochrome P-450 — was observed.     

Diurnal rhythmicity in plasma 24,25-dihydroxyvitamin D3: correlation with intestinal calcium transport and plasma minerals

Plasma 24,25-(OH)₂D₃ exhibits diurnal rhythmic variations in the adult rat. The decrease in 24,25-(OH)₂D₃ occurring at the onset of feeding during light-dark transition coincides with a decrease in plasma calcium but is inversely related to plasma phosphate and to increased intestinal calcium transport activity.     

The stimulation of purine nucleotide production by pyrroline-5-carboxylic acid in human erythrocytes

We previously reported that pyrroline-5-carboxylate (PC), the intermediate in the interconversions of proline, ornithine and glutamate markedly stimulates hexosemonophosphate-pentose pathway activity in human erythrocytes. The stimulation is mediated by pyrroline-5-carboxylate reductase which generates NADP⁺ accompanying the conversion of pyrroline-5-carboxylate to proline. We now report that the previously demonstrated effect of pyrroline-5-carboxylate on glucose oxidation through the hexose-monophosphate-pentose pathway is accompanied by increased phosphoribosyl-pyrophosphate production and increased formation of nucleotides via the salvage pathway. The demonstrated effect of pyrroline-5-carboxylate on purine processing may provide a regulatory link between amino acid and nucleotide metabolism.     

The effect of sodium ion on antinociception and opiate binding in vivo.

Large quantities of NaCl and CaCl₂ but not KCl given intrapertoneally decreased the antinociceptive activity of morphine. NaCl also antagonized the effect of morphine on the stereo-specific binding of opiates. This high dose of NaCl doubled the level of sodium in the brain but did not alter the specific gravity of brain tissue. These $\text{in}$$\text{vivo}$ effects of NaCl confirm the antagonistic effects of NaCl $\text{in}$$\text{vitro}$ that have been reported.