Activation of ribulose bisphosphate carboxylase in intact chloroplasts by CO2 and light

The activity of ribulose 1,5-bisphosphate (RuBP) car?ylase in intact spinach chloroplasts is shown to depend on light and CO₂. This activity was measured upon lysis of chloroplasts and assay of the initial activity using nonlimiting substrate concentrations. Incubation of chloroplasts at 25 °C in the absence of CO₂ results in a gradual inactivation of the RuBP car?ylase. In the presence of CO₂ the initial activity is preserved or increased. CO₂ is also able to reactivate the chloroplast car?ylase previously inactivated in the absence of CO₂. Upon illumination of the chloroplasts, additional activation was observed. This light activation results from an increased affinity for CO₂ of the chloroplast car?ylase. At pH 7.8, the enzyme in dark-adapted chloroplasts required 112 μ m CO₂ for half activation, while in the light it required 24 μ m CO₂. The light activation was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea, carbonylcyanide 3-chlorophenylhydrazone, or dl-glyceraldehyde. Part of the light activation is most likely due to increased Mg²⁺ in the stroma. dl-Glyceraldehyde inhibition also suggests that some intermediate of the photosynthetic carbon cycle is involved. These results suggest that photosynthetic CO₂ assimilation in the chloroplast depends upon the amount of activation of the RuBP car?ylase. This activation is regulated by CO₂ and light-induced changes in the chloroplast stroma such as pH, Mg²⁺, and intermediates of the photosynthetic carbon cycle.     

Physical properties of murine fibroblasts with altered acyl chain unsaturation.

Murine fibroblasts, LM cells, were cultured in suspension with laurate (12:0), myristate (14:0), palmitate (16:0), palmitoleate (16:1), or palmitate + palmitoleate (16:0 + 16:1) bound to fatty acid-free bovine serum albumin. Supplementation with saturated fatty acids decreased the ratio of unsaturated/saturated fatty acids in membrane phospholipids as much as 3.4-fold (palmitate-enriched cells). Concomitantly fluorescence polarization, absorption-corrected fluorescence, and relative fluorescence efficiency of the fluorescence probe molecule, β-parinaric acid, increased 1.5-, 2.9-, and 1.8-fold, respectively, in the membrane phospholipids. Unsaturated fatty acid (palmitoleate) increased the unsaturated/saturated fatty acid ratio by 20% but did not significantly alter the fluorescence parameters. When the cells were fed mixtures of palmitate and palmitoleate, the unsaturated/saturated fatty acid ratio of the membrane phospholipids and the above fluorescence parameters had values intermediate between those if each fatty acid had been fed separately. All fatty acid supplements caused a loss of two characteristic temperatures in Arrhenius plots of relative fluorescence efficiency. However, no shifts or appearance of new characteristic temperatures occurred. The break points at approximately 42, 37, and 22 °C were essentially un-altered. The data were consistent with the possibility that LM cells were unable to maintain constant fluidity, as indicated by fluorescence polarization, when supplemented with different fatty acids. A good correlation could be made between the phospholipid unsaturated/ saturated fatty ratio, the fluorescence polarization, and the toxicity elicited by different fatty acid supplements.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Isoform variants of troponin in skeletal and cardiac muscle cells cultured with and without nerves

Immunofluorescence microscopy shows that cultured skeletal and cardiac muscle cells of chicken embryos exhibit the same stainabilities with antibodies against skeletal and cardiac troponin components as do those in embryos. Muscle cells of each type cultured with motor or sympathetic nerves or in medium containing the nerve extract exhibit the same reactivities as do those in adult animals. Cardiac muscle cells incubated in the nerve-conditioned medium also change the form of troponin components to the adult type. It appears that the differentiation of individual muscle fibers to specific types is induced by nerves, and especially by the neurohumoral effect.     

A study of protein phosphorylation in shape change and Ca++-dependent serotonin release by blood platelets

Upon treatment with agents such as thrombin, collagen or concanavalin A, blood platelets change shape, secrete serotonin and phosphorylate two proteins having molecular weights of approximately 20,000 and 40,000. We have analyzed the relationship of this protein phosphorylation to shape change and release aided by the fact that while shape change occurs independently of extracellular calcium, release of serotonin displays a rather strict calcium requirement. Under limited calcium conditions, where virtually no serotonin release occurs, (Con A)-stimulated phosphorylation is uninhibited. Divalent cations (Mg⁺⁺, Co⁺⁺ and Zn⁺⁺) also inhibit release but not phosphorylation. The microtubule effectors colchicine and D₂O show concomitant effects on release and phosphorylation, indicating a microtubule involvement prior to phosphorylation. Papaverine inhibits release and phosphorylation while not strongly influencing shape change, suggesting that shape change does not require phosphorylation. We therefore conclude that phosphorylation of these proteins takes place after shape change but prior to release, and although it may be required for secretion to occur, the two processes are easily separated. Thus phosphorylation of these proteins is not likely to be an integral component of the release mechanism.     

Glucose-induced, calcium-mediated protein phosphorylation in intact pancreatic islets

Conditions for studying protein phosphorylation in intact pancreatic islets were developed in order to study the effects of glucose and other effectors. Islets were incubated in Krebs-Ringer bicarbonate buffer containing 5 mM malate and 5 mM pyruvate (metabolic fuels that are not insulin secretagogues) for 150 min to permit incorporation of 32Pi into islet phosphate pools. Glucose or other effectors were then added, and the incubation was terminated after 10 to 30 min. Glucose increased phosphorylation of four islet peptides with molecular weights of 20,000, 33,000, 43,000 and 57,000. The calcium channel blockers, verapamil and D-600, inhibited phosphorylation of each of the four proteins, and trifluoperazine inhibited phosphorylation of the proteins with molecular weights of 20,000 and 57,000. The results indicate that glucose-induced insulin release may be mediated in part by protein phosphorylation, and that calcium may act as an intracellular messenger in coupling the glucose stimulus to the secretory process.     

Enhanced insulin stimulation of sugar transport and DNA synthesis by glucocorticoids in cultured human skin fibroblasts

Glucocorticoids will enhance the growth of cultured human skin fibroblasts in serum-containing medium. In serum-free cultures hydrocortisone (5 X 10(-6) M) will enhance insulin stimulation of sugar transport and DNA synthesis (as measured by thymidine incorporation into trichloroacetic acid-precipitable material). The optimal concentration for the glucocorticoid effect on DNA synthesis was 5 X 10(-8) M for dexamethasone and 5 X 10(-7) M for hydrocortisone. In dexamethasone-treated cells, concentrations of insulin as low as 250 microU/ml (10 ng/ml) were effective in stimulating DNA synthesis. Further, hydrocortisone and dexamethasone (both at 5 X 10(-6) M) exhibited potentiating effects on insulin-stimulated sugar transport. These effects appeared to be mediated via inhibitory actions on the hexose transport system with the preservation of a functional insulin-receptor interaction resulting in insulin stimulation of deoxy-D-glucose transport at physiological insulin concentrations, 250 microU/ml (10 ng/ml). Hydrocortisone also enhanced specific [125I]insulin binding in these cells. The data indicate that the mechanism(s) of glucocorticoid enhancement of two actions of insulin may be different.     

Structure elucidation by one- and two-dimensional 360- and 500-MHz 1H NMR of the oligosaccharide units of two glycoproteins isolated from alveoli of patients with alveolar proteinosis

The structure of the oligosaccharide units of the glycoproteins of M_r 36,000 and 62,000 isolated from alveoli of patients with alveolar proteinosis have been determined by one- and two-dimensional ¹H NMR spectroscopy at 500 and 360 MHz. Bi-, tri-, and tetraantennary glycans of N-acetyllactosaminic type have been found in high percentage. They are 1 → 6 monofucosylated and fully sialylated, the ratio $\text{NeuAc}\text{(2 → 3)}\text{NeuAc}\text{× (2 → 6)}$ increasing with increasing degree of branching.     

Effect of chronic ethanol consumption on energy-linked processes associated with oxidative phosphorylation: Proton translocation and ATP-Pi exchange

Male rats were administered an ethanol-containing diet for 31 days during which time they demonstrated fatty liver. Mitochondria and submitochondrial particles were prepared from their livers (ethanol mitochondria, ethanol submitochondrial particles) and from their pair-fed partners (control mitochondria, control submitochondrial particles). The H⁺/coupling site ratio was not significantly different in ethanol and control mitochondria with succinate as electron donor. A 13% decrease in the H⁺/coupling site ratio was observed in ethanol mitochondria, however, when β-hydroxybutyrate was used as substrate. The rate of ATP-P_i exchange was decreased significantly in both ethanol mitochondria and submitochondrial particles as compared to control preparations. These observations demonstrate ethanol-elicited decreases in energy conservation in the site I region of the electron transport chain and in the activity of the ATP synthetase complex.     

Incorporation of a fatty acid containing a photosensitive group into lipids of cultured cardiac cells from chick embryo

Radioactive 12-(4-azido-2-nitrophenoxy)-stearic acid (NAP-stearate) was synthetized; it behaves as a competitive inhibitor of long-chain fatty acids for the entry into cultured cardiac cells from chick embryo. After uptake, [3H] NAP-stearate was incorporated by an energy-dependent process into neutral and polar lipids. Photoactivation as a function of time leads to a covalent labelling of the cells: up to 31 per cent of the radioactivity was recovered in the 105 000 g cell pellet, mainly in proteins. These experiments show that fatty acids containing photosensitive groups would potentially allow to localize the proteins involved in the binding and/or in the transport of fatty acids.     

The interaction of cyanocobalamin and some of its analogs with manganese(II) and gadolinium(III)

The influence of the paramagnetic ions Mn²⁺ and Gd³⁺ on the proton-decoupled ¹³C nuclear magnetic resonance spectra of three cyanocobalamin-monocarboxylic acids has been observed. The paramagnetic ions bind preferentially to the free carboxylate groups and cause the broadening of specific carbon resonances adjacent to these groups. These specific line-broadening effects have been used to assign the carbonyl carbons of the a-, f-, and g-acetamide side chains and have allowed us to confirm and/or correct the assignments of several carbon resonances that were assigned tentatively before.     

Measurement of DNA length by gel electrophoresis. I. Improved accuracy of mobility measurements using a digital microdensitometer and computer processing

The electrophoretic mobilities of DNA polymer fragments in an agarose gel have been measured from a photograph of the gel by different methods and converted to lengths by the reciprocal method. The method of measurement can introduce large errors in the length estimates. The use of a digital microdensitometer to obtain optical density profiles of gel tracks with subsequent computer processing to find peak positions was found to give the most accurate DNA lengths.     

Stimulation of protein phosphorylation during fertilization-induced maturation of Urechis caupo oocytes

Protein phosphorylation was studied during fertilization of Urechis caupo oocytes both in vivo, by measuring [³²P]phosphate incorporation into ³²P preloaded oocytes and in vitro, by measuring endogenous protein kinase and phosphatase activities in homogenates. During fertilization (and maturation) the rate of protein phosphorylation is dramatically increased. No change in the [³²P]phosphate uptake, or the nucleotide levels was observed at fertilization, so the increase cannot be attributed to changes in substrate availability. In vitro enzyme assays showed changes in protein kinase activity which approximately mirrored the changes in the in vivo phosphorylation pattern. As there were no changes in protein phosphatase activity, these results suggest the phosphorylation change results from an increase in protein kinase activity. The pattern of change, investigated by SDS-polyacrylamide gel electrophoresis, shows that proteins that were phosphorylated in the unfertilized egg become phosphorylated to a greater degree after fertilization. One protein (48 kd) undergoes an increase followed by a decrease of its phosphorylation level.