A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Adriamycin effects on transplasma membrane redox functions in porcine neutrophils

Transplasma membrane electron transport, as assayed by external ferricyanide reduction, has been related to control of growth and hormone response of cells. Elicitor-stimulated transmembrane NADPH oxidase is important for bacteriocidal superoxide production by neutrophils. Since adriamycin is myelosuppressive and can stimulate superoxide production, its effects on the two redox systems of porcine neutrophil plasma membranes were compared. Adriamycin inhibits transplasma membrane ferricyanide and stimulates superoxide production activated by phorbol myristate acetate (PMA). Ferricyanide reduction in PMA-treated cells becomes resistant to inhibition by adriamycin. These results provide evidence for an independent effect of adriamycin on transmembrane ferricyanide reduction and on superoxide generation.     

Plasma protein synthesis by isolated rat hepatocytes

A system of preparation of rat hepatocytes with extended viability has been developed to study the role of hormones and other plasma components upon secretory protein synthesis. Hepatocytes maintained in minimal essential medium reduced the levels of all amino acids in the medium except the slowly catabolized amino acids leucine, isoleucine, and valine, which steadily increase as the result of catabolism of liver protein. Although the liver cells catabolize 10-15% of their own protein during a 20-h incubation, the cells continue to secrete protein in a linear fashion throughout the period. The effects of insulin, cortisol, and epinephrine on general protein synthesis, and specifically on fibrinogen and albumin synthesis, have been tested on cells from both normal rats and adrenalectomized rats. Cells from normal animals show preinduction of tyrosine amino transferase (TAT), having at the time of isolation a high level of enzyme which shows only an increase of approximately 60% upon incubation with cortisol. In contrast, cells from adrenalectomized animals initially have a low level of enzyme which increases fourfold over a period of 9 h. The effects of both epinephrine and cortisol on protein synthesis are also much larger in cells from adrenalectomized animals. After a delay of several hours, cortisol increases fibrinogen synthesis sharply, so that at the end of the 20-h incubation, cells treated with hormone have secreted nearly 2.5 times as much fibrinogen as control cells. The effect is specific; cortisol stimulates neither albumin secretion nor intracellular protein synthesis. The combination of cortisol and epinephrine strongly depresses albumin synthesis in both types of cells. Insulin enhances albumin and general protein synthesis but has little effect on fibrinogen synthesis.     

Stimulation of photosynthesis by carbonyl compounds and chelators

A number of carbonyl compounds including bicarbonate, ethylene carbonate, dimethylcarbonate, propylene carbonate, bis-pentamethylene urea, and glycidol, and several chelators were tested for their effect on photosynthetic reactions in isolated spinach chloroplasts. It was found that carbonyl compounds inhibited the DCMU-insensitive silicomolybdate reduction by photosystem II but stimulated the O₂ evolution associated with ferricyanide reduction in presence of DBMIB and the H₂O→methylviologen reaction. Many chelators behaved in the same manner except 1,10-phenanthroline which shows the opposite effect. The carbonyl compounds did not uncouple because they stimulated the proton gradients associated with noncyclic photophosphorylation, whereas some chelators, such as bathocuproine or bathophenanthroline inhibited the proton gradients 100%. Electron transport in presence of ADP and inorganic phosphate showed a stimulation of rates beyond that obtained in presence of an uncoupler. The data are discussed in terms of inhibition of cyclic electron flow around PS II which leads to increased electron transport rates toward PS I.     

Different effects of inhibitors on two mutants of Escherichia coli K12 affected in the Fo portion of the adenosine triphosphatase complex.

The effects of the inhibitors dicyclohexyl-carbodiimide (DCCD), bathophenanthroline and tertiary octylcatechol, on some enzyme activities in membranes from strains of Escherichia coli carrying mutations in the uncB or uncC genes have been studied. Membranes prepared from uncC mutants retain a normal DCCD-sensitive Mg2+-stimulated adenosine triphosphatase (Mg-ATPase) activity whereas in uncB mutants this enzyme activity is insensitive to DCCD. The membrane-bound Mg-ATPase activity from the uncC mutant strain, as compared with that from the normal strain, is only partially sensitive to the inhibitors bathophenanthroline or tertiary-octylcatechol. Both of these inhibitors stimulate the membrane-bound Mg-ATPase from uncB mutant strains. A DCCD-insensitive Mg-ATPase activity is found in the cytoplasmic fraction following cell disruption of either the uncB or the uncC mutants. The lipophilic chelators bathophenanthroline and tertiary-octylcatechol stimulate the activity of the 'soluble' Mg-ATPase in the uncB mutant but partially inhibit the activity in the uncC mutant. The NADH oxidase activities in membranes from both mutant and normal strains are strongly inhibited by tertiary-octylcatechol and bathophenanthroline but not by DCCD.     

Comparison of antiviral and antitumor activity of activated macrophages.

The antiviral and antitumor activity in vitro of normal, stimulated, vaccinia virus “immune”, and activated peritoneal macrophages were compared. Activated (pyran or corynebacteria induced) PEC exhibited both antitumor and antiviral activity. Stimulated (thioglyocollate) and vaccinia virus “immune” PEC inhibited virus growth but did not possess antitumor activity. Normal (unstimulated) PEC were relatively ineffective in either activity. The antiviral activity was nonspecific, being expressed against herpes simplex and EMC viruses in addition to vaccinia. Although a possible role for interferon was suggested by the lack of activity of mouse PEC on vaccinia virus growth in heterologous FLK cells, definitive proof was not obtained. The activity was most pronounced against multiple cycles of viral infection initiated at a low multiplicity of infection. Single cycle virus growth was not affected, suggesting that the major inhibition was on subsequent cycles of virus growth.     

Transmembrane ferricyanide reduction in tobacco callus cells

Transmembrane ferricyanide reduction in whole cells of normal and of transformed tobacco (Nicotiana tabacum) callus tissue was compared. It was found that low concentrations of indoleacetic acid (IAA, 0.1 μM), gibberellic acid (GA, 0.3 μM), and benzyl adenine (BA, 0.03 μM) stimulate external ferricyanide reduction in normal tobacco callus cells, but inhibit this reaction up to 67% in transformed cells when hormones are applied to cells 10 min prior to assay. Higher concentrations of these growth regulators (1 μM or greater) inhibit transmembrane ferricyanide reduction in both types of cells, with the exception of IAA, giving an initial stimulation of the rate (12%), followed by 24% inhibition after 2 min. The observed external ferricyanide reduction by whole tobacco callus cells may be explained on the basis of a transplasmalemma redox system, which may be associated with the iron metabolism of these cells.     

Calmodulin antagonists inhibit electron transport in photosystem II of spinach chloroplasts

Chlorpromazine, phenothiazine and trifluoperazine, known as calmodulin antagonists, inhibit electron transport in Photosystem II of spinach chloroplasts in concentrations from 20–500 μM. The inhibition site is located on the diphenyl carbazide to indophenol pathway in Tris-treated chloroplasts, indicating that water oxidation is not affected by these drugs. Ca²⁺ ions, bound to chloroplast membranes before the addition of calmodulin antagonists, can protect against inhibition up to 25% of the electron transport rate. In presence of A23187, the Ca²⁺-specific ionophore, Ca²⁺ ions provide less protection against inhibition by the 3 calmodulin antagonists used. A possible role of a calmodulin-like protein in spinach chloroplasts is postulated.     

Ca2+ and calmodulin antagonists inhibit the proton gradients associated with non-cyclic and cyclic photophosphorylation in spinach chloroplasts

The synthesis of benzylpenicillin (BP) after mixing phenyl-acetyl-glycine(PAG), 6-aminopenicillanic acid (6-APA) and free or immobilized penicillin amidase (E.C.3.5.1.11.) was studied as a function of pH and ionic strength. Before the final equilibrium was reached a kinetically controlled synthesis of BP was observed. Then a transient maximum concentration in BP much larger than the final equilibrium content was synthesized in the acyl-transfer process. The factors influencing this maximum have been analyzed. Increasing ionic strength markedly decreased the maximum in BP and the rate of deacylation of phenyl-acetyl-penicillin amidase by 6-APA. The change was largest when the enzyme was immobilized in a positively charged support, where at low ionic strength the concentration of 6-APA around the enzyme is larger than the bulk concentration due to the partitioning of charged solutes.     

Proteolytic modification of mouse liver catalase

When catalase was immunoprecipitated from different subfractions of mouse liver homogenates, the enzyme which was obtained from extracts of the large granular fraction exhibited a lower molecular weight than that from either the cytosol or purified peroxisomal fractions, as judged by sodium dodecyl sulphate polyacrylamide gel electrophoresis. This modification of the enzyme could be prevented by the addition of proteolytic inhibitors to extraction buffers; and consequently, unmodified catalase was able to be purified in the presence of 5 mM iodoacetamide. Electrophoretic comparison of the catalases against standards of known molecular sizes indicated that the unmodified enzyme had a subunit mass approximately 2,000 daltons larger than the modified enzyme. The significance of these proteolytic modifications has been discussed in relation to the involvements of catalase and peroxisome turnover.