The modulation of membrane fluidity by hydrogenation processes. III. The hydrogenation of biomembranes of spinach chloroplasts and a study of the effect of this on photosynthetic electron transport

A method is reported for the in situ modification of the lipids of isolated spinach chloroplast membranes. The technique is based on a direct hydrogenation of the lipid double bonds in the presence of the catalyst, chlorotris(triphenylphosphine)rhodium (I). The pattern of hydrogenation achieved suggests that the catalyst distributes amongst all of the membranes. The polyunsaturated lipids within the membranes are hydrogenated at a faster rate and at an earlier stage than are the monoenoic lipids.Whilst addition of the catalyst to the chloroplast causes an initial 10–20% decrease in Hill activity, saturation of up to 40% of the double bonds present can be accomplished without causing further significant alterations in photosynthetic electron transport processes or marked morphological changes of the chloroplast structure as observed in the electron microscope.     

HCO 3 - and OH-transport across the plasmalemma ofChara

Internodal and whorl (branch) cells of the green alga,Chara corallina Klein ex Willd., em. R.D.W., were studied with the extracellular vibrating probe for measuring transmembrane ion currents, and with an extracellular pH microprobe for measuring the surface pH profile. Bands of positive inward current (OH^- efflux) 1–3 mm wide were separated by wider bands of outward current (HCO ₃ ^- influx) along the length of the cell. The measured peaks of inward current ranged from 20 to 60 A cm^-2 (98 m from the cell surface) which would correspond to a surface ionic flux of 270–800 pmol cm^-2 s^-1. The peaks of outward current (HCO ₃ ^- influx) ranged from 10 to 30 A cm^-2 which would correspond to a surface ionic flux of 140–400 pmol cm^-2 s^-1. The inward current bands matched the regions of surface alkalinity very well. The outward current (HCO ₃ ^- influx) was reduced at least 10-fold in low-HCO ₃ ^- medium, with a commensurate readjustment in the strength and pattern of inward current (OH^- efflux). (Although these experiments involved a manipulation of the external pH, it is felt that the main adjustment in current patterns was in response to the reduction in exogenous HCO ₃ ^- ). The presence of the vibrating probe perturbed the inward current region when vibrating with a 26-m amplitude, but this perturbation was eliminated when a 7-m amplitude was used. The perturbation was usually observed as a reduction in the number of inward current peaks with an increase (approximate doubling) in the amplitudes of the one or two remaining peaks. Both the inward and outward currents were light-dependent, falling off within seconds of light removal.     

The roles of nitrate and ammonium in the regulation of the development of nitrate reductase in Chlamydomonas reinhardii

The regulation of the development of nitrate reductase (NR) activity in Chlamydomonas reinhardii has been compared in a wild-type strain and in a mutant (nit-A) which possesses a modified nitrate reductase enzyme that is non-functional in vivo. The modified enzyme cannot use NAD(P)H as an electron donor for nitrate reduction and it differs from wild-type enzyme in that NR activity is not inactivated in vitro by incubation with NAD(P)H and small quantities of cyanide; it is inactivated when reduced benzyl viologen or flavin mononucleotide is present. After short periods of nitrogen starvation mutant organisms contain much higher levels of terminal-NR activity than do similarly treated wild-type ones. Despite the inability of the mutant to utilize nitrate, no nitrate or nitrite was found in nitrogen-starved cultures; it is therefore concluded that the appearance of NR activity is not a consequence of nitrification. After prolonged nitrogen starvation (22 h) the NR level in the mutant is low. It increases rapidly if nitrate is then added and this increase in activity does not occur in the presence of ammonium, tungstate or cycloheximide. Disappearance of preformed NR activity is stimulated by addition of tungstate and even more by addition of ammonium. The results are interpreted as evidence for a continuous turnover of NR in cells of the mutant with ammonium both stimulating NR breakdown and stopping NR synthesis. Nitrate protects the enzyme from breakdown. Reversible inactivation of NR activity is thought to play an insignificant rôle in the mutant.Abbreviations NR nitrate reductase - BV benzyl viologen     

Progestin metabolism in the rhesus monkey corpus luteum

For the purpose of describing the pathway by which estrogens are synthesized in the rhesus monkey ($\text{Macaca}$$\text{mulatta}$) corpus luteum (CL), CL were obtained during the midluteal phase of the menstrual cycle and fragments incubated with equimolar amounts of [7-³H]pregnenolone plus [4-¹⁴C]progesterone. Metabolites including ³H-progesterone, ³H, ¹⁴C-20α-dihydroprogesterone, ³H, ¹⁴C-17-hydroxyprogesterone, ³H-estrone and ³H-estradiol-17β appeared in the medium during the first 20 minutes of incubation, ³H, ¹⁴C-Androstenedione was not consistently noted until after 60 minutes. Despite the fact that the ¹⁴C/³H-17-hydroxyprogesterone ratio quickly approached a constant value in the medium, ¹⁴C-estrogens were not detected in the medium or tissue fragments suggesting that progesterone was not a principal precursor for estrogen synthesis. As evidenced by the observation that the ¹⁴C/³H-progesterone ratio was significantly higher in luteal fragments than the 17-hydroxyprogesterone ratio, 17-hydroxyprogesterone appeared to be synthesized from pregnenolone both by way of progesterone and by another route which did not include progesterone. C₂₁- and C₁₈-Steroids were more concentrated in tissue fragments after 120 minutes of incubation than in the medium indicating that these steroids were sequestered by luteal tissue.     

Enzymatic synthesis of β-lactam antibiotics: A thermodynamic background

The equilibrium constants and the respective standard Gibbs energy changes for hydrolysis of some β-lactam antibiotics have been determined. Native and immobilized penicillin amidase (EC 3.5.1.11) from Escherichia coli has been used as a catalyst. The values of standard Gibbs energy changes corresponding to the pH-independent product of equilibrium concentrations (ΔG⁰_c = ? RT ln K_c) have been calculated. The differences in the structure of the antibiotics nucleus hardly ever affect the value of the pH-independent component of the standard Gibbs energy change (ΔG⁰_c) and value of apparent standard Gibbs energy change at a fixed pH (ΔG^0′_c). At the same time, the value of ΔG⁰_c is more sensitive to the structure of the acyl moiety of the antibiotic; when ampicillin is used instead of benzylpenicillin, ΔG⁰_c increases by ～6.3 kJ mol^?1 (1.5 kcal mol^?1). pH-dependences of the apparent standard Gibbs energy changes for hydrolysis of β-lactam antibiotics have been calculated. The pH-dependences of ΔG^0′_c for hydrolysis of all β-lactam antibiotics have a similar pattern. The thermodynamic pH optimum of the synthesis of these compounds is in the acid pH range (pH < 5.0). The breakage of the β-lactam ring leads to a sharp decrease in the ΔG^0′_c value and a change in the pattern of the pH-dependence. For example, at pH 5.0 ΔG^0′_c decreases from 14.4 kJ mol^?1 for benzylpenicillin to ?1.45 kJ mol^?1 for benzylpenicilloic acid. The reason for these changes is mainly a considerable increase in the pK of the amino group of the nucleus of the antibiotic and, as a consequence, a decrease in the component of standard Gibbs energy change, corresponding to the ionization of the system. The thermodynamic potentials of the enzymatic synthesis of semisynthetic penicillins and cephalosporins on the basis of both free acids and their derivatives (N-acylated amino acids, esters) are discussed. It is shown that with esters of the acids, a high yield of the antibiotic can, in principle, be achieved at higher pH values.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.     

Hepatic and extra-hepatic glutathione-S-transferase activity in wild pigeons (Columba livia)

Glutathione-S-transferase (EC 2.5.1.18) activity was assayed in hepatic and extra-hepatic tissues of pigeons using l-chloro-2,4-dinitrobenzene and 1,2-dichloro-4-nitrobenzene as substrates. Gluthathione-S-transferase activity towards 1-chloro-2,4-dinitrobenzene in pigeon was in the order: kidney > liver > testes > brain > lung> heart. The enzyme activity with 1-chloro-2,4-dinitrobenzene as substrate was 40–44 times higher in pigeon liver and kidney than that observed with 1,2-dichloro-4-dinitrobenzene as substrate.K _m values of hepatic and renal glutathione transferase with l-chloro-2,4-dinitrobenzene as substrate were 2.5 and 3 mM respectively. Double reciprocal plots with varying reduced gluthathione concentrations resulted in biphasic curves with twoK _m values (liver 0.31 mM and 4mM; kidney 0.36 mM and 1.3 mM). The enzyme activity was inhibited by oxidized gluthathione in a dose-dependent pattern. 3-Methylcholanthrene elicited about 50% induction of hepatic glutathione transferase activity whereas phénobarbital was ineffective.     

Induction of DNA synthesis in adult rat hepatocytes cultured in a serum-free medium

Isolated hepatocytes from adult rats were cultured for 3 days in a serum-free synthetic medium. Supplementation with fibrinogen digests, glucagon and insulin remarkably increased DNA synthesis in hepatocytes. DNA synthesis began to increase at 35 h and reached a maximum at 41 to 54 h after plating. At this time, cells were morphologically identifiable as hepatocytes. Glucagon could be replaced by dibutyryl cyclic AMP or isobutyl-methyl-xanthine. Addition of amiloride (a Na⁺ influx inhibitor) during the initial 22 h completely inhibited DNA synthesis. These results suggest that influx of Na⁺ during early prereplicative period and increase in cellular cyclic AMP levels during late prereplicative period are necessary for the induction of DNA synthesis in hepatocytes.     

A difference in the affinity labeling of Ca2+, Mg2+-activated ATPases of normal and unc A strains of Escherichia coli by the 2',3'-dialdehyde derivative of adenosine 5'-diphosphate

The 2′,3′-dialdehyde of ADP, obtained by periodate oxidation of ADP, inhibited the hydrolytic activity of the purified Ca²⁺, Mg²⁺-activated ATPase of $\text{Escherichia}\text{coli}$. In the initial stages of the reaction inhibition was due to the reaction of 1 mol inhibitor/active site. When non-specific labelling of amino groups by the dialdehyde was lowered by the simultaneous presence of 15 mM ATP in the reaction mixture, 3 mol “ATP-protectable” binding sites/mol ATPase were found. “ATP-protectable” binding of the dialdehyde was not observed when the hydrolytically inactive ATPase of an $\text{unc A}$ mutant of $\text{E.}\text{coli}$ was used although binding of the inhibitor to non-protected amino groups still occurred. This suggests that the mutant ATPase is unable to bind ATP or that the amino groups with which the dialdehyde reacts in the native enzyme are absent or masked.