Ca2+ and Mg2+ as modulators of mitochondrial L-glycerol-3-phosphate dehydrogenase

1. Physiological concentrations of either Ca2+ or Mg2+ stimulated L-glycerol 3-phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. L-glycerol-3-phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations. 2. Substrate activation of mitochondrial L-glycerol-3-phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate-binding site functioning with high turnover. 3. Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies. 4. From the solubilized mitochondrial proteins L-glycerol-3-phosphate dehydrogenase was bound selectively to the hydrophobic phenyl-Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.     

Somatic embryogenesis and plantlet regeneration in the soybean Glycine max

A tissue culture procedure for the regeneration of somatic embryos and plantlets from somatic cells of the soybean Glycine max is described. Bean pods of soybean cv. TGM119 were immersed in liquid nitrogen for 20 minutes. Young embryos were excised from the immature seeds and cultured to form calli. Calli grown from the young embryos were incubated in liquid culture for two weeks. The liquid suspension culture was filtered to obtain single cells. The soybean cells were cultured for one month in a liquid medium in hanging drop cultures for development into proembryoids. The proembryoids were maintained on a solid growth medium for 40 days. The resultant callus tissue was transferred into MS media containing selected combinations and concentrations of 2,4-Dichlorophenoxyacetic acid, Naphthaleneacetic acid, Kinetin, Benzyladenine and Indoleacetic acid. In the presence of Benzyladenine (0.2 mg/l) and Indoleacetic acid (0.01 mg/l), globular and heart shaped somatic embryos were formed on the surface of the calli. Calli containing somatic embryos were transferred into liquid medium and incubated under low light conditions. After six months further incubation, more than 1,000 plantlets and a large number of somatic embryoids at various developmental stages were obtained per flask.Abbreviations KT kinetin - CM coconut milk - BA benzyladenine - NAA napthalene acetic acid - IAA indole acetic acid - 2,4-D 2,4 dichlorophenoxy acetic acid - MS Murashige and Skoog medium     

Photosynthetic characteristics of detached barley leaves during greening in the presence of SAN 9785

The effects of the pyridazinone compound SAN 9785 on the photosynthetic competence of leaves, on the photochemical activity of isolated thylakoids and on the formation and spectral properties of chlorophyll-protein complexes were studied during a 72-h greening period of detached etiolated leaves of barley (Hordeum vulgare L. cv. Horpácsi kétsoros). It was established that i) the photosynthetic capacity of the leaves decreased considerably (by 80 and 90%, as determined by¹⁴CO₂ fixation and fast fluorescence induction measurements, respectively); ii) the photochemical activity of isolated thylakoids from water to potassium ferricyanide and from dichlorophenol indophenol/ascorbate to methylviologen exhibited only slight reductions when expressed on a chlorophyll basis compared with the control; iii) the slow fluorescence induction curves of the treated leaves demonstrated the presence of a peculiar fluorescence component interrupting the quenching of fluorescence at around 1 min illumination; iv) a shortage of the chlorophyll-protein complex of photosystem I (CPI) occurred with a higher content of the monomer of the light harvesting complex in the thylakoids of treated leaves; and v) the fluorescence spectrum of the CPI band present in treated leaves indicates the destruction of the structural integrity of this complex during isolation from the membrane.Abbreviations Chl chlorophyll - CPI, CPII chlorophyll-protein complexes of the reaction centres of PSI and PSII - DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DPIP 2,6-dichlorophenol indophenol - DPIPH₂ chemically reduced form of DPIP - F _o fluorescence of constant yield - F _v fluorescence of variable yield - F _i ,F _m mitial and maximum yield of fluorescence - LHCP³ monomer of the light-harvesting complex - LHCP² and LHCP¹ oligomers of the light-harvesting complex LHCP³ - PSI, PSII photosystems I, II - SAN 9785 4-chloro-5-(dimethylamino)-2-phenyl-3(2H)-pyridazinone, also known as BASF 13-338 - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis     

In vivo definition of the functional origin of replication (ori(+ )) of the promiscuous plasmid pLS1

The nocR gene of Agrobacterium tumefaciens Ti plasmid pTiT37 is the regulatory gene of the nopaline catabolism (noc) operon of pTiT37. We have cloned and sequenced nocR, which encodes a DNA-binding protein. The deduced amino acid sequence is similar to those of members of the LysR family of prokaryotic activator proteins. Gel retardation experiments demonstrated that the NocR protein binds to the nocR promoter in both the presence and absence of nopaline. The increased mobility of the complex and alterations in the DNase I footprints revealed a nopaline-induced conformational change in the NocR-DNA complex. Sequence analysis of the NocR binding site indicated the presence immediately downstream of the –10 sequence of the nocR promoter of a 12 by putative operator overlapping a consensus gyrase recognition sequence and an 18 by long alternating purine-pyrimidine sequence. These results suggest that nopaline-induced alterations in the NocR protein-nocR promoter complex might control gene expression in the noc operon.