Study of the amino acid residue topography of the adenosine triphosphatase center of (Ca--Mg)-dependent sarcoplasmic reticulum adenosine triphosphatase by kinetic and spectral methods

The topography of HS- and NH2-groups and tryptophane residues in ATPase centre of (Ca--Mg)-ATPase on sarcoplasmic reticulum (SR) was investigated by kinetics, electron spectroscopy and spectrofluorimetry method. Both o-phthalaldehyde interacting with lysine or arginine residue or with end amino acid and fluorescein dimercuric acetate interaction with cysteine residue of HS-groups make (Ca--Mg)-ATPase both in SR and the pure enzyme completely inactive at molar ratio enzyme: inhibitor equal to 1 : 1. A 500 molar ATP surplus reduces drastically the enzyme inactivation rate by both inhibitors. The data supplied by the spectrofluorimetry and the induction-resonance theory were used to calculate the distances between nearest tryptophane residues and chromophore (o-FTC) generated by o-phthalaldehyde interaction with NH2-group the protein amino acid residue (17 A) and o-FTC and fluorescein dimercuric acetate (19 A) attached to enzyme HS-group. Because o-FTC is inside the protein pocket it is not accessible to J- ions up to 2.5 M KJ. However some tryptophane resudies and fluorescein dimercuric acetate attached to HS-group are near to the macromolecule surface. Lysine (or arginine residues) or end amino acid NH2-group and cysteine residues HS-group, and some tryptophane residues are at ATPase centre of (Ca--Mg)-ATPase from sarcoplasmic reticulum. Possible topography of the centre is discussed.     

A bioenergetic study of a benthic nematode,plectus palustris de man 1880, throughout its life cycle

Summary Growth and reproduction of the parthenogenetic freshwater nematode,Plectus palustris, were studied at different controlled levels of food densities at 20° C. A bacteria-sloppy agar mixture was used as substrate and food medium. No growth or reproduction occurred at the lowest food density (8.10⁷ bacterial cells ml^-1). At 8.10⁸ cells ml^-1, the larval duration was 18.5 days, the instantaneous growth rate (g) of young larvae 0.2 d^-1 and the daily fecundity rate during a prolonged period of constant egg production 12.6 eggs·d^-1. At a food density of 8.10⁹ cells ml^-1, the corresponding values are 12.5 days, 0.4 d^-1 and 37.7 eggs d^-1.By including the data on respiration from a previous paper (Klekowski et al., 1979), the energetics of the species at different food densities can be discussed: production processes are apparently more dependent on food supply than respiration. However, prolongation of the larval phase in lower food densities greatly increases the cumulated respiratory costs per unit production. A second point is the ability to produce smaller-sized primiparous females in sub-optimal food which shortens the immature life period and serves to reduce the burden of cumulated metabolic costs for attaining sexual maturity.A comparison of the range of food densities used in the experiments with bacterial densities known from lake sediments of different trophic type suggests that food is likely to be the main factor governing the population dynamics of bacterivorous species under field situations.     

Photosynthesis as a function of short day induction and gibberellic Acid treatment in bougainvillea "san diego red"

Short day induction in Bougainvillea “San Diego Red” increases photosynthetic rates in mature leaves; gibberellic acid treatments, which inhibit flowering, cancel the short day effect. These results lend support of a nutritional hypothesis that suggests that in Bougainvillea assimilate supply to the reproductive axis increases before floral initiation and during flower development.     

Distribution of Assimilates from Various Source Leaves During the Development of Gladiolus grandiflorus

The distribution pattern of the products of photosynthesis wasstudied in gladiolus plants (Gladiolus grandiflorus cv Eurovision)in four stages of development I, plants having a very younginflorescence still enclosed between the leaves; II, plantswith a young inflorescence just emerged from the leaves, III,plants at full bloom, IV, plants with young fruits. The first,third or sixth foliage leaf was labelled with ¹⁴CO₂, and subsequentdistribution in the plant was determined Results were expressedas a percentage of translocated ¹⁴C accumulated by each partof the plant which gives a measure of its ‘sink strength’,or as ‘relative sink activity’ (RSA) which is independentof the size of the indicated organ. There are two competing sinks in the developing gladiolus—theinflorescence and the new corm. When RSA is the criterton theinflorescence constitutes the main sink irrespective of thesource leaf from the first stage until flowering. With the subsequentwilting of the flowers and fruit set RSA of the inflorescencedeclines rapidly and the new corm becomes the main sink When‘sink strength’ is the criterton it appears thatthe inflorescence acts as a very weak sink when it is youngand becomes increasingly stronger until flowering and then declinessteeply. Sink strength of the corm declines during the developmentof the inflorescence and then increases again steeply with wiltingof the flowers and fruit set. There are small differences betweenthe various source leaves. The young new corm acts as a strongsink for the lower foliage leaf and progressively weaker forupper leaves. Gladiolus grandiflorus, flower development, corm, assimilates distribution, sink strength, relative sink activity     

Electron-cytochemical localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31) in fungal cells

Summary New cytochemical method, based on biochemical experiments, was elaborated for the ultrastructural localization of phospho(enol)pyruvate carboxylase (EC 4.1.1.31). The procedure was used to study the saprophytic submerged mycelium of the ascomycetous fungusClaviceps purpurea Tul. producing clavine alkaloids. The pelleted mycelium was fixed in ice cold 3% glutaraldehyde in 50 mM cacodylate buffer pH 7.2 and washed repeatedly in the same cold buffer. The reaction mixture contained 100 mM Tris-HCl buffer pH 9.0, 10 mM phospho(enol)pyruvate, 30 mM sodium potassium tartrate, 3 mM Pb(NO₃)₂, 60 mM MgCl₂ and 30 mM NaHCO₃. Enzyme activity was localized in vacuoles, particularly inside lipid globules (spherosomes) and less frequently in membranous vesicles. Acetyl-CoA activated PEP-carboxylase both in cell free extracts and in the cytochemical staining. Aspartate inhibited the enzyme in the biochemical assay with coupled malate dehydrogenase system; the cytochemical reaction was not influenced, probably due to the interference of asparagine synthase (EC 6.3.1.1).     

Requirement for the location of both appropriate and irrelevant H-2 antigens on the same stimulator cell for unspecific DNA-synthesis inhibition by the H-2-antigen-primed,specific suppressor T cells

Specific suppressor T cells (SSTC), primed in vivo with H-2 antigens, have been shown previously to inhibit DNA synthesis in the one-way, three-cell mixed lymphocyte reaction (MLR) provided that (a) the stimulator cells bear the priming H-2 antigens, and (b) the responder cells possessIC+S regions homologous to those of the SSTC. Anti-B10.A BlO.A(2R) SSTC (anti-D^d) and anti-A.AL A.TL SSTC (anti-K^k) are shown here to be able to inhibit the DNA synthesis triggered in MLR, not only by the corresponding antigens, D^d and K^k, respectively, but also by irrelevant, third-party H-2 and Mls products provided that the corresponding and third-party antigens are presented on the same stimulator cell. If stimulatorH-2 regions, whose products interact with SSTC and responders, are located on different stimulator cells within the particular MLR, SSTC activity is not elicited. Participation of cytotoxic T lymphocytes in DNA-synthesis suppression is ruled out. Direct contact or location of the inhibited responder cell very close to SSTC is considered to be required for the development of SSTC activity.     

Identification on I-Ak molecules of a functional site recognized by proliferating T-lymphocytes

The inhibitory capacity of 17 monoclonal antibodies (m.Ab.) specific for the products of the I-A ^k subregion was evaluated in proliferative responses of B10.BR T-lymphocytes to GAT, Keyhole limpet hemocyanin, and ovalbumin. Considered in isolation, each m.Ab. mediated inhibitory effects of comparable magnitude on these three different proliferative responses. On the other hand, clear differences were observed when the magnitude of the inhibitory effects was compared from one m.Ab. to another. The m.Ab. were consequently classified as strong or moderate-to-weak inhibitors of T-cell proliferative responses. Evidence was simultaneously gained indicating the following: (a) the determinants recognized by different m.Ab. were expressed on the same molecules; (b) the differences in affinity of the m.Ab. for I-A^k positive cells did not explain their differences in inhibitory capacities; (c) conversely, the inhibitory capacity of each m.Ab. followed its ability to inhibit the cell surface fixation of Ia.17-specific 10-2.16 m.Ab.; (d) the strong inhibitory capacity of some m.Ab. was not related to a special ability to modulate cell surface Ia molecules. These results suggest that antigen recognition by T lymphocytes is preferentially restricted by a functional site of the I-A^k molecules related to the Ia.17 and Ia.1 specificities.Abbreviations EDTA Ethylenediamine-tetraacetic acid disodium salt - EHAA Eagle's Hanks' amino acids medium - FCS fetal calf serum - in polypeptide G is glutamate, A, alanine, T, tyrosine - HEPES N-2-hydroxy-piperazine-N-2-ethane sulfonic acid - kd dissociation rate constant - KLH Keyhole limpet hemocyanin - LPS lipopolysaccharide - m.Ab. monoclonal antibodies - NP-40 nonidet P-40 - PBS phosphate buffered saline - PBS-BSA PBS supplemented with 1% bovine serum albumin - PBS-BSA-NP-40 PBS-BSA supplemented with 0.5% NP-40 - RT room temperature - SEM standard error of the mean - s.c. spleen cells     

beta-Adrenergic receptors stimulated peroxidase secretion from rat lacrimal gland

Incubation of rat extraorbital lacrimal gland slices with the beta-agonist isoproterenol caused peroxidase secretion but no K+ release. The peroxidase secretion was inhibited by propranolol. Addition of dibutyryl cyclic AMP or adenosine 3'5'-cyclic phosphorothioate to lacrimal slices produced peroxidase secretion at a higher rate than that obtained with optimal concentration of isoproterenol. Methyl isobutylxanthine is also a strong stimulator of peroxidase secretion. Peroxidase activity was determined by a modified sensitive guaiacol method. Membrane fraction of lacrimal cells was shown to contain an isoproterenol-stimulated adenylate cyclase activity. It is therefore suggested that there is a beta-adrenergic receptor in the rat lacrimal gland and that its stimulation causes activation of an adenylate cyclase which leads to peroxidase secretion.     

Influence of C-protein on mechano-chemical properties and structure of reconstituted actomyosin

C-protein on the mechano-chemical properties (ATPase activity and superprecipitation) of actomyosin systems has been investigated. The presence of C-protein in AM-complexes has been shown to decrease the rate of superprecipitation (SPP) and simultaneously increase the ATPase activity. Both effects of C-protein are dependent on its quantity in the system. Tropomyosin decreased considerably but does not eliminate completely the inhibitory influence of C-protein on the SPP. Electron microscopy does not reveal considerable structural differences in the initial AM-complexes depending on the presence or absence of C-protein. It is supposed that the discovered effects of C-protein on the behaviour of AM-systems are determined by the fine local structural and (or) charge changes produced by C-protein in the region of myosin cross-bridges, which in its turn results in a modification of the actin-myosin interaction. Possible participation of C-protein in the regulation of the interaction of thin and thick filaments in the muscle is discussed.