THE INORGANIC CARBON REQUIREMENTS OF CHLAMYDOMONAS SEGNIS (CHLOROPHYCEAE) FOR CELL DEVELOPMENT IN SYNCHRONOUS CULTURES1

The time in the cell cycle when CO₂ provision was required for cell development and division was determined in synchronous cultures of Chlamydomonas segnis Ettl bubbled with air (0.03% CO₂) or air enriched with 5% CO₂ under continuous light at 25°C and pH 7. Provision of CO₂ (% in air v/v) during the G₁-phase was found to be essential for the completion of the cell cycle. There was no demand for CO₂ supply throughout the S-phase and mitosis. Using cultures adapted to CO₂ concentrations ranging from 0.03 to 5% in air, the apparent CO₂ concentration (K_m) required for the cells to develop during the G_-1-phase and to attain one half the maximal rates of photo-synthetic O₂ evolution was calculated as 0.05%. This value increased to 0.1 and 0.5% during the S-phase. For total protein and carbohydrate accumulation, which would reflect inorganic carbon (CO₂+ HCO₃^?) assimilation, the K_m (% CO₂) were ca. 0.1 and 0.14 throughout the cell cycle, respectively. The CO₂ concentration at which the cells exhibited the shortest generation time (6.7 h) was 0.1%. These results showed that during development, cells photosynthesizing (evolving O₂) at maximal rates but accumulating protein and carbohydrate at one half the maximal rates or less would complete their vegetative life cycle in the shortest time.     

Enzymes of starch metabolism in developing grains of high lysine barley mutant

The absolute activities of ADPG(UDPG)-pyrophosphorylase, starch phosphorylase, ADPG(UDPG)-starch synthetase, NDP-kinase and inorganic pyrophosphatase have been studied in high lysine mutant barley Notch-2 and its parent NP 113 grains during development. In general, mutant Notch-2 grains had higher average activities of UDPG-pyrophosphorylase and starch phosphorylase and lower activity of ADPG(UDPG)-starch synthetase per grain than the parent NP 113 during grain development. Activities of NDP-kinase, ADPG-pyrophosphorylase and inorganic pyrophosphatase differed only to a small extent between the mutant Notch-2 and NP 113. It is suggested that the lower activity of ADPG(UDPG)-starch synthetase might be responsible for the reduced accumulation of starch in the mutant Notch-2 grain as compared with parent NP 113 during development.     

Investigation of the operational stabilities and kinetics of glucoamylase immobilized on alkylamine derivatives of titanium(IV)-activated porous inorganic supports

Glucoamylase (exo-1,4-α-d-glucosidase, EC 3.2.3.1) was coupled to several porous silica matrices by an improved metal-link/chelation process using alkylamine derivatives of titanium(IV)-activated supports. In order to select the titanium activation procedure which gave stable enzyme preparations, long-term stability tests were performed. The immobilized glucoamylase preparations, in which the carrier was activated to dryness with a 15% w/v TiCl₄ solution, displayed very stable behaviour, with half-lives of ～60 days. The optimum operating conditions were determined for these preparations. There are significant differences between the behaviour of the immobilized enzyme and the free enzyme. The apparent K_m increased on immobilization due to diffusional resistances. The pH optimum for the immobilized preparation showed a slight shift to acid pH relative to that of the soluble enzyme. Also, the optimum temperature descreased to 60°C after immobilization. In order to test Michaelis-Menten kinetics at high degrees of conversion, time-course analysis of soluble starch hydrolysis was performed. It was observed that simple Michaelis-Menten kinetics are not applicable to the free/immobilized glucoamylase-starch system at high degrees of conversion.     

DDT inhibition of Ca-Mg ATPase from peripheral nerves and muscles of lobster, Homarus americanus

Sensitivity of CaMg ATPase from axonic plasma membrane (APM) and sarcoplasmic reticulum (SR) of lobster, $\text{Homarus}\text{,}\text{americanus}$, to DDT was studied. The CaMg ATPase found in SR with the high Ca²⁺ affinity is sensitive to DDT while the portion of ATPase related to the low Ca²⁺ affinity site is not inhibited by DDT. Also, DDT is more inhibitory against the CaMg ATPase prepared from APM than the one obtained from SR. The relationship between inhibition of the CaMg ATPase by DDT in the axonic nerve membrane and $\text{in}\text{,}\text{vivo}$ poisoning symptoms of the nervous system is discussed.     

The use of inorganic substances to stimulate gut evacuation in Daphnia magna

Observations were done on the effect of inorganic substances on the gut evacuation process in Daphnia magna. Procedures which accelerate this process are described.     

Inhibition by polyamines of lipid peroxide formation in rat liver microsomes.

Both NADPH- and ascorbic acid-dependent lipid peroxidations were inhibited by spermine, the degree of inhibition being greater with the former peroxidation. The effective concentration of spermine required for inhibition was higher when larger amounts of microsomes were used. However, the activities of NADPH-cytochrome c reductase and NADPH-peroxidase were not influenced by spermine. These results suggest that spermine inhibits lipid peroxidation by binding to phospholipids in the microsomes.     

Relative toxicities of particulate and soluble forms of beryllium to a rat liver parenchymal cell line in culture and possible mechanisms of uptake.

The relative toxicities of particulate beryllium phosphate, soluble beryllium sulphate and a beryllium sulphosalicylate complex to a rat liver parencymal derived cell line have been examined in culture. Due to the propensity of beryllium salts to form beryllium phosphate in solution the incubation medium used was free of inorganic phosphate. Cell death measured by the loss of cellular lactate dehydrogenase into the medium can be produced within 76 h from beryllium phosphate and beryllium sulphosalicylate or 48 h from beryllium sulphate provided the cells have, irrespective of the form of added beryllium, taken up a minimum of 2--5 nmol Be/10(6) cells. Whilst beryllium phosphate was readily taken up as a particle, beryllium complexed with excess sulphosalicylate was not so markedly accumulated by the cells except possibly by formation of small amounts of beryllium phosphate in the medium as a result of inorganic phosphate lost from the cells. The extent of beryllium uptake from beryllium sulphate quantitatively most resembled that observed for beryllium phosphate but was largely independent of beryllium phosphate formation in the medium and not accompanied by the uptake of the SO42- anion. However, the accumulation of beryllium derived from beryllium sulphate did appear to be associated with the production of a sedimentable from believed most probably to be colloidal beryllium hydroxide. The uptake of all forms of beryllium was temperature sensitive and metabolic inhibitor studies and treatment of the cells with trypsin or neuraminidase supported the view that the distinct behaviour of beryllium derived from beryllium sulphate may be related to the enhanced toxicity of this form both under the conditions used and when administered to experimental animals.     

Role in hemolysis of the interaction of tellurium compounds with glutathione

We have shown that tellurite and tellurate require the interaction with reduced glutathione (GSH) to hemolyze human erythrocytes. The study of the nature of this interaction is the main object of this paper. The degree of hemolysis was determined by the method of Angelone. The addition of extracellular 1 mM GSH or cysteine increased the rate of hemolysis. Concanavalin A (0.3 mg/mL) and/or 4 mg/mL adenosine did not affect the hemolysis by 0.1 mM tellurite. One tenth to 1 mM 4-acetamido-4′-isothiocyanostilbene-2,2′-disulfonate (SITS) inhibited this hemolysis by 60–100%. Millimolar GSH released this inhibition. Incubation of 0.1 mM tellurite with 1 mM GSH for 90 min at 37°C, produced a hemolytic agent when prepared and tested under nitrogen, but one that was not active when prepared in air. The hemolysis byp-hydroxymercuribenzoate orp-hydroxymercuriphenylsulfonate did not involve GSH. Scanning electron micrographs showed a sphero-echinocyte transformation, in the pre-hemolytic stage, with all the agents tested. The rate of penetration of tellurite plays a role in determining the rate of hemolysis, as shown by the effect of SITS. The release by GSH of the inhibition by SITS poses questions concerning the site of action and cell membrane penetration of the hemolytic agent. Telluride or some intermediate in the interaction of GSH with tellurite is the actual hemolytic agent.     

Generation of electric current by chromatophores of Rhodospirillum rubrum and reconstitution of electrogenic function in subchromatophore pigment-protein complexes

Lipoprotein complexes, containing (1) bacteriochlorophyll reaction centers, (2) bacteriochlorophyll light-harvesting antenna or (3) both reaction centers and antenna, have been isolated from chromatophores of non-sulphur purple bacteria Rhodospirillum rubrum by detergent treatments. The method of reconstituting the proteoliposomes containing these complexes is described. Being associated with planar azolectin membrane, proteoliposomes as well as intact chromatophores were found to generate a light-dependent transmembrane electric potential difference measured by Ag/AgCl electrodes and voltmeter. The direction of the electric field in proteoliposomes can be regulated by the addition of antenna complexes to the reconstitution mixture. The reaction center complex proteoliposomes generate an electric field of a direction opposite to that in chromatophores, whereas proteoliposomes containing reaction center complexes and a sufficient amount of antenna complexes produce a potential difference as in chromatophores. ATP and inorganic pyrophosphate, besides light, were shown to be usable as energy sources for electric generation in chromatophores associated with planar membrane.