Heterologous hybridization of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) restores the enzyme activities

The catalytic core (A8) and small subunit (B) of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were isolated from two species of cyanobacteria (Aphanothece halophytica and Synechococcus ACMM 323) as well as from the photosynthetic purple sulfur bacterium, Chromatium vinosum. The subunit B is essential for the activity of all three enzymes. The heterologous hybridization of RuBisCO molecules from the three organisms was attempted and the reconstitution of the catalytically active hybrid was achieved between A8 derived from either Aphanothece or Synechococcus and subunit B from Aphanothece, Synechococcus or Chromatium. However, reconstitution of the enzymically active hybrid between A8 from Chromatium and B subunits from the cyanobacteria could not be achieved. Experiments by using high performance liquid column chromatography also showed the formation of a heterologous hybrid possessing RuBP carboxylase activity.     

Immunolocalization of β-1-4-galactan and its relationship with lignin distribution in developing compression wood of Cryptomeria japonica

Compression wood (CW) contains higher quantities of β-1-4-galactan than does normal wood (NW). However, the physiological roles and ultrastructural distribution of β-1-4-galactan during CW formation are still not well understood. The present work investigated deposition of β-1-4-galactan in differentiating tracheids of Cryptomeria japonica during CW formation using an immunological probe (LM5) combined with immunomicroscopy. Our immunolabeling studies clearly showed that differences in the distribution of β-1-4-galactan between NW (and opposite wood, OW) and CW are initiated during the formation of the S₁ layer. At this stage, CW was strongly labeled in the S₁ layer, whereas no label was observed in the S₁ layer of NW and OW. Immunogold labeling showed that β-1-4-galactan in the S₁ layer of CW tracheids significantly decreased during the formation of the S₂ layer. Most β-1-4-galactan labeling was present in the outer S₂ region in mature CW tracheids, and was absent in the inner S₂ layer that contained helical cavities in the cell wall. In addition, delignified CW tracheids showed significantly more labeling of β-1-4-galactan in the secondary cell wall, suggesting that lignin is likely to mask β-1-4-galactan epitopes. The study clearly showed that β-1-4-galactan in CW was mainly deposited in the outer portion of the secondary cell wall, indicating that its distribution may be spatially consistent with lignin distribution in CW tracheids of Cryptomeria japonica.     

Determination of Intracellular Chloride Ion Concentration in a Halotolerant Cyanobacterium Aphanothece halophytica

Salt inhibition of RuBP carboxylase activity from Aphanothecehalophytica was caused by Cl^-, but not by K⁺ nor Na⁺. The intracellularCl^- concentration increased about 4-fold from 35 mM to 150 mM,when NaCl concentration in the culture medium was increasedfrom 0.5 M to 2.0 M. ¹Permanent address: Department of Biochemistry, Faculty of Science,Chulalongkorn University, Bangkok, Thailand (Received February 12, 1988; Accepted June 1, 1988)     

Uptake of Neutral Red by the Vacuoles of a Green Alga, Micrasterias pinnatifida

Neutral red (NR) in the culture medium entered the vacuolesof a green alga, Micrasterias pinnatifida, at a higher rateat pH 8 than at pH 5. NR remained soluble in vacuoles of cellscultured at pH 5, while it precipitated and formed granulesin cells cultured at pH 8. The vacuoles of cells cultured atpH 8 contained fibrils, but those of cells cultured at pH 5did not. The amount of NR that entered the cells was markedlyreduced by the addition to the medium of nigericin at 10^-5M,monensin at 10^-5M, bafilo-mycin A₁ at 10^-5M, or ammonium chlorideat 50 mM. The formation of NR granules in vacuoles were stronglyinhibited and the disorganization of NR granules were acceleratedby the addition of nigericin at 10^-5M, or bafilomycin A₁ at10^-5M to the culture medium. The possibility is discussed thatNR which enters vacuoles might become positively charged (NRH⁺)by protons brought into vacuoles by proton pumps and that NRH⁺might combine with some negatively charged macromolecules toform aggregates or granules. (Received April 18, 1996; Accepted May 27, 1996)     

Distribution of Glycinebetaine in Old and Young Leaf Blades of Salt-Stressed Barley Plants

In barley plants exposed to stepwise salt-stress (up to 200mM NaCl), sodium and chloride ions accumulated preferentiallyin old rather than in young leaf blades. Furthermore, the levelof glycinebetaine in young leaf blades was approximately threetimes that in old leaf blades. ³Present address: Department of Biochemistry, Faculty of Science,Kasesart University, Bangkok, 10900 Thailand     

Formation and decomposition of vacuoles inBotryococcus in relation to the trans-Golgi network

Summary The formation and the decomposition of vacuoles in a member of Xanthophyceae,Botryococcus braunii, were examined by light and electron microscopy. Particles around the nucleus were identified as vacuoles from their stainability with neutral red. These particles disappeared during cell division. They reacted positively in an activity test for acid phosphatase. Electron microscopy revealed the presence of spherical vacuoles around the nucleus. During cell division, these vacuoles seemed to be decomposed by the ER which surrounded the vacuoles. Soon after this decomposition, many immature multivesicular bodies (MVBs) appeared to develop from the trans-Golgi network (TGN) and were pinched off from the TGN. These immature MVBs took up small vesicles in them as they grew into the mature MVBs. Mature MVBs took up and digested the surrounding cytoplasm, fused with one another, and eventually became the vacuoles.Abbreviations MVB multivesicular body - TGN trans-Golgi network     

Loose association of ribulose 1,5-bisphosphate carboxylase/oxygenase with chloroplast thylakoid membranes

The intra-chloroplastic distribution of ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO) between thylakoid membranes and stroma was studied by determining the enzyme activities in the two fractions, obtained by the rapid centrifugation of hypotonically disrupted chloroplast preparations of spinach and pea leaf tissues. The membrane-associated form of RuBisCO was found to increase in proportion to the concentration of MgCl₂ in the disrupting medium; with 20 mM MgCl₂ approximately 20% of the total RuBisCO of spinach chloroplasts and 10% of that of pea chloroplasts became associated with thylakoid membranes. Once released from membranes in the absence of MgCl₂, addition of MgCl₂ did not cause reassociation of the enzyme. The inclusion of KCl in the hypotonic disruption buffer also caused the association of RuBisCO with membranes; however, up to 30 mM KCl, only minimal enzyme activities could be detected in the membranes, whereas above 40 mM KCl there was a sharp increase in the membrane-associated form of the enzyme.Higher concentrations of chloroplasts during the hypotonic disruption, as well as addition of purified preparations of RuBisCO to the hypotonic buffer, resulted in an increase of membrane-associated activity. Therefore, the association of the enzyme with thylakoid membranes appears to be dependent on the concentration of RuBisCO. P-glycerate kinase and aldolase also associated to the thylakoid membranes but NADP-linked glyceraldehyde-3-P dehydrogenase did not. The optimal conditions for enzyme association with the thylakoid membranes were examined; maximal association occurred at pH 8.0. The association was temperature-insensitive in the range of 4° to 25° C. RuBisCO associated with the thylakoid membranes could be gradually liberated to the soluble form upon shaking in a Vortex mixer at maximal speed, indicating that the association is loose.Abbreviations DTT dithiothreitol - RuBP ribulose 1,5-bisphosphate - RuBisCO ribulose 1,5-bisphosphate carboxylase/oxygenase - MES 2-(N-morpholino) ethane sulfonic acid     

Glycolate formation catalyzed by spinach leaf transketolase utilizing the superoxide radical

A homogeneous preparation of transketolase was obtained from spinach leaf; the specific enzyme activity was 9.5 mumolo of glyceraldehyde-3-P formed (mg of protein)-1 min-1, when xylulose-5-P and ribose-5-P were used as the donor and acceptor, respectively, of the ketol residue. Transketolase catalyzed the formation of glycolate from fructose-6-P coupled with the O2- -generating system of xanthine-xanthine oxidase. The addition of superoxide dismutase (145 units) or 1,2-dihydroxybenzene-3,5-disulfonic acid (Tiron) (5 mM), both O2- scavengers, to the reaction system inhibited glycolate formation 72 and 58%, respectively. The reacton was not inhibited by catalase. Mannitol, an .OH scavenger, and beta-carotene and 1,4-diazobicyclo[2.2.2]octane, 1O2 scavengers, showed little or no inhibitory effects. The rate of glycolate formation catalyzed by the transketolase system was measured in a coupled reaction with a continuous supply of KO2 dissolved in dimethyl sulfoxide, used as an O2- -generating system. The optimum pH of the reaction was above pH 8.5. The second-order rate constant for the reaction between transketolase and O2-, determined by the competition for O2- between nitroblue tetrazolium (NBT) and transketolase, was 1.0 X 10(6) M-1 s-1. Transketolase showed an inhibitory effect on the O2- -dependent reduction of NBT only if the reaction mixture was previously incubated with ketol donors such as fructose-6-P, xylulose-5-P, or glycolaldehyde. The results suggest the possibility that transketolase catalyzes O2- -dependent glycolate formation under increased steady-state levels of O2- in the chloroplast stroma.     

Immunological Studies of Betaine Aldehyde Dehydrogenase in Barley

The changes in the level of the protein for betaine aldehydedehydrogenase, which catalyzes the last step in the synthesisof glycinebetaine, were analyzed with antiserum raised againstSDS-denatured betaine aldehyde dehydrogenase from spinach. Inbarley leaves, the levels of betaine aldehyde dehydrogenaseprotein were found to be enhanced by the addition of 200 mMNaCl to the growth medium. These changes in the level of theenzyme protein corresponded to those in the activity of theenzyme, as described in our previous study (Arakawa et al. 1990).The extent of this enhancement was reduced when barley plantswere relieved from salt stress. An increase in the level ofthe protein was also induced by water stress, such as the withholdingof water or the addition of polyethylene glycol 6000. Betainealdehyde dehydrogenase protein was detected in etiolated leavesand roots, as well as in green leaves. In etiolated leaves,the level of betaine aldehyde dehydrogenase protein was notaffected by salt stress. ¹ This work was supported by a grant from the Bio-Media Projectof the Japanese Ministry of Agriculture, Forestry and Fisheries(BMP92-III-l-1).