A390 zyxwvutsrqpo zyxwvu zyxwvutsrqpo zyxwvutsrqpo zyxwvutsrqp Biochemical Society Transactions (2000) Volume 28, Part 5 zyxwvu zyxwvutsrqpo 400 Sucrose Uptake in Leaves of Cassava Manihot esculentacrantz 1402Calcium Binding and Translocation b y VDAC: a possible 3;Eksittiku11,T.Limpaseni1 and M.ChulavatnatoI* 1 Department of Biochemistry,Faculty of Science,Chulalongkorn University,Phayathai Road,Bangkok 10330 Thailand. 2 Department of Biochemistry, Faculty of Science, Mahidol University,Rama 6 Road, Bangkok 10400,Thailand. Sucrose is a major translocated form of carbon assimilates in most plant species.It has been shown to be actively transported across membrane by transport protein to heterotrophic organs for its growth development and storage.In cassava,sucrose is transported from leaves and stored as starch in the roots.In this study,cassava leaf disc,l2 mm.in diameteswere incubated in the medium containing I4Csucrose.1t was found that the sucrose uptake was linear with time and exhibited Michaelis-Menten characteristics with K, 1.31 mM and regulatory mechanism in mitochondrial function. D. GinceL H. Zaid, V. Shoshan-Barmatz Department of L;fe Sciences Ben-Gurion University, Beer- Sheva, Israel. gincel@bgurnail. bgu.ac.il Mitochondria play a central role in energy production, Ca2+ signaling, aging and cell death. To control cytosolic or mitochondrial calcium concentration, mitochondria possess several influx and efflux Ca2+ transport systems in the inner membrane. However, the pathway for Ca2+ crossing the outer mitochondrial membrane has yet n o t been identified. Our results indicate that the Voltage-Dependent Anion Channel (VDAC), an outer mitochondrial membrane protein, provides that pathway. V D A C is a large channel that transports anions, cations, ATP and other metabolites. In this study w e show that: (i) Purified V D A C reconstituted into a planar lipid bilayer or lipo- zyxwvuts Vmax of 3.7nmole/hour/cm2 indicating that sucrose transport in cassava leaves was carrier mediated.The sucrose uptake was inhibited by N-ethylmaleimide,p-chloromercuribenzenesulfonic acid and iodoacetic acid,suggesting involvement of thiol groups in the pr0cess.h was strongly inhibited by dinitropheno1,carbonyl cyanide m-chlorophenylhydrazone, vanadate,erythrosin B and KCN,suggesting that the uptake involved proton transport and was energy dependent. Cassava is a cyanophoric plant.All cassava tissues,with the exception of seeds,contain the cyanogenic glycosides linamarin(>93% total IO%total cyanogen). They were syncyanogen) and lotaustralin (i thesized in leaves.When effect of cyanogenic glycosides on sucrose uptake was studied,it was found that linamarin showed a prominent inhibition on the process.In presence of linamarin,sucrose transport was significantly reduced.Other mono and disaccharides tested showed little effect.It appears that cyanogenic glycosides may regulate the sucrose transport in cassava. Supported by National Science and Technology Development Agency somes is highly permeable t o Ca2+; (ii) VDAC contains Ca2+ binding sites; (iii) La3+ and the polycationic d y e ruthenium red completely close the V D A C channel and inhibit Ca2+ transport in isolated mitochondria. V D A C permeability t o Ca2+ and binding of Ca2+ provide the answers to how the inner membrane located Ca2+ transport systems sense cytosolic Ca2+. V D A C is a component of the mitochondrial Permeabilite Transition Pore (PTP), a large, high conductence, non-specific channel spanning both the inner and outer mitochondrial membranes. We suggest that VDAC as a Ca2+ binding protein has a role in the regulation of the PTP activity, and in intracellular Ca2+ signaling. ,401 Leucine uptake into membrane vesicles from midge larvae. A. Pugliese, M. F o r c e h F. Lissoni, R. Giacchini, P.Parenti, G.M. Hanozet Department of Environmental Sciences, Pzza della Scienza 1 , 20126 Milano Italy Membrane vescicles obtained from the brush border membranes of the midge larvae Chironomus riparzus (Insecta, Diptera) were used to study the patways for neutral amino acid transport. Larvae were collected in the wild and then either maintained in a cold room or stored in liquid nitrogen without significant changing their physiological properties. Larval size ranged between 4 to 10 mm. Vesicles were purified by homogenisation in hypotonic Hepes-Tris buffer, two precipitations with 12 mM MgCl2 and differential centrifugation as described by Biber et al. (Biochim. Biophys. Acta1981, v. 647, p.169). The resulting preparation was 16-fold enriched of leucine aminopeptidase and alkaline phosphatase, not enriched of enzime markers from basolateral membranes and free of contaminant of mitochondrial membranes. For the high activities of typical brush border marker enzymes our preparation was judged consisting mainly of midgut luminal membranes. Membranes exhibited pH-dependent, Na +/gradient coupled transport phenomenon for L-leucine. Initial rate or leucine uptake at varying leucine concentration was a function of a saturable component plus a linear one. Fitting the former with the Michaelis-Menten equation gave a high affinity, low capacity transport system (Km = 0.01 mM and Vmax = 400 pmoli/ 1Os/ mg protein). Leucine uptake was strongly inhibited by all neutral amino acid, acid (substrate for the including the 2-amino-2-norbornanecarboxylic mammalian leucine transport system) and by some polar amino acid, such as serine and histidine. For this inhibition pattern the system much resembles the broad-scope high affinity, high capacity, K+dependent neutral amino acid transport system from lepidopteran larvae. This represents the first report on amino acid uptake in dipteran larvae. 0 2000 Biochemical Society ,404 Metabotropic Glutamate receptors expressed in bone cells Y.G u and S. Publicover School of Biosciences, University of Birmingham, Birmingham Blli 2TT Glutamate is an important neurotransmitter in the CNS. O n the Basis of pharmacological, electrophysiological and biochemical studies, glutamate receptors can be categorized into t w o groups termed ionotropic and metabotropic. RT-PCR, antibody labeling and electrophysiological investigations have demonstrated expression of N M D A receptors in rat osbeoblasts, MG63 cells and rat osteoclasts. Recent evidence suggests that bones may receive glutamatergic innervation. We have found discrepancies between the electrophysiological effects, o n osteoblasts, of glutamate and N M D A , suggesting that other (possibly metabotropic) glutamate receptors may be present in these cells. Using RT-PCR we have detected expression of metabotropic glutamate receptor subunits in rat femoral cells in primary Culture. Osteoblasts express m R N A for mGluRlb (but not mGluRla), but no mGluR2, mGluR4 and mGluR6, whereas in marrow stromal cells we detected mGluR6 and (apparently) low levels of mGluRl a and b, but n o mGluR2 or mGluR4 . Further more, we observed glutamate induced Nitric Oxide reduction in marrow cells which expressed mGluR6. This effect would be blocked by P K C inhibitor. These finding support a signaling role for glutamate in bone, possibly associated with cell differentiation.