Article
Reference
The behaviour of the chloroplast ATPase activity in an apolar media
KERNEN, Peter, et al.
Abstract
Spinach thylakoids were transferred into a reverse micellar phase (detergent CTAB, 1-hexanol and n-octane (CHO)). The macrocomplexes transferred into CHO displayed ion-dependent and methanol-enhanced ATPase activities similar to the membrane-bound enzyme in aqueous buffer. Both latent and methanol-modulated enzyme activities were highly dependent on the amount of water present, e.g. reducing water content from 7.2%, to 3.6%
decreased the specific ATPase activity 3 fold. Decreasing concentrations of water in CHO increased the fraction of bound water as shown by ¹H-Nuclear Magnetic Resonance. However the observed water-dependent ATPase activities did not fully follow the measured activity of water in the low water system, indicating that other regulatory factors are involved.
KERNEN, Peter, et al . The behaviour of the chloroplast ATPase activity in an apolar media.
Experientia , 1993, vol. 49, 25th Annual Meeting of the Swiss Societies for Experimental Biology (USGEB/USSBE), March 25/26, 1993, University of Lausanne-Dorigny, Lausanne, p.
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293
QUANTITATIVE TISSUE DlSTRlBUTJON OF HUMAN PLASMA MEMBRANE CALCIUM PUMP ISOFORMS
Stauffer, T.P., Hilfiker, H., Carafoli, E., Sttehlcr, E.E.•, Institute of Biochemistry, Swiss Federal lnsritutc of Technology (ETH), CH-8092 Zurich, Switzerland, • Dcpanment of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905, U.S.A.
The human plasma membrane calcium A TPase (hPMCA) gene family consists of at least four members; isofonn variability is funher increased by alternative RNA splicing al a number of "hot spots". We have used quantitlldvc polymerase chaii1 reaction (PCR) methodology to assess the disaibution of the human PMCAl, 2, 3 and 4 mRNAs in various tissues.
The results show !hat hPMCAl e.nd 4 correspond to ubiqujtous isofonns whereas the expression of significant amounts of hPMCA2 and 3 is rcslricted to cerebral concx and skele!lll muscle. ln contrast to the relative non-specific pauem of overall hPMCA gene expression, the different alternative spliced sub-forms of each gene c:anscript show a highly tissue.
specific pauem of expression with a predominance of complex splicing options perfonned in the human cerebral concx. Novel alternative spliced human PMCA mRNAs were detected by RT-PCR for isoforms 2, 3 and 4.
A new nomenclanm: for !he precise description of PMCA isoforms has been developed that Ulkes alternative splicing at all "hot spots" imo accounL Supponed by Swiss NF gTants Nos. 31-27103.89 and 31-28772.90
294
CHARACTERIZATIO OF A CALMODULIN-STJMULATED Ca2• TRANSPORT AC'rrVITY JN TONOPLAST VESICLES FROM MAIZE ROOTS
Gavin, 0., Pilet, P.E. and Chanson, A., Institute of Plant Biology and Physiology, Biolop Building, CH-1015 Lausanne
Maize (Zea mays L .. cv· LG 11) root membranes were fractionated by dexttan density gradient cenaifugaiion. Marker enzymes were used 10 smdy the distribution of the different membranes in the gradients and a filtration technique was developed to mea. ure 4~C3l• uanspon in sealed vesicles. Most of the Cal· transport activity was associated with the ER.
However. a small pan or this activity was associated with the 1onoplas1 (corresponding 10 the activiry of !he H"'/Ca2+-antipon) and 10 the plasma membrane. When the e;i2~ trnnspon was measured in the presence of exogenous calmoduHn (I µM), the cnlmodulin-s1imulatcd activity was associated with !he 1.onoplas1 vesicles only. This calmodulin·stimulated Cal+ transpon was insensitive LO monensin, a proton ionophorc, ruling ou1 a direct effect or calmodulin on the H"/Ca2• amipon. We propose that a calmodulin-stimulated
ea2-
·A TPase is associated with the tonoplru;t of young maize root cells. Data will be presented on the characterization of this calmodulin-srimula1ed Ca2+ -transpon activity.295
FUNCTION AND EXPRESSION OF MULTIPLE DRUG RESISTANCE-LIKE GENES IN ARABIDOPSIS THALIANA Siedler M., Ringli. C., •nd Dudler R., Insticut fiir Pnanzenbiologie, Universitiit Zurich, Cl:l-8008 Zurich
The products of multiple drug resis.tance CMDR)-like genes are inicgral membrane proteins that function as energy-dependanl transporters of a diverse array of substrates in many eukaryotes. Best known are the mammalian P-glycoproteio drug transporters which are re-sponsible for the multiple drug resistance phenomenon of many tumor cells. While some P-glycoproteins hnve recently been shown to have dual functions as drug transpor\ers and chloride channels, other related proteins ore.
involved in signal sequence-independent transport of peptide.~ across membranes and heavy metal de1oxifica1ion. To explore the possible function of MOR-like gene products in processes such as herbicide detoxification and signal-molecule 1.ransport in plants, we decided to analyze such genes in Arabid11p:ns 1ha/im1a. The genome of this species appeors to contain a small number of MOR-like genes, two of which we have cloned and analyzcd (atpgpl and atpgp2). We have studied the spacial und tempQrBI expression pattern of a1pgpl in 1ran·sgenlc plants using promoter/reporter gone chimeric construct~. To explore the function Of this gene \YC have constructed transgenic plants
overexpressing either the sense str3nd or the anti·sense $!rand of a1pgp/.
The results of tnese experiments will be presented.
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296
CHARACTERIZATION OF AMINOPHOSPOLIPID TRANSLOCATION IN RED CELL VESICLES
#zs. Belefnay, •A. Zachowski, *P. F. Devaux and
#p, Ott;Inst. Biochem. & Mol. Biol., University of Bern, Bllhlstrasse 28, CH-3012 Bern/Switzerland;
*rnst. Biol. Physico-Chimique., 13 rue
P. M.
Curie, F-75005 Paris/France
A'!'P·dependent ami:nophospholipid translocation was determined by EPR in the membranes of vesicles released from red blood cells. from the initial rates of phospolipid translocation and the concomitant ATP consumption, a stoichiometry of approximately one aminophospholipid molecule tra:nslocated pe!' ATP molecule hydrolyzed could be obtalued. The c.ransloc:at j on was i:nhibitoo by vanadate in
a
concentrat:ion dependent way by up to 95%
for the spi~-labelled phosphatidylserine and 59%
for the phosphatidylethanolamine analogue.Suramin ( 400 µMl also inhibited translocation, but only by 45 '!; o::: 32 '!;, respectively. Since both agents are know~ to be inhibitors of P-type ATPases the results strongly svaaest that the aminophospholipid translocase of the red cell membrane acts vie formation of a phosphoenzyme intermediate.
297
SOLUBILIZATIO . AND RECONSTITUTION OF THE TWO TONOPLAST PROTON PUMPS FROM Rubus lrispidus CELL CULTURE
Perotti, E. and Chanson, A., Institute of Plon1 Biology and Physiology, Biology Building, CH-1015 Lausanne
Two pro1on-translocaling activities are present on lhc tonoplast of higher plam cells: an anion- ensitive ATPasc and a cation-sensitive pyrophosphatase (PPnse). Both pumps generate an electrochemical potential difference 'Of protons. serving as the driving force for several transpon processes (unlport. anrlpon and sympon) across the vacuolar membrane. To enhance our understanding of the molecular properties and tr3.nsport mechanism of these two pumps. it was necessary 10 rcconstilute them inio liposomes and to compare 1he H~-pumping ac1ivh)' with that of native tonoplast vesicles. The A TP· and pyrophosphate- dependent prot0n pumps from 1onoplast·enriched vesicles prcpartd from R11b1.~f hispid1~i cell cuhurts were solubilized in the presence of Trhon X-100 and reconstituted into liposomes of soybean phosphilipids, using Bio-Bc3ds SM·2 10 remove the detergent. The specific activity of the two pumps wa greatly increased by the solubilization-rcconstitution procedure. Identical charnc1eris1ics were found for pyrophosphate·
dependent proton transport in native and reconstituted vesicles.
However, the ATP·dependent proton transport of the reconstituted vesicle. was no longer inhibited by KN03•
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I
THE BEHJ\VIOUR OF THE CHLOROPLAST ATPase AC'rl Vl'r~
IN AN APOLAR MEDIA
Kernan, p,l~ Degli Agos i, R.1, Greppin, H.1, Darszon,, A.~, l and Strasser, R. J .1, Uni versi te de Geneve, Laboratoire. de Bioenergetique, Jussy- Lullier and 2uNAM, lnsti tuto de Biotechnologia, cuernavaca, Mexico
Spinach thylakoids were transferred into a reverse micel.lar phase (detergent CTAB, 1-hexanol and n-octane (CHO) ) . The macrocomple.xes transferred into CHO displayed ion-dependent and methanol-enhanced ATPase activities similar to the membrane-bound enzyme in aqueous buffer. Both latent and methanol-modulated enzyme activities were highly dependent on the amount of water present, e.g. reducing water content from 7.2, to 3.6\ decreased the specific ATPase activity J fold. Decreasing. concentrations of water in CHO increased the frac ion of bound water as shown by lH-Nuclear Magnetic Resonance. However the observed water-dependent ATPase activities did not fully follow the measured activity of water in the l.ow water syste.m, indicating that other regull.ltory factors are involved.
