In vitro analysis of ion channels in periaxolemmal-myelin and white matter clathrin coated vesicles: Modulation by calcium and GTPγS

This study reports the analysis of K⁺ channel activity in bovine periaxolemmal-myelin and white matter-derived clathrin-coated vesicles. Channel activity was evaluated by the fusion of membrane vesicles with phospholipid bilayers formed across a patch-clamp pipette. In periaxolemmal myelin spontaneous K⁺ channels were observed with amplitudes of 25–30, 45–55, and 80–100 pS, all of which exhibited mean open-times of 1–2 msec. The open state probability of the 50 pS channel in periaxolemmal-myelin was increased by 6-methyldihydro-pyran-2-one. Periaxolemmal-myelin K⁺ channel activity was regulated by Ca²⁺. Little or no change in activity was observed when Ca²⁺ was added to thecis side of the bilayer. Addition of 10 M total Ca²⁺ also resulted in little change in K⁺ channel activity. However, at 80 M total Ca²⁺ all K⁺ channel activity was suppressed along with the activation of a 100 pS Cl^– channel. The K⁺ channel activity in periaxolemmal myelin was also regulated through a G-protein. Addition of GTPS to thetrans side of the bilayer resulted in a restriction of activity to the 45–50 pS channel which was present at all holding potentials. Endocytic coated vesicles, form in part through G-protein mediated events; white matter coated vesicles were analyzed for G proteins and for K⁺ channel activity. These vesicles, which previous studies had shown are derived from periaxolemmal domains, were found to be enriched in the subunits of G₀, G_s, and G_i and the low molecular weight G protein,ras. As with periaxolemmal-myelin treated with GTPS, the vesicle membrane exhibited only the 50 pS channel. The channel was active at all holding potentials and had open times of 1–6 msec. Addition of GTPS to the bilayer fused with vesicle membrane appeared to suppress this channel activity at low voltages yet induced a hyperactive state at holding potentials of 45 mV or greater. The vesicle 50 pS K⁺ channel was also activated by the 6-methyl-dihydropyron-2-one (20 M).Abbreviations CNPase 2–3 cyclic nucleotide phosphohydrolase - EDTA ethylenediamine N,N,N,N-tetraacetic acid - G-protein GTP(guanosine triphosphate) binding protein - GTPS guanosine 5-O-(3-thiotriphosphate) - MAG myelin associated glycoprotein - Na⁺ K⁺ ATPase, Na⁺ and K⁺ stimulated adenosine triphosphatase - PLP myelin proteolipid protein Special issue dedicated to Dr. Majorie B. Lees.     

Differential sensitivity of cardiac K(ATP) + channels to guanine nucleotides — evidence for a heterogeneous channel population

In cell-free patches from cultured neonatal rat cardiocytes, the cytosolic presence of GTP--S (100 µmol/l) or GDP-\-S (100 µmol/1) activated K_(ATP) ⁺ channels. GTP--S required cytosolic Mg⁺⁺, suggesting that an activated G-protein causes the increase in open probability. The great variations of the channel response to GTP--S and GDP-\-S indicates that cardiac K_(ATP) ⁺ channels represent a heterogeneous family. Correspondence to: M. Kohlhardt     

NMR structure of the ribosomal protein L23 from Thermus thermophilus

The ribosomal protein L23 is a component of the large ribosomal subunit in which it is located close to the peptide exit tunnel. In this position L23 plays a central role both for protein secretion and folding. We have determined the solution structure of L23 from Thermus thermophilus. Uncomplexed L23 consists of a well-ordered part, with four anti-parallel -strands and three -helices connected as ------, and a large and flexible loop inserted between the third and fourth -strand. The observed topology is distantly related to previously known structures, primarily within the area of RNA biochemistry. A comparison with RNA-complexed crystal structures of L23 from T. thermophilus, Deinococcus radiodurans and Haloarcula marismourtui, shows that the conformation of the well-ordered part is very similar in the uncomplexed and complexed states. However, the flexible loop found in the uncomplexed solution structure forms a rigid extended structure in the complexed crystal structures as it interacts with rRNA and becomes part of the exit tunnel wall. Structural characteristics of importance for the interaction with rRNA and with the ribosomal protein L29, as well as the functional role of L23, are discussed.     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.     

Quantitation of movement of the phosphoryl group during catalytic transfer in the arginine kinase reaction: 31P relaxation measurements on enzyme-bound equilibrium mixtures

³¹P nuclear spin relaxation measurements have been made on enzyme-bound equilibrium mixtures of lobster-muscle arginine kinase in the presence of substituent activating paramagnetic cation Co(II) (in place of Mg(II)), i.e., on samples in which the reaction, ECoATParginine ECoADPP-arginine, is in progress. The results have been analyzed on the basis of a previously published theory (Nageswara Rao, B.D. (1995) J. Magn. Reson., B108, 289–293) to determine the structural changes in the reaction complex accompanying phosphoryl transfer. The analysis enables the determination of the change in the Co(II)-³¹P (-P(ATP)) vector as the transferable phosphoryl group moves over and attaches to arginine to form P-arginine. It is shown that the Co(II)-³¹P distance of 3.0 Å, representing direct coordination of Co(II) to -P(ATP), changes to 4.0 Å when P-arginine is formed in the enzyme-bound reaction complex. This elongation of the Co(II)-³¹P vector implies an excursion of at least 1.0 Å for the itinerant phosphoryl group on the surface of the enzyme.     

Sodium-transport NADH-quinone reductase of a marineVibrio alginolyticus

The respiratory chain of a marine bacterium,Vibrio alginolyticus, required Na⁺ for maximum activity, and the site of Na⁺-dependent activation was localized on the NADH-quinone reductase segment. The Na⁺-dependent NADH-quinone reductase extruded Na⁺ as a direct result of redox reaction. It was composed of three subunits, , , and , with apparentMr of 52, 46, and 32 KDa, respectively. The reduction of ubiquinone-1 to ubiquinol proceeded via ubisemiquinone radicals. The former reaction was catalyzed by the FAD-containing subunit. This reaction showed no specific requirement for Na⁺. For the formation of ubiquinol, the presence of the subunit and the FMN-containing subunit was essential. The latter reaction specifically required Na⁺ for activity and was strongly inhibited by 2-n-heptyl-4-hydroxyquinolineN-oxide. It was assigned to the coupling site for Na⁺ transport. The mode of energy coupling of redox-driven Na⁺ pump was compared with those of decarboxylase- and ATP-driven Na⁺ pumps found in other bacteria.     

Correlation between 15N NMR chemical shifts in proteins and secondary structure

Summary An empirical correlation between the peptide ¹⁵N chemical shift, ¹⁵N_i, and the backbone torsion angles _i, _i–1 is reported. By using two-dimensional shielding surfaces (_i1–1), it is possible in many cases to make reasonably accurate predictions of ¹⁵N chemical shifts for a given structure. On average, the rms error between experiment and prediction is about 3.5 ppm. Results for threonine, valine and isoleucine are worse (4.8 ppm), due presumably to ₁-distribution/-gauche effects. The rms errors for the other amino acids are 3 ppm, for a typical maximal chemical shift range of 15–20 ppm. Thus, there is a significant correlation between ¹⁵N chemical shift and secondary structure.     

In vitro regeneration of evergreen azalea from leaves

Rhododendron simsii Hellmut Vogel was regenerated using different types of explants, auxins and cytokinins. After a callus induction phase, with 2,4-dichlorophenoxyacetic acid or -naphthaleneacetic acid, adventitious shoot regeneration was obtained on a medium supplemented with thidiazuron or zeatin. With thidiazuron shoots were small and a subsequent elongation step was required before rooting. An elongation step was not required when zeatin was used. The duration of the callus induction phase was negatively correlated with the regeneration capacity.Abbreviations 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - 2iP 6-(--dimethylallylamino)purine - NOA ß-naphthoxyacetic acid - BA 6-benzyladenine - IBA 1-H-indole-3-butyric acid - IAA 1-H-indole-3-acetic acid - TDZ thidiazuron - WPM woody plant medium     

Selection of isoresponsive genotypes in toria (Brassica campestris L.) based on the pattern of response to environmental variations: a proposed method

Summary Thirty-one toria genotypes were compared with three well-established cultivars, Ludhiana Composite-2, K-1 and TCSU-2 (standard testers). The genotypes, which were almost identical to a standard tester in response to environmental variations and which also had other desirable characteristics, were considered to be acceptable for commercial cultivation. Using this criterion, TCSU-7, TH-5 and TH-4 were found to be acceptable for commercial cultivation. TH-4 and TCSU-7 were found superior to TH-5 if r² can be considered as a measure of the agronomical manipulations expected in environmental variations.     

Synthesis of chemically modified sialic acid-containing sialyl-LeX ganglioside analogues recognized by the selectin family

Sialyl Lewis X ganglioside analogues containing 5-acetamido-3,5-dideoxy-l-arabino-2-heptulopyranosylonic acid (C7-Neu5Ac), 5-acetamido-3,5-dideoxy-d-galacto-2-octulopyranosylonic acid (C8-Neu5Ac), and 5-acetamido-3,5-dideoxy-l-glycero-d-galacto-1-2-nonulopyranosylonic acid (8-epi-Neu5Ac) in place ofN-acetylneuraminic acid (Neu5Ac) have been synthesized. Glycosylation of 2-(trimethylsilyl)ethyl 6-O-benzoyl--d-galactopyranoside with the phenyl or methyl 2-thioglycoside derivatives of the respective sialic acids, usingN-iodosuccinimide (NIS)-trifluoromethanesulfonic acid as a promoter in acetonitrile, gave the three required 2-(trimethylsilyl)ethyl (2S)-sialyl-(2 3)--galactopyranosides. These were converted viaO-benzoylation, selective transformation of the 2-(trimethylsilyl)ethyl group to acetyl, and introduction of the methylthio group with methylthiotrimethylsilane into the corresponding glycosyl donors. Glycosylation of 2-(trimethylsilyl)ethylO-(2,3,4-tri-O-benzyl--l-fucopyranosyl)-(1 3)-O-(2-acetamido-6-O-benzyl-2-deoxy--d-glucopyranosyl)-(1 3)-2,4,6-tri-O-benzyl--d-galactopyranoside with these donors in the presence of dimethyl(methylthio)sulfonium triflate (DMTST) afforded the expected -glycosides, which were converted into the corresponding -trichloroacetimidates, and these, on coupling with (2S, 3R, 4E)-2-azido-3-O-benzoyl-4-octadecene-1,3-diol, gave the required -glycosides. Finally, these were transformed via selective reduction of the azide group, condensation with octadecanoic acid,O-deacylation, and de-esterification into the target compounds in good yields.     

A 500-MHz1H-NMR study on theN-linked carbohydrate chain of bromelain

The 500-MHz¹H-NMR characteristics of theN-linked carbohydrate chain Man1-6[Xyl1-2]Man1-4GlcNAc1-4[Fuc1-3]GlcNAc1-NAsn of the proteolytic enzyme bromelain (EC 3.4.22.4) from pineapple stem were determined for the oligosaccharide-alditol and the glycopeptide, obtained by hydrazinolysis and Pronase digestion, respectively. The¹H-NMR structural-reporter-groups of the (1–3)-linked fucose residue form unique sets of data for the alditol as well as for the glycopeptide.     

Preparation of a series of sulfated tetrasaccharides from shark cartilage chondroitin sulfate D using testicular hyaluronidase and structure determination by 500 MHz1H NMR spectroscopy

Six tetrasaccharide fractions were isolated from shark cartilage chondroitin sulfate D by gel filtration chromatography followed by HPLC on an amine-bound silica column after exhaustive digestion with testicular hyaluronidase. Their structures were determined unambiguously by one- and two-dimensional 500 MHz¹H NMR spectroscopy in conjunction with HPLC analysis of chondroitinase AC-II digests of the tetrasaccharides. One fraction was found to contain two tetrasaccharide components. All the seven tetrasaccharides shared the common core structure GlcA1-3GalNAc1-4GlcA1-3GalNAc with various sulfation profiles. Four were disulfated comprising of two monosulfated disaccharide units GlcA1-3GalNAc(4-sulfate) and/or GlcA1-3GalNAc(6-sulfate), whereas the other three were hitherto unreported trisulfated tetrasaccharides containing a disulfated disaccharide unit GlcA(2-sulfate)1-3GalNAc(6-sulfate) and a monosulfated disaccharide unit GlcA1-3GalNAc(4-or 6-sulfate). These sulfated tetrasaccharides were demonstrated to serve as appropriate acceptor substrates for serum -N-acetylgalactosaminyltransferase, indicating their usefulness as authentic oligosaccharide substrates or probes for the glycobiology of sulfated glycosaminoglycans.Abbreviations NFU National formulary unit - COSY correlation spectroscopy - HOHAHA homonuclear Hartmann-Hahn - 1D or 2D one- or two-dimensional - IdoA l-iduronic acid - GlcA d-gluco-4-enepyranosyluronic acid - Di-0S GlcA1-3GalNAc - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-4S GlcA1-3GalNAc(4-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-6S GlcA1-3GalNAc(6-sulfate) - Di-diS _d GlcA(2-sulfate)1-3GalNAc(6-sulfate) - Di-diS_E GlcA1-3GalNAc(4, 6-disulfate) - U G, U, 2S, 4S, and 6S represent GlcA, GalNAc, GlcA, 2-O-sulfate, 4-O-sulfate, and 6-O-sulfate, respectively     

The α/β Interfaces of α1β1, α3β3, and F1: Domain Motions and Elastic Energy Stored during γ Rotation

ATP synthase (F_oF₁) consists of F₁ (ATP-driven motor) and F_o (H⁺-driven motor). F₁ is a complex of ₃₃ subunits, and is the rotating cam in ₃₃. Thermophilic F₁ (TF₁) is exceptional in that it can be crystallized as a monomer and an ₃₃ oligomer, and it is sufficiently stable to allow refolding and reassembly of hybrid complexes containing 1, 2, and 3 modified or . The nucleotide-dependent open–close conversion of conformation is an inherent property of an isolated and energy and signals are transferred through / interfaces. The catalytic and noncatalytic interfaces of both mitochondrial F₁ (MF₁) and TF₁ were analyzed by an atom search within the limits of 0.40 nm across the interfaces. Seven (plus thermophilic loop in TF₁) contact areas are located at both the catalytic and noncatalytic interfaces on the open form. The number of contact areas on closed increased to 11 and 9, respectively, in the catalytic and noncatalytic interfaces. The interfaces in the barrel domain are immobile. The torsional elastic strain applied through the mobile areas is concentrated in hinge residues and the P-loop in . The notion of elastic energy in F_oF₁ has been revised. X-ray crystallography of F₁ is a static snap shot of one state and the elastic hypotheses are still inconsistent with the structure, dyamics, and kinetics of F_oF₁. The domain motion and elastic energy in F_oF₁ will be elucidated by time-resolved crystallography.     

The use of BRM-activated killer cells in adoptive immunotherapy: A pilot study with nine advanced cancer patients

Adoptive immunotherapy using MHC-nonrestricted-lymphocytes, peripheral blood T cells and NK cells was devised. Peripheral blood mononuclear cells (3 x 107) were selected by immobilization to anti-CD3 monoclonal antibody for 4 days and cultured for 2 weeks in the presence of IL-2. Thereafter they were reactivated by 500 U/ml of IFN- and 1000 U/ml of IL-2 for 1 hour. Enhancement of NK and LAK activities was confirmed. Peripheral blood T cells proliferated in response to immobilized anti-CD3 antibody (3% to 30%). Approximately 6 x 109 BRM-activated killer (BAK) cells composed of CD56+ T cells and CD56+ NK cells, were dispensed to cancer patients via intravenous drip infusion. Nine patients were treated with BAK cells every 2 weeks or every month on an outpatient basis. During the course of adoptive immunotherapy, the crossed affinity immunoelectrophoresis (CAIE) pattern of serum immunosuppressive acidic protein (IAP) was analysed. Both the production and glycosylation pattern of IAP is changed in response to tumor enlargement and may therefore act as a marker of the disease progression. During the course of BAK therapy, the glycosylation IAP pattern of 6 patients changed from tumor (T) to normal (N). In addition, the performance status of all patients was maintained at 90–100% of the Karnofsky scale and any side effects including fever were not observed during treatments with BAK cells. Moreover, the overall quality of life (QOL) of the patients, scored at the Face scale was favorable. In addition, blood levels of activated T cells producing IFN- were assayed as an indication marker of BAK therapy. The normal range of IFN- producing T cells comprised 6.9 ± 0.9% of peripheral blood mononuclear cells (PBMC), according to a single cell FACScan analyses of PBMCs derived from normal individuals. IFN- producing T cells of Patients No. 8 and 9, who received extensive chemotherapy before initiation of BAK therapy, comprised only 0.2% and 2% of PBMC, respectively. These patients died 3 and 6 months after beginning BAK therapy. Peripheral blood T cells of Patients Nos. 1–7 proliferated in response to immobilized anti-CD3 antibody and the frequency of IFN- producing T cells in PBMC preparation of these patients were over 3% before initiation of BAK therapy. Since our data show a positive correlation between survival time and initial T cell counts, a low frequency of these cells may contraindicate BAK therapy.     

The frequency of the γ chain variant AγT in different populations,and its use in evaluating γ gene expression in association with thalassemia

Summary The occurrence of the ^AT chain (i.e. ^A75 Ile Thr) in different populations was evaluated through a study of 4250 cord blood samples and blood samples from more than 350 SS¹ patients. High frequencies were observed in Italy, Yugoslavia, Turkey, Holland, but also in Japan, Vietnam, and India. The chain is (nearly) absent in the Black population of Ghana and Kenya, and low frequencies were observed in China and Australian aborigines. Only a few adult SS patients (18 out of 357) were ^AT heterozygotes. The chromosomes with the ^AT globin gene were mapped through an evaluation of the presence of 10 different restriction sites. The ^AT chromosomes from different populations were closely related and had the same subhaplotypes of [--++-+] (Hinc II 5 to ; Xmn I 5 to ^G; Hind III in ^G and ^A; Hinc II in and 3 to ), quite different from the subhaplotypes seen for ^AT negative chromosomes.² This suggests a common ancestor which may have originated in Southern Europe. An evaluation of the chain production by both chromosomes in SS patients and -thalassemia heterozygotes was possible for subjects with an ^AT heterozygosity. It was concluded that in -thalassemia trait, the chain synthesis is directed for about two-thirds by the thalassemic chromosome and for about onethird by the normal chromosome; the contribution by the normal chromosome decreases with a decrease in total chain production.This is contribution #0890 of the Department of Cell and Molecular Biology, Medical College of Georgia, Augusta, GA 30912, USA     

Sparse distribution of γ/δ T lymphocytes around human epithelial tumors predominantly infiltrated by primed/memory T cells

Summary Evidence from the mouse system has suggested that T lymphocytes accumulating in non-lymphoid tissue, in particular epithelia, may preferentially express the T cell receptor (TCR) . In this study, we characterize the T cell receptor or phenotype of lymphocytes infiltrating human tumors of epithelial origin using monoclonal antibodies (mAb) for immunohistology and flow cytometry on cells extracted by enzyme digestion. This report shows that the majority of CD3⁺ tumor-infiltrating lymphocytes are TCR ⁺ but a small percentage of TCR can be clearly defined scattered throughout the tumor tissue with apparently no microanatomical selection. So far there has been little evidence for an accumulation of activated T cells in human tumor tissues as defined by mAb against molecules appearing transiently during the acute phase of activation. Now mAb are available that can identify primed or memory T cells such as mAb UCHL-1 recognizing the CD45RO antigen. Here we show that CD3⁺ tumor-infiltrating lymphocytes have a statistically significant accumulation of primed T cells, as compared to the autologous peripheral blood lymphocytes, suggesting their immune stimulation by tumor cells.     

Purification and characterization of (1→3, 1→4)-β-glucan endohydrolases from germinated wheat (Triticum aestivum)

A (13, 14)--glucan 4-glucanohydrolase [(13, 14)--glucanase, EC 3.2.1.73] was purified to homogeneity from extracts of germinated wheat grain. The enzyme, which was identified as an endohydrolase on the basis of oligosaccharide products released from a (13, 14)--glucan substrate, has an apparent pI of 8.2 and an apparent molecular mass of 30 kDa. Western blot analyses with specific monoclonal antibodies indicated that the enzyme is related to (13, 14)--glucanase isoenzyme EI from barley. The complete primary structure of the wheat (13, 14)--glucanase has been deduced from nucleotide sequence analysis of cDNAs isolated from a library prepared using poly(A)⁺ RNA from gibberellic acid-treated wheat aleurone layers. One cDNA, designated LW2, is 1426 nucleotide pairs in length and encodes a 306 amino acid enzyme, together with a NH₂-terminal signal peptide of 28 amino acid residues. The mature polypeptide encoded by this cDNA has a molecular mass of 32085 and a predicted pI of 8.1. The other cDNA, designated LW1, carries a 109 nucleotide pair sequence at its 5 end that is characteristic of plant introns and therefore appears to have been synthesized from an incompletely processed mRNA. Comparison of the coding and 3-untranslated regions of the two cDNAs reveals 31 nucleotide substitutions, but none of these result in amino acid substitutions. Thus, the cDNAs encode enzymes with identical primary structures, but their corresponding mRNAs may have originated from homeologous chromosomes in the hexaploid wheat genome.     

Assignment of aliphatic side-chain 1HN/15N resonances in perdeuterated proteins

Summary The perdeuteration of aliphatic sites in large proteins has been shown to greatly facilitate the process of sequential backbone and side-chain ¹³C assignments and has also been utilized in obtaining long-range NOE distance restraints for structure calculations. To obtain the maximum information from a 4D ¹⁵N/¹⁵N-separated NOESY, as many main-chain and side-chain ¹H_N/¹⁵N resonances as possible must be assigned. Traditionally, only backbone amide ¹H_N/¹⁵N resonances are assigned by correlation experiments, whereas slowly exchanging side-chain amide, amino, and guanidino protons are assigned by NOEs to side-chain aliphatic protons. In a perdeuterated protein, however, there is a minimal number of such protons. We have therefore developed several gradient-enhanced and sensitivity-enhanced pulse sequences, containing water-flipback pulses, to provide through-bond correlations of the aliphatic side-chain ¹H_N/¹⁵N resonances to side-chain ¹³C resonances with high sensitivity: NH₂-filtered 2D ¹H-¹⁵N HSQC (H₂N-HSQC), 3D H₂N(CO)C_/ and 3D H₂N(COC_/)C_/ for glutamine and asparagine side-chain amide groups; 2D refocused H(N_/)C_/ and H(N_/C_/)C_/ for arginine side-chain amino groups and non-refocused versions for lysine side-chain amino groups; and 2D refocused H(N)C and nonrefocused H(N_.)C for arginine side-chain guanidino groups. These pulse sequences have been applied to perdeuterated ¹³C-/¹⁵N-labeled human carbonic anhydrase II (²H-HCA II). Because more than 95% of all side-chain ¹³C resonances in ²H-HCA II have already been assigned with the C(CC)(CO)NH experiment, the assignment of the side-chain ¹H_N/¹⁵N resonances has been straightforward using the pulse sequences mentioned above. The importance of assigning these side-chain H_N protons has been demonstrated by recent studies in which the calculation of protein global folds was simulated using only ¹H_N-¹H_N NOE restraints. In these studies, the inclusion of NOE restraints to side-chain H_N protons significantly improved the quality of the global fold that could be determined for a perdeuterated protein [R.A. Venters et al. (1995) J. Am. Chem. Soc., 117, 9592–9593].To whom correspondence should be addressed.     

Malate regulation of Mg2+-ATPase of chloroplast coupling factor 1

The regulatory effects of malate on chloroplast Mg²⁺-ATPase were investigated and the mechanism was discussed. Malate stimulated methanol-activated membrane-bound and isolated CF₁ Mg²⁺-ATPase activity. The subunit of CF₁ may be involved in malate regulation of the enzyme function. Modification of subunit at one site of the peptide by NEM may affect malate stimulation of ATPase while at another site may have no effect. The effect of malate on the Mg²⁺-ATPase was also controlled by the Mg²⁺/ATP ratio in the reaction medium. The enhancing effect of malate on Mg²⁺-ATPase activity depended on the presence of high concentrations of Mg²⁺ in the reaction mixture. Kinetic study showed that malate raised the V_max of catalysis without affecting the K_m for Mg²⁺ ATP. The experiments imply that the stimulation of Mg²⁺-ATPase by malate is probably correlated with the P_i binding site on the enzyme. The regulation of ATPase activity by malate in chloroplasts may be relevant to its function in vivo.Abbreviations CF₁ chloroplast coupling factor 1 - CF₁ (-) and CF₁ (-) CF₁ deficient in the and subunit - MF₁ mitochondria coupling factor 1 - NEM N-ethylmaleimide - PMS phenazine methosulfate - OG n-octyl--d-glucopyranoside     

Expression of the Arabidopsis G-protein GPα1: purification and characterisation of the recombinant protein

The Arabidopsis G subunit, GP1, was expressedwithin Escherichia coli by co-transformation with the expressionvector and the dnaY gene which encodes tRNA^Arg _AGA/AGG. Isolation of the recombinant GP1 in a highly pureform could be achieved by a combination of anion exchange and dyeaffinity chromatography or by a single step affinity procedure viachromatography on 4-amino-anilido-GTP agarose. The recombinant proteinyielded by both procedures was highly active and bound GTPS withan apparent K_d in the nM range. GTPS binding wasstimulated two-fold in the presence of Zn²⁺ compared with that inthe presence of Mg²⁺, Mn²⁺ or Ca²⁺.Abbreviations: 4aaGTP, 4-amino-anilido-GTP; GTPS,guanosine- 5-(3-O-thiotriphosphate), PMSF,phenylmethylsulphonyl fluoride; PVDF, polvinylidene fluoride;rGP1, recombinant GP1