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51.
The structural polarity of statocytes of Lepidium sativum L. is converted to a physical stratification by a root-tip-directed centrifugal acceleration. Sedimentation of amyloplasts and nucleus to the centrifugal (distal) cell pole and the lateral displacement of the distal endoplasmic reticulum (ER) complex occur after centrifugation for 20 min at an acceleration of 50 g. With higher doses (20 min, 100-2,000 g), smaller organelles become increasingly displaced. From the centrifugal to the centripetal cell pole, the following stratification is observed: 1) amyloplasts with mitochondria; 2) nucleus with mitochondria and a few dictyosomes, as well as laterally located ER; 3) dictyosomes with a few mitochondria; 4) vacuoles; and 5) lipid droplets. Within the first 7.5 min, after the roots have been returned to 1 g, the original arrangement of the amyloplasts sedimented on the underlying ER complex is reestablished in 66% of the statocytes. When roots previously centrifuged in an apical direction are exposed in a horizontal position to 1 g, the latent period of the graviresponse is increased by 7.5 min relative to the non-centrifuged controls. The kinetics of the response are identical to the controls. Roots centrifuged first in an apical direction and then for 2 h in a lateral direction (1,000 g) have statocytes with a physical stratification perpendicular to the root axis. A gravitropic curvature does not take place during the lateral centrifugation. These results support the hypothesis that the distal ER complex is necessary and sufficient for graviperception. 相似文献
52.
Brain tubulin was labeled in vitro by post-translational incorporation of [14C]-tyrosine or in vivo by intra-cranial injection of [3H]-leucine. The labeled protein was purified by ion-exchange chromatography. After incubating at 37 degrees C with a microsomal membrane preparation from rat brain, part of the labeled soluble tubulin became sedimentable at high-speed centrifugation. This was independent of the native configuration of tubulin, the state of tyrosination of the COOH-terminus, or the presence of 100 microM colchicine in the mixture. In addition, the double-labeled tubulin-colchicine complex obtained from the binding of [3H]-colchicine to [14C]-tyrosinated tubulin, bound to the membrane preparation to the same extent as [14C]-tyrosinated tubulin. The data show that either tubulin or the complex resulting from its binding to colchicine distributed between the soluble and the membrane fractions when mixed at 37 degrees C with a microsome preparation. Seemingly, the site for colchicine binding to tubulin needs not to be free for the protein-membrane association. 相似文献
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56.
Andreas Faissner Jan Kruse Klaus Kühn Melitta Schachner 《Journal of neurochemistry》1990,54(3):1004-1015
The J1 glycoproteins can be obtained in multiple forms in the soluble fraction of developing and adult mouse brain tissue. They are recovered as two forms of apparent molecular weights of 160,000 and 180,000 (J1-160) from adult mouse brain and as forms of apparent molecular weights of 200,000 and 220,000 (J1-220) from developing brain. J1-160 and J1-220 share common epitopes but are considered as separate entities, with J1-220 being immunochemically closely related if not identical to tenascin. Based on the observation that J1 immunoreactivity appears on basement membrane and interstitial collagens after denervation of the neuromuscular junction in adult rodents, we became interested in investigating the binding properties of J1 glycoproteins to extracellular matrix constituents in vitro. Both J1-160 and J1-220 bound to collagens type I-VI and IX but not to laminin, fibronectin, bovine serum albumin, or gelatin under hypotonic buffer conditions. Under isotonic buffer conditions, J1-220 bound to all collagen types, whereas J1-160 bound only to collagen types V and VI with values that could be examined by Scatchard analysis. Binding of J1-220 to collagens displayed two binding constants (KD) between 1.5 and 4.4 X 10(-9) and 1.8 and 5.5 X 10(-8) M, respectively, under hypotonic buffer conditions and a single KD of 2.1-8.0 X 10(-8) M under isotonic buffer conditions. Binding of J1-160 to collagens had an apparent KD of 1.9-8.0 X 10(-9) M under hypotonic buffer conditions. Under isotonic buffer conditions, binding constants of J1-160 to collagen types V and VI were approximately 2 X 10(-8) M. Binding of J1-220 to collagen type I could be inhibited by J1-220, J1-160, and collagen type VI but not by fibronectin or gelatin. Conversely, binding of J1-160 was inhibited by J1-220, J1-160, and collagen type VI (in order of decreasing efficacy of competition). J1-160 and J1-220 were retained on a heparin-agarose column and eluted in a salt gradient at approximately 0.5 M NaCl. The formation of the J1-heparin complexes was inhibited 100-fold more efficiently by heparin than by chondroitin sulfate. These experiments show that J1 glycoproteins resemble in many respects the extracellular matrix constituents fibronectin, laminin, vitronectin, and von Willebrand factor. 相似文献
57.
Andreas Schmid Gerhard Burckhardt Heinz Gögelein 《The Journal of membrane biology》1989,111(3):265-275
Summary Endocytotic vesicles from rat kidney cortex, isolated by differential centrifugation and enriched on a Percoll gradient, contain both an electrogenic H+ translocation system and a conductive chloride pathway. Using the dehydration/rehydration method, we fused vesicles of enriched endosomal vesicle preparations and thereby made them accessible to the patch-clamp technique. In the fused vesicles, we observed Cl– channels with a single-channel conductance of 73±2 pS in symmetrical 140mm KCl solution (n=25). The current-voltage relationship was linear in the range of –60 to +80 mV, but channel kinetic properties dependended on the clamp potential. At positive potentials, two sublevels of conductance were discernible and the mean open time of the channel was 10–15 msec. At negative voltages, only one substate could be resolved and the mean open time decreased to 2–6 msec. Clamp voltages more negative than –50 mV caused reversible channel inactivation. The channel was selective for anions over cations. Ion substitution experiments revealed an anion permeability sequence of Cl–=Br–=I–>SO
4
2–
F–. Gluconate, methanesulfonate and cyclamate were impermeable. The anion channel blockers 4,4-diisothiocyanatostilbene-2,2-disulfonic acid (DIDS, 1.0mm) and 5-nitro-2-(3-phenylpropylamino)-benzoic acid (NPPB, 0.1mm) totally inhibited channel activity. Comparisons with data obtained from radiolabeled Cl–-flux measurements and studies on the H+ pump activity in endocytotic vesicle suspensions suggest that the channel described here is involved in maintenance of electroneutrality during ATP-driven H+ uptake into the endosomes. 相似文献
58.
Creatine Transport in Cultured Cells of Rat and Mouse Brain 总被引:7,自引:3,他引:4
Astroglia-rich cultures derived from brains of newborn rats or mice use a transport system for the uptake of creatine. The uptake system is saturable, Na+-dependent, and highly specific for creatine and Na+. Kinetic studies on rat cells revealed a Km value for creatine of 45 microM, a Vmax of 17 nmol x h-1 x (mg of protein)-1, and a Km value of 55 mM for Na+. The carrier is competitively inhibited by guanidinopropionate (Ki = 15 microM). No such transport system was found in neuron-rich primary cultures from embryonic rat brain. It is hypothesized that creatine transport is an astroglial rather than a neuronal function. 相似文献
59.
60.
Andreas Pries Alexander Steinbüchel Hans G. Schlegel 《Applied microbiology and biotechnology》1990,33(4):410-417
Summary Transposon Tn 951-encoded -galactosidase was expressed in Pseudomonas saccharophila and enabled this bacterium to grow on lactose as sole carbon source. In contrast, -galactosidase was not expressed in Alcaligenes eutrophus even if the lacZ gene of Tn 951 was separated from the lacI gene. However, -galactosidase was expressed in A. eutrophus, if a DNA fragment, which was suspected to harbour the promoter of the A. eutrophus poly(3-hydroxybutyric acid)-synthetic genes, was ligated to the promoter probe vector pMC1403, which employs lac Z, Y as reporter genes. Plasmid pPL76, which harboured one of the promoter-lac fusions, enabled A. eutrophus not only to express -galactosidase but also to grow slowly on lactose (doubling time = 25–30 h). Subsequently, the promoter-lac fusion was ligated to Tn5 in pSUP5011 and was inserted into the genome of A. eutrophus H16 and of the glucose-utilizing mutant H16-G+1 by applying the suicide plasmid technique. Two recombinant strains, H16-cPL and H16-G+1-cPL, which grow with a doubling time of 16–23 h on lactose, were investigated in detail. The cells only utilized the glucose residue of lactose as a carbon source for grouth and excreted galactose into the medium. Only after the Escherichia coli gal operon had been cloned in vector pVK101 and had been mobilized to H16-cPL or H16-G+1-cPL, was lactose completely utilized; no galactose was detected in the medium and the growth yields increased twofold. Depending on the orientation of the gal operon in pVK101, the expression of galactokinase seems to be dependent either on the promoter of aminoglycoside phosphotransferase gene (kan) or on the promoter of the tetR gene.
Offprint requests to: A. Steinbüchel 相似文献