Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.6.3.14 (ATP synthase)
 7,042 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A technique for selecting mutants of Escherichia coli in which the proton-translocating sector of the adenosine triphosphatase (ATPase) complex has been inactivated is reported. The procedure uses a strain of E. coli (NR-70) lacking the extrinsic (F1) sector of the ATPase complex and which in consequently permeable to protons (B. P. Rosen, J. Bacteriol. 116:1124--1129, 1973). After growing strain NR-70 under noninducing conditions for the lac operon, cells were mutagenized and plated on minimal medium containing low concentrations of lactose. Several mutants of strain NR-70 were isolated as large colonies on these plates, apparently because they could concentrate lactose more efficiently. A description of one of the mutants, strain KW-1, is reported here. The most distinguishing difference in growth properties of the two strains was that, when transferred to medium containing low concentrations of lactose, strain KW-1 induced the lac operon with a shorter lag time than strain NR-70. The mutation in strain KW-1 leading to more rapid growth on lactose was cotransducible with the asn and unc loci, at 83 min on the E. coli genetic map. Intact cells of strain KW-1 actively transported L-proline as well as did wild-type cells, whereas cells of strain NR-70 were markedly deficient in L-proline transport. The improvement in the transport capacity of strain KW-1 correlated with a marked decrease in proton permeability relative to that of strain NR-70. Based on an acid-base pulse technique that measured the proton conductance of the membranes of intact cells, strain NR-70 was at least 10 times more permeable to protons than was the wild type, whereas strain KW-1 was only 2 times more permeable. The transport properties and proton conductance were also compared with membrane vesicles prepared by osmotic shock. With either D-lactate or ascorbate-N-methylphenazonium methosulfate as respiratory substrates, vesicles of strain KW-1 transported L-proline much more rapidly than did vesicles of strain NR-70, but still at rates less rapid than those of the wild type. The passive proton conductance of the membrane vesicles was quantitated by measuring the rate of H+ influx into vesicles in response to a valinomycin-generated K+ diffusion potential. The proton permeability of vesicles of strain KW-1 was reduced 1.5-fold relative to vesicles of strain NR-70, but these vesicles were still four times more permeable to protons than was the wild type. Vesicles of strain KW-1 corresponded to wild-type vesicles treated with 0.5 micrometer carbonylcyanide m-chlorophenylhydrazone (CCCP) and vesicles of strain NR-70 corresponded to wild-type vesicles treated with 1.4 micrometer CCCP. Treatment of wild-type vesicles with these concentrations of CCCP caused decreases in transport comparable to those observed in the mutants. Strain KW-1 lacked ATPase activity. Cross-reacting material to F1-ATPase was not found in strain KW-1 by double immunodiffusion analysis.
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PMID:Method for isolation of Escherichia coli mutants with defects in the proton-translocating sector of the membrane adenosine triphosphatase complex. 15 9

The real size of platinum-carbon (Pt-C) replicated particles is not directly equivalent to either its metal-coated diameter or its shadow width. This paper describes two indirect methods, shadow widths and coated particle diameters, for determining a particle's actual size beneath a Pt-C replication film. Both produce equivalent measurements using the same standardized conditions: 2.3 nm Pt-C films deposited at a 45 degree angle on an approximately -100 degrees C surface in a 10(-6) torr vacuum. For the first method, gold balls nucleated in a partial pressure of helium and deposited on flat indirect carbon films (root mean square roughness of 0.8 nm) on 400 mesh grids were used as test particles for calibrating shadow widths as a function of particle size. The gold ball test specimens were replicated, and a distribution of Pt-C shadow widths orthogonal to the Pt-C deposition direction was measured and averaged for gold balls 1.5 +/- 0.25 nm, 2.0 +/- 0.25 nm, etc. The diameter of each gold ball was measured within the Pt-C film along with its shadow width because the Pt-C did not obscure or adhere well to the gold. The shadow width distributions for each gold size do not differ significantly from log normal. Two proteins, the lactose repressor and the mitochondrial ATPase, F1, were also used as replication test objects. Negative staining of both proteins was conducted to measure their average diameters. In the second method, a distribution of Pt-C-coated lac repressor diameters perpendicular to the shadow direction was measured. The Pt-C film thickness measured on the quartz crystal monitor was subtracted from the average metal-coated protein diameter to obtain the lac repressor's diameter. The Pt-C-coated particle diameter distributions also did not differ significantly from log normal. While doing this work it was discovered that outgassing the Pt-C electron gun greatly affected Pt-C film granularity: 19 sec produced a high contrast, granular Pt-C film, whereas 120 sec yielded a low contrast, less granular Pt-C film. Both gold balls and protein particles were subjected in separate experiments to either 19 or 120 sec of outgassing of the Pt-C gun prior to Pt-C replication. Outgassing had a profound effect on the average size of the Pt-C shadow widths on both gold and protein particles. The Pt-C gun outgassing procedure also determined the smallest replicated particle that could be resolved. The frequency of some smaller gold ball sizes detected after replication was reduced disproportionately with 19 sec vs. 120 sec outgassing. However, Pt-C gun outgassing did not affect the average measured diameter of the Pt-C-coated protein particles. The "geometric assumption" that each metal-coated particle creates a shadow width the same size as the metal-coated particle diameter was tested using a globular protein. Pt-C replication of protein particles at a 45 degree and 20 degree angle could not confirm the geometric assumption because an average shadow width was always significantly larger than its average Pt-C-coated particle diameter. A model for how the large shadow widths are formed is presented. Gold balls were also replicated at a 45 degree angle with current high resolution conditions at a substrate temperature of -185 degrees C, and the results of these replicas were compared to the results reported here at approximately -100 degrees C.
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PMID:Quantification of particle sizes with metal replication under standard freeze-etching conditions: a gold ball standard for calibrating shadow widths was used to measure freeze-etched globular proteins. 857 81

Integrins are integral membrane proteins that mediate cell-matrix and cell-cell adhesion. They are important for vascular development and hematopoiesis, immune and inflammatory responses, and hemostasis. Integrins are also signaling receptors that can transmit information bidirectionally across plasma membranes. Research in the past 2 decades has made progress in unraveling the mechanisms of integrin signaling and brings the field to the moment of attempting synthetic reconstruction of the signaling pathways in vitro. Reconstruction of biologic processes provides stringent tests of our understanding of the process, as evidenced by studies of other biologic machines, such as ATP synthase, lactose permease, and G-protein-coupled receptors. Here, we review recent progress in reconstructing integrin signaling and the insights that we have gained through these experiments.
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PMID:Reconstruction of integrin activation. 2192 Oct 44