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151.
The fumarate transport system of the bacterium Helicobacter pylori was investigated employing radioactive tracer analysis. The transport of fumarate at micromolar concentrations was saturable
with a K
M
of 220 ± 21 μm and V
max of 54 ± 2 nmole/min/mg protein at 20°C, depended on temperature between 4 and 40°C, and was susceptible to inhibitors, suggesting
the presence of one or more fumarate carriers. The release of fumarate from cells was also saturable with a K
M
of 464 ± 71 μm and V
max of 22 ± 2 nmol/min/mg protein at 20°C. The rates of fumarate influx at millomolar concentrations increased linearly with
permeant concentration, and depended on the age of the cells. The transport system was specific for dicarboxylic acids suggesting
that fumarate is taken up via dicarboxylate transporters. Succinate and fumarate appeared to form an antiport system. The
properties of fumarate transport were elucidated by investigating the effects of amino acids, monovalent cations, pH and potential
inhibitors. The results provided evidence that influx and efflux of fumarate at low concentrations from H. pylori cells was a carrier-mediated secondary transport with the driving force supplied by the chemical gradient of the anion. The
anaerobic C4-dicarboxylate transport protein identified in the genome of the bacterium appeared to be a good candidate for the fumarate
transporter.
Received: 11 December 1997/Revised: 7 May 1998 相似文献
152.
Arvan L. Fluharty William E. Meek Hayato Kihara 《Biochemical and biophysical research communications》1983,112(1):191-197
Analysis of arylsulfatase A from pseudo arylsulfatase A deficiency fibroblasts by sodium dodecyl sulfate polyacrylamide gel electrophoresis and immunoradiochemical nitrocellulose blot radiography revealed two subunit bands which migrated faster than subunit bands of enzyme from normal fibroblasts. Immunoreactive material was present only at levels comparable to enzyme activity. These findings imply that arylsulfatase A in pseudodeficiency is structurally altered, but it is catalytically equivalent to normal arylsulfatase A. This altered enzyme must be the product of the pseudodeficiency gene since no immunoreactive product of the metachromatic leukodystrophy gene could be detected in metachromatic leukodystrophy cells by the procedure employed. It is not clear from the present data if the attenuated arylsulfatase A activity in pseudodeficiency results from a decreased rate of synthesis or an increased lability of the mutant enzyme. 相似文献
153.
The appearance in the electron microscope of mechanically-dispersed corneal collagen has been observed after positive staining with phosphotungstic acid and/or uranyl acetate and after negative staining with phosphotungstate ions. The distributions of positive stains (both cationic and anionic) were similar to those observed in other type I collagens (e.g. skin, tendon). A high correlation was found between charge density in the fibril and the distribution of charged amino acids predicted from the sequence of calf skin collagen. This correlation could be improved by including type III sequence data, suggesting the presence of 20% type III collagen within each fibril. Negative staining showed the usual collagen D-periodicity but without a clear gap/overlap structure. Detailed analysis revealed at least six sites where stain penetration was inhibited. Specific staining of glycosides using N,N,N′,N′-tetramethylethylenediamine(TEMED)-osmate suggested that these sites identify the covalent attachment of disaccharides to the collagen. Using synchroton X-ray diffraction from TEMED-osmate stained corneas we have determined the locations of the stain ions (and hence the glycosides) in the moist tissue. The results demonstrate that even though the detailed charge distribution and axial molecular packing in corneal collagen are similar to other type I collalgens, carbohydrate material, probably disaccharide, is attached at fairly regular intervals. This does not occur in other type I collagens. In particular, the presence of glycoside in the overlap region may play a role in producing the narrow uniform fibrils which are essential for the transparency of the cornea. 相似文献
154.
The pH dependence of the peptidolytic reaction of recombinant human immunodeficiency virus type 1 protease has been examined over a pH range of 3-7 for four oligopeptide substrates and two competitive inhibitors. The pK values obtained from the pKis vs pH profiles for the unprotonated and protonated active-site aspartyl groups, Asp-25 and Asp-25', in the monoprotonated enzyme form were 3.1 and 5.2, respectively. Profiles of log V/K vs pH for all four substrates were "bell-shaped" in which the pK values for the unprotonated and protonated aspartyl residues were 3.4-3.7 and 5.5-6.5, respectively. Profiles of log V vs pH for these substrates were "wave-shaped" in which V was shifted to a constant lower value upon protonation of a residue of pK = 4.2-5.2. These results indicate that substrates bind only to a form of HIV-1 protease in which one of the two catalytic aspartyl residues is protonated. Solvent kinetic isotope effects were measured over a pH (D) range of 3-7 for two oligopeptide substrates, Ac-Arg-Ala-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2 and Ac-Ser-Gln-Asn-Tyr-Pro-Val-Val-NH2. The pH-independent value for DV/K was 1.0 for both substrates, and DV = 1.5-1.7 and 2.2-3.2 at low and high pH (D), respectively. The attentuation of both V and DV at low pH (D) is consistent with a change in rate-limiting step from a chemical one at high pH (D) to one in which a product release step or an enzyme isomerization step becomes partly rate-limiting at low pH (D). Proton inventory data is in accord with the concerted transfer of two protons in the transition state of a rate-limiting chemical step in which the enzyme-bound amide hydrate adduct collapses to form the carboxylic acid and amine products. 相似文献
155.
S K Grant I C Deckman M D Minnich J Culp S Franklin G B Dreyer T A Tomaszek C Debouck T D Meek 《Biochemistry》1991,30(34):8424-8434
156.
Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration. 总被引:5,自引:3,他引:2
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K M Meek N J Fullwood P H Cooke G F Elliott D M Maurice A J Quantock R S Wall C R Worthington 《Biophysical journal》1991,60(2):467-474
The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern. 相似文献
157.
The structure and content of the extracellular stromal matrix of several oedematous human corneas was investigated using electron microscopy, X-ray diffraction and biochemical techniques. Electron microscopy revealed the presence of wavy lamellae and various sized collagen-free 'lakes' within the stroma of the oedematous corneas, with their posterior sections containing by far the largest 'lakes'. The existence of 'lakes' was supported by the equatorial X-ray diffraction evidence. Staining the oedematous corneas with Cuprolinic blue prior to electron microscopical and meridional X-ray diffraction studies demonstrated a loss of stromal proteoglycans normally associated with collagen. Immunochemical evidence demonstrated reduced levels of antigenic keratan sulphate in the oedematous corneas while biochemical techniques revealed constant chondroitin sulphate levels in the same corneas. 相似文献
158.
159.
Peptide substrates and inhibitors of the HIV-1 protease 总被引:11,自引:0,他引:11
M L Moore W M Bryan S A Fakhoury V W Magaard W F Huffman B D Dayton T D Meek L Hyland G B Dreyer B W Metcalf 《Biochemical and biophysical research communications》1989,159(2):420-425
Oligopeptides containing the consensus retroviral protease cleavage sequence Ser/Thr-X-Y-Tyr/Phe-Pro are substrates for purified recombinant HIV-1 protease with Km's in the millimolar range. The minimum sequence containing the consensus pentapeptide which serves as a good substrate is a heptapeptide spanning the P4-P3' residues. Substitution of reduced Phe-Pro or Tyr-Pro dipeptide isosteres or the statine analog 3-hydroxy-4-amino-5-phenylpentanoic acid for the scissile dipeptide afforded inhibitors of HIV-1 protease with Ki values in the micromolar range, three orders of magnitude better in affinity than the corresponding substrates. Inhibitors of HIV-1 protease may provide a novel and potentially useful therapeutic approach to the treatment of acquired immune deficiency syndrome (AIDS). 相似文献
160.
Zehra Sayers Michel H.J. Koch Susan B. Whitburn Keith M. Meek Gerald F. Elliott Arnold Harmsen 《Journal of molecular biology》1982,160(4):593-607
Using a synchrotron X-ray source, it has been possible to record a low-angle diffraction pattern from fresh bovine corneal stroma.The pattern can be interpreted as arising from the short-range order packing of collagen fibrils in lamellae. Model calculations suggest that the positions of the fibrils remain correlated over distances corresponding to, at most, three fibril diameters (~ 120 nm). These results support theories of transparency of the cornea based on short-range order.Further, a study of the fibril spacing as a function of hydration confirms that water uptake occurs largely between the lamellae and in regions devoid of collagen fibrils, and shows that the fibril diameter increases with hydration. 相似文献