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K Fisher

Publications and source records attributed to K Fisher.

At least 19 recordsLinked to original sources
PubMed1995

Involvement of the P cluster in intramolecular electron transfer within the nitrogenase MoFe protein.

Nitrogenase is the catalytic component of biological nitrogen fixation, and it is comprised of two component proteins called the Fe protein and MoFe protein. The Fe protein contains a single Fe4S4 cluster, and the MoFe protein contains two metallocluster types called the P cluster (Fe8S8) and FeMo-cofactor (Fe7S9Mo-homocitrate). During turnover, electrons are delivered one at a time from the Fe protein to the MoFe protein in a reaction coupled to component-protein association-dissociation and MgATP hydrolysis. Under conditions of optimum activity, the rate of component-protein dissociation is rate-limiting. The Fe protein's Fe4S4 cluster is the redox entity responsible for intermolecular electron delivery to the MoFe protein, and FeMo-cofactor provides the substrate reduction site. In contrast, the role of the P cluster in catalysis is not well understood although it is believed to be involved in accumulating electrons delivered from the Fe protein and brokering their intramolecular delivery to the substrate reduction site. A nitrogenase component-protein docking model, which is based on the crystallographic structures of the component proteins and which pairs the 2-fold symmetric surface of the Fe protein with the exposed surface of the MoFe protein's pseudosymmetric alpha beta interface, is now available. During component-protein interaction, this model places the P cluster between the Fe protein's Fe4S4 cluster and FeMo-cofactor, which implies that the P cluster is involved in mediating intramolecular electron transfer between the clusters. In the present study, evidence supporting this idea was obtained by demonstrating that it is possible to alter the rate of substrate reduction by perturbing the polypeptide environment between the P cluster and FeMo-cofactor without necessarily disrupting the metallocluster polypeptide environments or altering component-protein interaction.

Amino Acid Sequence↗
PubMed1994

Identification of a nitrogenase protein-protein interaction site defined by residues 59 through 67 within the Azotobacter vinelandii Fe protein.

During nitrogenase catalysis the Fe protein and the MoFe protein associate and dissociate in a MgATP-dependent process involving electron transfer from the Fe protein to the MoFe protein. A docking model, based primarily on the crystal structures of the separate components from Azotobacter vinelandii, was previously proposed in which the 2-fold symmetric surface of the homodimeric Fe protein interacts with the exposed surface of a MoFe protein pseudosymmetric alpha beta-unit interface. In this model, a loop, which is included within residues 59 through 67 of the Fe protein primary sequence, is likely to interact with the MoFe protein during component protein docking. In the present study, evidence supporting the component protein docking model was obtained by construction of an A. vinelandii strain that produces a hybrid Fe protein for which residues 59 through 67 have been replaced by the corresponding residues from the Fe protein of Clostridium pasteurianum. Biochemical analyses of the hybrid Fe protein revealed the following features when compared with the unaltered Fe protein. First, the hybrid Fe protein exhibited half the maximum specific activity of the normal Fe protein and was insensitive to inhibition by low levels of NaCl. Second, the hybrid Fe protein activity was hypersensitive to a molar excess of MoFe protein, which also resulted in the uncoupling of MgATP hydrolysis from substrate reduction. Third, stopped-flow spectrophotometry experiments showed that during catalysis the hybrid Fe protein dissociates from the MoFe protein at only half the normal rate of Fe protein-MoFe protein dissociation. Thus, the salient feature of the hybrid Fe protein is that it appears to form a relatively tighter complex with the MoFe protein. This property is in line with previous biochemical reconstitution experiments where it was shown that a heterologous mixture of Fe protein from C. pasteurianum and MoFe protein from A. vinelandii form a tight, inactive complex and supports the proposal that a region defined by residues 59 through 67 within the Fe protein is involved in component protein interaction.

Adenosine Triphosphate↗
PubMed1994

Fatty acid composition of erythrocytes and plasma triglyceride and cardiovascular risk in Asian diabetic patients.

The excess of coronary heart disease in Indian Asians compared with Europeans is unexplained by conventional risk factors, although the high prevalence of diabetes may play a part. To explore the contribution of diet we compared the fatty acid composition of erythrocyte membrane phospholipid and plasma triglyceride in 36 Gujarati Asians and 24 Europeans with non-insulin-dependent diabetes. Erythrocytes from Asian subjects contained higher proportions of linoleic, dihomogammalinolenic, and arachidonic acids, and lower proportions of oleic and n-3 series fatty acids; triglycerides contained higher linoleic and lower oleic acid levels. For example, mean percentage (SE) of oleic acid (18:1n-9) in erythrocytes was 16.7 (0.2) in Asians and 20.5 (0.6) in Europeans (p = 0.0001), and total n-6:n-3 ratio was, respectively, 12.8 (0.7) and 6.7 (0.7) (p = 0.0001). A high dietary intake of linoleic acid may not be cardioprotective unless balanced by significant intakes of oleic and n-3 series fatty acids, at least in diabetic Indian Asians. By itself, the conventional recommendation to substitute polyunsaturated for saturated fat in the diet may be inadequate to reduce thrombogenesis, and the overall balance of fatty acids, including monounsaturates, should be considered.

Arachidonic Acids↗
PubMed1992

Evidence for a third transcript from the human factor VIII gene.

Intron 22 of the human factor VIII gene was recently found to contain a gene, associated with a CpG island, which is transcribed in the direction opposite to factor VIII. We now report that another transcript emanates from the island and is transcribed in the same direction as factor VIII. The divergent transcripts originate within 122 bases of each other. The newly identified 5' exon in intron 22 potentially codes for eight amino acids and is spliced to exons 23-26, with the factor VIII reading frame maintained. The protein encoded by this transcript would include the factor VIII C2 domain, responsible for phospholipid binding and essential for coagulant activity.

Amino Acid Sequence↗
PubMed1991

Klebsiella pneumoniae nitrogenase. The pre-steady-state kinetics of MoFe-protein reduction and hydrogen evolution under conditions of limiting electron flux show that the rates of association with the Fe-protein and electron transfer are independent of the oxidation level of the MoFe-protein.

The pre-steady-state kinetics of H2 evolution from Klebsiella pneumoniae nitrogenase functioning at 23 degrees C, pH 7.4, under conditions of extremely low electron flux through the MoFe-protein exhibited a lag phase of several minutes duration. The approach to a steady-state rate of H2 evolution was accompanied by a 50% decrease in the amplitude of the MoFe-protein e.p.r. signal. These kinetics have been simulated using our published kinetic model for nitrogenase [Lowe & Thorneley (1984) Biochem. J. 224, 877-886], which was developed using data obtained with nitrogenase functioning at high electron fluxes. The e.p.r. data showed that the rate of complex-formation between reduced Fe-protein and the MoFe-protein (k+1 = 5 x 10(7) M-1.s-1) is the same for the resting (E0) and one-electron-reduced (E1H) states of the MoFe-protein. Stopped-flow spectrophotometry also showed that electron transfer from the Fe-protein to the MoFe-protein in states E0 and E1H occurs at the same rate (kobs. = 140 s-1). These data support our previous assumption that the rate constants that define the 'Fe-protein cycle' are independent of the level of reduction of the MoFe-protein.

Computer Simulation↗
PubMed1991

Safe performance of difficult laparoscopic cholecystectomies.

Laparoscopic cholecystectomy has been advocated for the treatment of uncomplicated symptomatic gallstone disease, but has not been widely advocated for the management of more complicated gallbladder disease such as acute cholecystitis, previous surgery, or common duct stones. During the last 9 months, 360 patients underwent laparoscopic removal of their gallbladder. A total of 138 had a complicated presentation, making surgery more difficult. This article discusses the management of these patients with acute cholecystitis, previous surgery, or common duct disease. Using the described techniques, there were no complications or mortality. Laparoscopic management of difficult gallbladder problems is safe and effective.

Acute Disease↗
PubMed1991

Multiple carriers for dipeptide transport: carrier-mediated transport of glycyl-L-proline in renal BBMV.

To determine whether multiple carriers are responsible for luminal uptake of glycyl-L-proline (Gly-Pro) in the renal proximal tubule, transport of Gly-[3H]Pro was measured in brush-border membrane vesicles (BBMV). A Line-weaver-Burk analysis of Michaelis-Menten kinetics revealed the presence of two carriers: a lower affinity, higher capacity carrier (Km = 1.3 x 10(-2) M; Vmax = 4.6 x 10(-8) mol.mg-1.min-1) and a higher affinity, lower capacity carrier (Km = 2.7 x 10(-7) M; Vmax = 7.8 x 10(-13) mol.mg-1.min-1). The dipeptides Gly-Sar, beta Ala-His, and pyroGlu-His competitively inhibited the low-affinity carrier. No effect on the Km or Vmax of Gly-Pro transport in this range was seen in the presence of the dipeptides Gly-Gly or cycloHis-Pro. The high-affinity carrier exhibited a different inhibition spectrum. Competitive inhibition of Gly-Pro transport was demonstrated for the dipeptides Gly-Gly and Gly-Sar. However, none of the other peptides tested above altered Gly-Pro transport in the high-affinity range, including pyroGlu-His, which is transported by a high-affinity carrier. At both low (4 x 10(-8) M) and high (4 x 10(-3) M) concentrations, uptake of Gly-Pro was stimulated in the presence of an inwardly directed H+ gradient but was unaffected by the presence of an inward Na+ gradient. In addition, measurements in the presence of valinomycin and an outwardly directed K+ gradient strongly suggest that H(+)-stimulated uptake at both concentrations is electrogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1990

Klebsiella pneumoniae nitrogenase. Mechanism of acetylene reduction and its inhibition by carbon monoxide.

The electron flux through the MoFe-protein of nitrogenase from Klebsiella pneumoniae determines the absolute and relative rates of 2H+ reduction to H2 and acetylene (C2H2) reduction to ethylene (C2H4) at saturating levels of reductant (Na2S2O4) and MgATP. High electron flux, induced by a high Fe-protein (Kp2)/MoFe protein (Kp1) ratio, favours C2H2 reduction. These data can be explained if ethylene, the two-electron reduction product of C2H2, is not released until three electrons have been transferred from Kp2 to Kp1. This explanation is also consistent with a pre-steady-state lag phase for C2H4 formation of 250 ms observed when functioning enzyme is quenched with acid. Electron flux through nitrogenase is inhibited by C2H2 at high protein concentrations. This is because the association rate between Kp1 and oxidized Kp2 is enhanced by C2H2, leading to an increased steady-state concentration of the inhibitory complex Kp2oxKp1C2H2. This effect is not relieved by CO. Thus CO and C2H2 (or C2H4) must be bound at the same time to distinct sites, presumably at Mo or Fe centres, on the enzyme.

Acetylene↗
PubMed1990

A protocol for management of temporomandibular joint ankylosis.

A management protocol for temporomandibular joint (TMJ) ankylosis consisting of 1) aggressive resection, 2) ipsilateral coronoidectomy, 3) contralateral coronoidectomy when necessary, 4) lining of the TMJ with temporalis fascia or cartilage, 5) reconstruction of the ramus with a costochondral graft, 6) rigid fixation, and 7) early mobilization and aggressive physiotherapy is presented. The protocol was retrospectively evaluated in the first 14 patients (18 involved TMJs) treated and followed postoperatively for at least 1 year. The facial asymmetries present in all unilateral cases remained corrected. The mean maximum postoperative interincisal opening at 1 year was 37.5 mm (292.36% mean increase), lateral excursions were present in 16 of 18 joints (vs 0 of 18 joints preoperatively), and pain was present in 2 of 18 joints (vs 13 of 18 preoperatively). The results of this study indicate that this protocol is effective for treatment of TMJ ankylosis.

Adolescent↗
PubMed1990

Routine quality assurance and results of a diagnostic radiology quality assurance programme.

Routine testing of diagnostic radiology equipment performance has shown that more than 10% of equipment has not been properly setup. It also identifies equipment which is deteriorating in use as well as faults which need immediate correction. There is little doubt it is cost-effective and has also been shown to be effective in reducing the collective dose to the population from diagnostic radiology.

England↗
PubMed1989

Kinetics and mechanism of the reaction of cyanide with molybdenum nitrogenase from Azotobacter vinelandii.

The steady-state kinetic behavior of the six-electron reduction of N2 by nitrogenase is known to differ markedly from the six-electron reduction of cyanide in two ways. First, on extrapolation to infinite concentration of cyanide, the H2 evolution reaction is almost completely suppressed whereas at extrapolated infinite concentration of N2, H2 evolution continues. Second, as the ratio of the Fe protein to the MoFe protein increases, the reduction of N2 is favored over H2 evolution, whereas the reduction of cyanide becomes less favored relative to H2 evolution. We have extended these steady-state experiments with Azotobacter vinelandii nitrogenase to include a third observation, that the six-electron reduction of N2 is favored over H2 evolution at high total protein concentrations whereas cyanide reduction is less favored over H2 evolution at high total protein concentrations. All three steady-state observations can be explained by a model whereby cyanide is proposed to bind to a redox state of the MoFe protein more oxidized than that reactive toward H2 evolution and N2 reduction. To test this model, we have examined the pre-steady-state kinetic behavior of both cyanide reduction by A. vinelandii nitrogenase and cyanide inhibition of total electron flow through nitrogenase. The data show that in the presence or absence of cyanide there is a short lag of 100 ms before H2 is detected, followed by a linear phase of H2 evolution lasting for about 3 s, during which time no effects of cyanide are observable. After 3 s electron flow is finally inhibited by cyanide, and the cyanide reduction product CH4 is finally formed.(ABSTRACT TRUNCATED AT 250 WORDS)

Azotobacter↗
PubMed1989

Low-affinity transport of pyroglutamyl-histidine in renal brush-border membrane vesicles.

These studies were performed to determine if a low-affinity carrier is present in the luminal membrane of proximal tubular cells for the transport of the dipeptide, pyroglutamyl-histidine (pGlu-His). We have previously described the existence of a specific, high-affinity, low-capacity [transport constant (Kt) = 9.3 X 10(-8) M, Vmax = 6.1 X 10(-12) mol.mg-1.min-1] carrier for pGlu-His in renal brush-border membrane vesicles. In the present study, we sought to demonstrate that multiple carriers exist for the transport of a single dipeptide by determining whether a low-affinity carrier also exists for the uptake of pGlu-His. Transport of pGlu-His into brush-border membrane vesicles was saturable over the concentration range of 10(-5)-10(-3) M, yielding a Kt of 6.3 X 10(-5) M and a Vmax of 2.2 X 10(-10) mol.mg-1.min-1. Uptake was inhibited by the dipeptides glycyl-proline, glycyl-sarcosine, and carnosine but not by the tripeptide pyroglutamyl-histidyl-prolinamide. We conclude that 1) pGlu-His is transported across the luminal membrane of the proximal tubule by multiple carriers and 2) the lower affinity carrier, unlike the higher affinity carrier, is nonspecific with respect to other dipeptides.

Biological Transport↗
PubMed1988

Monolayer coupling in phosphatidylserine bilayers: distinct phase transitions induced by magnesium interacting with one or both monolayers.

We have investigated the thermotropic behavior of phospatidylserine bilayers interacting with Mg2+ either on one side or both sides, using differential scanning calorimetry. Large unilamellar vesicles (LUV) of phosphatidylserine exposed to Mg2+ on the external side only displayed an upward shift of the gel-liquid transition temperature (Tm) of about 6-8 degrees C relative to the Tm of LUV in Na+. Mg2+ was shown not to enter the vesicle interior, by means of fluorescence measurements on encapsulated 8-hydroxyquinoline-5-sulfonate. Multilamellar vesicles prepared in the presence of Mg2+, or vesicles prepared by Mg2+-induced fusion of small unilamellar vesicles, had Tm values that were shifted upward by about 16-17 C degrees. When the latter preparation was treated with EDTA to produce vesicles with Mg2+ inside and Na+ outside, the Tm was found to be shifted again by only 6-8 degrees C. These observations indicate that the monolayer interacting with Na+ fluidizes the monolayer interacting with Mg2+, and that the latter tends to solidify the former. The two monolayers thus appear to be coupled, possibly by hydrocarbon chain interdigitation.

Animals↗
PubMed1988

Regulation of reconstituted renal Na+/H+ exchanger by calcium-dependent protein kinases.

Studies were performed to determine the effect of protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and calcium-phospholipid-dependent protein kinase on Na+/H+ exchange activity. Proteins from the apical membrane of the proximal tubule of the rabbit kidney were solubilized in octyl glucoside and incubated in phosphorylating solutions containing the protein kinase. 22Na+ uptake was determined subsequently after reconstitution of the proteins into proteoliposomes. Calcium-calmodulin-dependent multifunction protein kinase II inhibited the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The inhibitory effect of this kinase had an absolute requirement for calmodulin, Ca2+, and ATP. Calcium-phospholipid-dependent protein kinase stimulated the amiloride-sensitive component of proton gradient-stimulated Na+ uptake in a dose-dependent manner. The stimulating effect of this kinase had an absolute requirement for ATP, Ca2+, and an active phorbol ester. These experiments indicate that Na+/H+ exchange activity of proteoliposomes reconstituted with proteins from renal brush-border membranes are inhibited by protein phosphorylation mediated by calcium-calmodulin-dependent multifunctional protein kinase II and stimulated by that mediated by calcium-calmodulin-dependent protein kinase.

Adenosine Triphosphate↗