The structure and function of the ATP-sensitive K+ channel in insulin-secreting pancreatic beta-cells.
(17/4423)
ATP-sensitive K+ channels (KATP channels) play important roles in many cellular functions by coupling cell metabolism to electrical activity. The KATP channels in pancreatic beta-cells are thought to be critical in the regulation of glucose-induced and sulfonylurea-induced insulin secretion. Until recently, however, the molecular structure of the KATP channel was not known. Cloning members of the novel inwardly rectifying K+ channel subfamily Kir6.0 (Kir6.1 and Kir6.2) and the sulfonylurea receptors (SUR1 and SUR2) has clarified the molecular structure of KATP channels. The pancreatic beta-cell KATP channel comprises two subunits: a Kir6.2 subunit and an SUR1 subunit. Molecular biological and molecular genetic studies have provided insights into the physiological and pathophysiological roles of the pancreatic beta-cell KATP channel in insulin secretion. (+info)
Interaction between the nucleotide exchange factor Mge1 and the mitochondrial Hsp70 Ssc1.
(18/4423)
Function of Hsp70s such as DnaK of the Escherichia coli cytoplasm and Ssc1 of the mitochondrial matrix of Saccharomyces cerevisiae requires the nucleotide release factors, GrpE and Mge1, respectively. A loop, which protrudes from domain IA of the DnaK ATPase domain, is one of six sites of interaction revealed in the GrpE:DnaK co-crystal structure and has been implicated as a functionally important site in both DnaK and Ssc1. Alanine substitutions for the amino acids (Lys-108 and Arg-213 of Mge1) predicted to interact with the Hsp70 loop were analyzed. Mge1 having both substitutions was able to support growth in the absence of the essential wild-type protein. K108A/R213A Mge1 was able to stimulate nucleotide release from Ssc1 and function in refolding of denatured luciferase, albeit higher concentrations of mutant protein than wild-type protein were required. In vitro and in vivo assays using K108A/R213A Mge1 and Ssc1 indicated that the disruption of contact at this site destabilized the interaction between the two proteins. We propose that the direct interaction between the loop of Ssc1 and Mge1 is not required to effect nucleotide release but plays a role in stabilization of the Mge1-Ssc1 interaction. The robust growth of the K108A/R213A MGE1 mutant suggests that the interaction between Mge1 and Ssc1 is tighter than required for function in vivo. (+info)
Evidence for an inducible nucleotide-dependent acetone carboxylase in Rhodococcus rhodochrous B276.
(19/4423)
The metabolism of acetone was investigated in the actinomycete Rhodococcus rhodochrous (formerly Nocardia corallina) B276. Suspensions of acetone- and isopropanol-grown R. rhodochrous readily metabolized acetone. In contrast, R. rhodochrous cells cultured with glucose as the carbon source lacked the ability to metabolize acetone at the onset of the assay but gained the ability to do so in a time-dependent fashion. Chloramphenicol and rifampin prevented the time-dependent increase in this activity. Acetone metabolism by R. rhodochrous was CO2 dependent, and 14CO2 fixation occurred concomitant with this process. A nucleotide-dependent acetone carboxylase was partially purified from cell extracts of acetone-grown R. rhodochrous by DEAE-Sepharose chromatography. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis suggested that the acetone carboxylase was composed of three subunits with apparent molecular masses of 85, 74, and 16 kDa. Acetone metabolism by the partially purified enzyme was dependent on the presence of a divalent metal and a nucleoside triphosphate. GTP and ITP supported the highest rates of acetone carboxylation, while CTP, UTP, and XTP supported carboxylation at 10 to 50% of these rates. ATP did not support acetone carboxylation. Acetoacetate was determined to be the stoichiometric product of acetone carboxylation. The longer-chain ketones butanone, 2-pentanone, 3-pentanone, and 2-hexanone were substrates. This work has identified an acetone carboxylase with a novel nucleotide usage and broader substrate specificity compared to other such enzymes studied to date. These results strengthen the proposal that carboxylation is a common strategy used for acetone catabolism in aerobic acetone-oxidizing bacteria. (+info)
Inhibition of ATPase, GTPase and adenylate kinase activities of the second nucleotide-binding fold of the cystic fibrosis transmembrane conductance regulator by genistein.
(20/4423)
In the presence of ATP, genistein, like the ATP analogue adenosine 5'-[beta,gamma-imido]triphosphate (pp[NH]pA), increases cystic fibrosis transmembrane conductance regulator (CFTR) chloride currents by prolonging open times. As pp[NH]pA is thought to increase CFTR currents by interfering with ATP hydrolysis at the second nucleotide-binding fold (NBF-2), the present study was undertaken to investigate the effects of genistein on a fusion protein comprising maltose-binding protein (MBP) and NBF-2 (MBP-NBF-2). MBP-NBF-2 exhibited ATPase, GTPase and adenylate kinase activities that were inhibited by genistein in a partial non-competitive manner with respect to ATP or GTP. Ki values for competitive and uncompetitive inhibition were respectively 20 microM and 63 microM for ATPase, 15 microM and 54 microM for GTPase, and 46 microM and 142 microM for adenylate kinase. For ATPase activity, genistein reduced Vmax by 29% and Vmax/Km by 77%. Additional evidence for complex-formation between genistein and MBP-NBF-2 was obtained by the detection of genistein-dependent alterations in the CD spectrum of MBP-NBF-2 that were consistent with the formation of a higher-ordered state. Addition of MBP-NBF-2 increased the fluorescence intensity of genistein, consistent with a change to a less polar environment. pp[NH]pA partially eliminated this enhanced fluorescence of genistein. These observations provide the first direct biochemical evidence that genistein interacts with CFTR, thus inhibiting NBF-2 activity, and suggest a similar mechanism for genistein-dependent stimulation of CFTR chloride currents. (+info)
Motor proteins of the kinesin family. Structures, variations, and nucleotide binding sites.
(21/4423)
Microtubule-dependent motors of the kinesin family convert the energy from ATP hydrolysis into mechanical work in order to transport vesicles and organelles along microtubules. The motor domains of several kinesins have been solved by X-ray diffraction, but the conformational changes associated with force development remain unknown. Here we describe conformational properties of kinesin that might be related to the mechanism of action. First, we have evaluated the conformational variability among all known kinesin structures and find they are concentrated in six areas, most of which are functionally important either in microtubule binding or in linking the core motor to the stalk. Secondly, we show that there is an important difference between kinesins when compared with myosins or GTPases (with which kinesin motor domains bear structural and catalytic similarities); in the diphosphate-state (with bound ADP), all kinesins show a 'tight' nucleotide-binding pocket, comparable with myosin or GTPases in the triphosphate state, whose nucleotide-binding pockets become open, or 'loose', following nucleotide hydrolysis. Thus, kinesin-ADP appears to be in a tense state, resembling that observed in myosin-ATP or p21ras-GTP. (+info)
Difference between active and inactive nucleotide cofactors in the effect on the DNA binding and the helical structure of RecA filament dissociation of RecA--DNA complex by inactive nucleotides.
(22/4423)
The RecA protein requires ATP or dATP for its coprotease and strand exchange activities. Other natural nucleotides, such as ADP, CTP, GTP, UTP and TTP, have little or no activation effect on RecA for these activities. We have investigated the activation mechanism, and the selectivity for ATP, by studying the effect of various nucleotides on the DNA binding and the helical structure of the RecA filament. The interaction with DNA was investigated via fluorescence measurements with a fluorescent DNA analog and fluorescein-labeled oligonucleotides, assisted by linear dichroism. Filament structure was investigated via small-angle neutron scattering. There is no simple correlation between filament elongation, DNA binding affinity of RecA, and DNA structure in the RecA complex. There may be multiple conformations of RecA. Both coprotease and strand exchange activities require formation of a rigid and well organized complex. The triphosphate nucleotides which do not activate RecA, destabilize the RecA-DNA complex, indicating that the chemical nature of the nucleotide nucleobase is very important for the stability of RecA-DNA complex. Higher stability of the RecA-DNA complex in the presence of adenosine 5'-O-3-thiotriphosphate or guanosine 5'-O-3-thiotriphosphate than ATP or GTP indicates that contact between the protein and the chemical group at the gamma position of the nucleotide also affects the stability of the RecA-DNA complex. This contact appears also important for the rigid organization of DNA because ADP strongly decreases the rigidity of the complex. (+info)
Role of nucleotides and peptide substrate for stability and functional state of the human ABC family transporters associated with antigen processing.
(23/4423)
The transporters associated with antigen processing (TAP) belong to the family of ATP-binding cassette (ABC) transporters which share structural organization and use energy provided by ATP to translocate a large variety of solutes across cellular membranes. TAP is thought to hydrolyze ATP in order to deliver peptides to the endoplasmic reticulum where they can assemble with major histocompatibility complex class I molecules. However, initial binding of peptide substrates to TAP has been suggested to be ATP-independent. In this study, the effect of temperature, energetic nucleotides, and peptide on conformation and functional capacity of TAP proteins was examined. Incubation of insect cell microsomes overexpressing human TAP complexes or of human B cell microsomes at 37 degrees C induced a rapid and irreversible structural change that reduced dramatically TAP reactivity with antibodies to transmembrane and nucleotide-binding domains and abolished peptide binding and transport by TAP. These alterations were inhibited almost completely by di- or trinucleotides, and partially by high affinity peptides, suggesting that complete nucleotide dissociation inactivates TAP complexes. Experiments with isolated TAP subunits and fragments suggested that TAP complex stabilization by nucleotides may depend on their binding to the TAP1 subunit. Thus, the cellular level of functional TAP complexes may be regulated by nucleotide concentrations. It is speculated that this regulation may serve to prevent induction of autoimmunity by stressed cells with low energy levels. (+info)
Potent inhibition of mammalian ribonucleases by 3', 5'-pyrophosphate-linked nucleotides.
(24/4423)
Molecular modeling based on the crystal structure of the complex of bovine pancreatic RNase A with the inhibitor 5'-diphosphoadenosine 3'-phosphate (ppAp) (Leonidas, D. D., Shapiro, R., Irons, L. I., Russo, N., and Acharya, K. R. (1997) Biochemistry 36, 5578-5588) was used to design new inhibitors that extend into unoccupied regions of the enzyme active site. These compounds are dinucleotides that contain an unusual 3',5'-pyrophosphate linkage and were synthesized in solution by a combined chemical and enzymatic procedure. The most potent of them, 5'-phospho-2'-deoxyuridine 3'-pyrophosphate, P' --> 5'-ester with adenosine 3'-phosphate (pdUppAp), binds to RNase A with Ki values of 27 and 220 nM at pH 5.9 and 7, respectively. These values are 6-9-fold lower than those for ppAp and 50-fold lower than that for the transition state analogue, uridine vanadate. pdUppAp has broad specificity; it is an effective inhibitor of at least two other members of the pancreatic RNase superfamily, human RNase-2 (eosinophil-derived neurotoxin) and RNase-4, which share only 36-44% sequence identity with the pancreatic enzyme. The potency of pdUppAp and the other inhibitors described here depends critically on the extended internucleotide linkage; the pyrophosphate group enhances dinucleotide binding to the three RNases by 2.1-2.9 orders of magnitude, as compared with a monophosphate. These data give further insight into the organization of the catalytic centers of the various RNases. Moreover, the new class of inhibitors provides a useful means by which to probe the biological actions of these and other related enzymes. (+info)