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R S Lee

Publications and source records attributed to R S Lee.

At least 19 recordsLinked to original sources
PubMed1992

Investigation of the aurovertin binding site of Escherichia coli F1-ATPase by fluorescence spectroscopy and site-directed mutagenesis.

(1) Previous mutational analyses have shown that residue beta R398 of the beta-subunit is a key residue for binding of the inhibitory antibiotic aurovertin to Escherichia coli F1Fo-ATP synthase. Here, we studied purified F1 from the beta R398C and beta R398W mutants. ATPase activity in both cases was resistant to aurovertin inhibition. The fluorescence spectrum (lambda exc = 278 or 295 nm) of beta R398W F1 showed a significant red-shift as compared to wild-type and beta R398C enzymes, indicating that residue beta R398 lies in a polar environment. On the basis of this and previous evidence, we propose that aurovertin binding to F1-ATPase involves a specific charged donor-acceptor H-bond between residue beta R398 and the 7-hydroxyl group of aurovertin. (2) The fluorescent substrate analog lin-benzo-ADP was shown to bind to beta R398W F1 catalytic sites with the same Kd values as to wild-type F1, and with the same quenching of the fluorescence of the analog. Fluorescence energy transfer was seen between the beta R398W residue and bound lin-benzo-ADP. Analysis of transfer efficiency at varying stoichiometry of bound lin-benzo-ADP showed that interaction occurred between one beta R398W residue and one catalytic-site-bound analog molecule at a distance of approximately 23 A. The relationships of the aurovertin and catalytic sites in the primary and tertiary structure are discussed.

Adenosine Diphosphate↗
PubMed1992

On the location and function of tyrosine beta 331 in the catalytic site of Escherichia coli F1-ATPase.

1) Using a combination of site-directed mutagenesis and fluorescence spectroscopy we have studied the location and function of residue beta Y331 in the catalytic site of Escherichia coli F1-ATPase. The fluorescent analog lin-benzo-ADP was used as a catalytic-site probe, and was found to bind to three sites in normal F1, with Kd1 = 0.20 microM and Kd2,3 = 5.5 microM. lin-Benzo-ATP was a good substrate for hydrolysis. 2) The mutants investigated were beta Y331F, L, A and E. kcat/KM for ATP hydrolysis in purified F1 was reduced according to the series Y greater than or equal to F greater than L greater than A greater than E, with E being severely impaired; concomitant decreases in binding affinity for lin-benzo-ADP were seen. 3) Fluorescence properties of lin-benzo-ADP bound to F1 differed widely, depending on the residue present at position beta 331. Red shifts of excitation and emission spectra occurred with F and L residues, but not with Y, A, or E. There was strong quenching of fluorescence with wild-type (Y), partial quenching with A, and no quenching with F, L, or E. 4) We conclude that (a) the environment around the bound adenine moiety in the catalytic site is nonpolar, (b) residue beta 331 is part of the adenine-binding subdomain and when tyrosine is the residue, the phenolic hydroxyl makes direct interaction with the fluorophore, (c) an aromatic residue is not absolutely required at position beta 331 for catalytic function, but an increase in polarity leads to functional impairment, and (d) in terms of fluorescence response of bound lin-benzo-ADP all three catalytic sites behaved the same. 5) F1 from mutant beta Y297F bound lin-benzo-ADP with the same fluorescence and binding characteristics as normal F1, and catalytic properties were similar to normal. Therefore, there was no reason to conclude that residue beta Y297 is involved in binding the adenine moiety of ATP.

Base Sequence↗
PubMed1992

F1-ATPase with cysteine instead of serine at residue 373 of the alpha subunit.

Escherichia coli strain AN718 contains the alpha S373F mutation in F1F0-ATP synthase which blocks ATP synthesis (oxidative phosphorylation) and steady-state F1-ATPase activity. The revertant strain AN718SS2 containing the mutation alpha C373 was isolated and shown to confer a phenotype of higher growth yield than that of the wild type in liquid medium containing limiting glucose, succinate, or LB. Purified F1 from strain AN718SS2 was found to have 30% of wild-type steady-state ATPase activity and 60% of wild-type oxidative phosphorylation activity. Azide sensitivity of ATPase activity and ADP-induced enhancement of bound aurovertin fluorescence, both of which are lost in alpha S373F mutant F1, were regained in alpha C373 F1. N-Ethylmaleimide (NEM) inactivated alpha C373 F1 steady-state ATPase potently but had no effect on unisite ATPase. Complete inactivation of alpha C373 F1 steady-state ATPase corresponded to incorporation of one NEM per F1 (mol/mol), in just one of the three alpha subunits. NEM-inactivated enzyme showed azide-insensitive residual ATPase activity and loss of ADP-induced enhancement of bound aurovertin fluorescence. The data confirm the view that placement at residue alpha 373 of a bulky amino acid side-chain (phenylalanyl or NEM-derivatized cysteinyl) blocks positive catalytic cooperativity in F1. The fact that NEM inhibits steady-state ATPase when only one alpha subunit of three is reacted suggests a cyclical catalytic mechanism.

Amino Acid Sequence↗
PubMed1992

Catalytic properties of Escherichia coli F1-ATPase depleted of endogenous nucleotides.

Nucleotide-depleted Escherichia coli F1 was prepared by the procedure of Wise et al. (1983, Biochem. J. 215, 343-350). This enzyme had high rates of steady-state ATPase and GTPase activity. When "unisite" ATP hydrolysis was measured using an F1/ATP concentration ratio of 10, all of the substoichiometric ATP became bound to the high-affinity catalytic site and none became bound to noncatalytic sites. The association rate constant for ATP binding was 7 x 10(5) M-1 s-1 and the KdATP was 7.9 x 10(-10) M, as compared to values of 3.8 x 10(5) M-1 s-1 and 1.9 x 10(-10) M, respectively, in native (i.e., nucleotide-replete) F1. Rate constants for bound ATP hydrolysis, ATP resynthesis, and P(i) release, and the reaction equilibrium constant, were similar in nucleotide-depleted and native F1. Therefore, we conclude that occupancy of the noncatalytic sites is not required for formation of the high-affinity catalytic site of F1 and has no significant effect on unisite catalysis. In further experiments we looked for the occurrence of inhibitory, catalytic-site-bound MgADP in E. coli F1. Such an entity has been reported for chloroplast and mitochondrial F1. However, our experiments gave no indication for inhibitory MgADP in E. coli F1.

Adenosine Diphosphate↗
PubMed1991

Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain.

(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.

Aurovertins↗
PubMed1989

Identification of a mutation in Escherichia coli F1-ATPase beta-subunit conferring resistance to aurovertin.

A mutation conferring aurovertin resistance on Escherichia coli F1-ATPase was identified as R398----H in the F1 beta-subunit. Beta-subunit from the mutant does not bind aurovertin; therefore our results suggest the region of sequence around residue beta-398 is involved in aurovertin binding. Since nucleotide and aurovertin binding to isolated beta-subunit are not mutually exclusive, the data further suggest that the beta-subunit catalytic nucleotide-binding domain does not include residue 398. The mutation prevented aurovertin inhibition of ATPase at pH 6 and 8.5, implying charge on the arginine side-chain is not a major determinant of aurovertin binding or that the pK of R398 is shifted due to a peculiar environment. The equivalent residue is usually arginine in F1 beta-subunits of different species; notably in the aurovertin-insensitive thermophilic bacterium PS3 F1-ATPase, this residue is phenylalanine.

Aurovertins↗
PubMed1989

Loss in transformed cells of cell cycle regulation of expression of a nuclear protein recognized by SLE patient antisera.

The identification of cellular proteins involved in the control of cell proliferation in normal cells is essential for understanding the mechanism underlying growth regulation and cellular transformation. A nuclear protein termed Ki antigen with a relative mobility of 32,000 (Mr 32K) and which is recognized by SLE patient antisera has been identified in cells of human, bovine, and murine origin. Recently, cDNA clones for the bovine and human Ki antigens have been isolated using SLE patient antisera (T. Nikaido, et al., in preparation). The nucleotide sequence predicted a protein of 239 amino acids with a possible nuclear localization signal resembling that identified in SV40 T antigen and other nuclear proteins. Here we show that the expression of Ki antigen is regulated in the normal cell, but not in the transformed cell. Furthermore, in the K-ras temperature-sensitive mutant cell line, ts 371 normal rat kidney (NRK), Ki antigen expression increases several-fold at the permissive temperature relative to the nonpermissive temperature. These results suggest that expression of Ki antigen might be correlated with cellular transformation as well as with cell growth regulation.

Animals↗
PubMed1988

5'-Nucleotidase of human placental trophoblastic microvilli possesses cobalt-stimulated FAD pyrophosphatase activity.

An enzyme with FAD pyrophosphatase activity was extracted from human placental syncytiotrophoblast microvilli and purified to near-homogeneity. The enzyme has been identified as 5'-nucleotidase by several criteria. Throughout purification, parallel increases in the specific activities of FAD pyrophosphatase and AMP phosphatase were observed. The enzyme was a glycoprotein with a subunit molecular weight of 74,000. EDTA treatment resulted in a marked decline in both activities, and restoration of FAD pyrophosphatase activity but not 5'-nucleotidase activity was accomplished by the addition of Co2+ or, to a lesser extent, Mn2+. The substrate specificity of the 5'-nucleotidase activity that we observed agreed closely with the results of others. The pyrophosphatase activity was relatively specific for FAD. ADP, ATP, NAD(H), and FMN were not hydrolyzed, and ADP strongly inhibited both activities. For FAD pyrophosphatase activity, a Km of 1.2 x 10(-5) M and a Vmax of 1.1 mumol/min/mg protein were determined in assays performed in the presence of Co2+. In the absence of added Co2+, the Vmax declined but the Km was unchanged. For 5'-nucleotidase (AMP as substrate) the Km was 4.1 x 10(-5) M and the Vmax 109 mumol/min/mg protein. Hydrolysis of FMN to riboflavin was observed in partially purified detergent extracts of microvilli that contained alkaline phosphatase activity and lacked FAD pyrophosphatase and 5'-nucleotidase activity. The presence of both FAD pyrophosphatase and FMN phosphatase activities in syncytiotrophoblast microvilli supports the view that the placental uptake of vitamin B2 involves the hydrolysis of FAD and FMN to riboflavin which is then absorbed, a sequence postulated for intestinal absorption and liver uptake.

5'-Nucleotidase↗
PubMed1988

Vertigo in the hyperviscosity syndrome.

Diseases that cause hyperviscosity of the blood can result in otologic symptoms--especially vertigo. These symptoms are predominantly produced by peripheral vestibular involvement. The pathophysiology probably involves vascular obstruction in the venules. Specific changes in the microvasculature are presumed to be comparable to those that occur with hyperviscosity disorders in the retina. Maintenance of a normal blood viscosity will prevent damage to the ear, as well as other organs. Additionally, reduction of the blood viscosity can greatly improve otologic symptoms. Case studies and a review of the pertinent literature are included.

Adult↗
PubMed1988

Size and genetic composition of long terminal repeat sequences in the mouse intracisternal A-particle gene 81.

The intracisternal A-particle (IAP) genome is a retrovirus-like gene family. Over 1000 copies of IAP genes are dispersed throughout the mouse genome. The purpose of this study was to determine the nucleotide sequence of both ends of the long terminal repeat (LTR) units which bind the IAP gene 81. The results obtained showed that, whereas the structural domain and several genomic characteristics of the IAP 81 LTR units and other LTR structures of a given IAP gene were almost identical, the nucleotide sequences of these LTR units associated with different IAP gene were sometimes very different from each other. In the comparisons of different LTR structures, the transcriptional signals for promotion, initiation, polyadenylation and termination as well as several characteristic sequences of the Z-DNA region, enhancer core sequence and glucocorticoid-responsive element were all found to be present in IAP 81 LTRs. The possible roles of the primary nucleotide sequence of an LTR structure are discussed.

Animals↗
PubMed1987

Assessment of a method for the prediction of mandibular rotation.

A new method to predict mandibular rotation developed by Skieller and co-workers on a sample of 21 implant subjects with extreme growth patterns has been tested against an alternative sample of 25 implant patients with generally similar mean values, but with less extreme facial patterns. The method, which had been highly successful in retrospectively predicting changes in the sample of extreme subjects, was much less successful in predicting individual patterns of mandibular rotation in the new, less extreme sample. The observation of a large difference in the strength of the predictions for these two samples, even though their mean values were quite similar, should serve to increase our awareness of the complexity of the problem of predicting growth patterns in individual cases.

Cephalometry↗
PubMed1985

Enhanced sensitivity of cerebellar Purkinje cells to iontophoretically-applied serotonin in thiamine deficiency.

Electrophysiological aspects of thiamine depletion in the rat induced by dietary deficiency are described. Behavioral changes as well as qualitative and quantitative alterations in the sensitivity of cerebellar Purkinje cells to iontophoretically-applied 5-hydroxytryptamine (5-HT) were observed. Thiamine-deficient rats were characterized essentially by ataxia, piloerection, paresis, apparent weakness, and hypothermia after 4-6 weeks on a thiamine-free diet. Basal Purkinje cell firing frequency was unaffected by thiamine deficiency. The response of Purkinje cells to iontophoretically-applied 5-HT was solely inhibitory in deficient rats. In control rats, however, responses to 5-HT were excitatory, biphasic, or inhibitory. Neurons in the thiamine-deficient animals were more sensitive to the inhibitory effects of 5-HT, as demonstrated by a significant parallel shift to the left of the dose-response curve. Durations of 5-HT effects were similar in both groups. Dose-response relationships for GABA-induced inhibition of Purkinje cell firing from thiamine deficient and control rats did not differ from one another. These data demonstrate a relatively selective effect of thiamine depletion on cerebellar serotonergic neurotransmission assessed electrophysiologically. We believe there is up-regulation of 5-HT receptors on Purkinje cells caused by thiamine deficiency-induced impairment of indoleamine input to the cerebellum from raphe and related nuclei.

Animals↗
PubMed1978

The emergence of candidosis. The dominant postmortem cerebral mycosis.

Comparable human postmortem surveys in central Kentucky and southern Florida have demonstrated an altered pattern of cerebral mycoses due primarily to therapeutic manipulations. From both states 8,975 complete autopsies yielded 39 patients with histologically verified cerebral mycoses. The most common infection was candidosis (49%), characterized by intraparenchymal microabscesses without significant leptomeningitis in hospitalized patients compromised by antibiotic therapy for infection with gram-negative organisms. The remaining 20 patients with noncandidal cerebral mycoses included 9 (23%) with cryptococcosis, 5 (13%) with zygomycosis, 2 (5%) with aspergillosis, 2 (5%) with histoplasmosis, 1 (2.5%) with blastomycosis and 1 (2.5%) with curvulariosis. These compromised patients had leptomeningitis when infected with yeasts and cerebral infarcts with cerebritis when infected with hyphal fungi. In contrast to human cerebral candidosis, the non-candidal cerebral mycoses precipitated the patient's terminal hospitalization. These infections seemed to be contracted outside the hospital. Therapy for gram-negative bacterial infections has enabled Candida species to overtake Cryptococcus neoformans as the most common cause of postmortem cerebral mycosis.

Adolescent↗
PubMed1977

Hysteroscopy.

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Adenocarcinoma↗