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C K Wang

Publications and source records attributed to C K Wang.

At least 19 recordsLinked to original sources
PubMed1992

Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.

Animals↗
PubMed1992

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Time-resolved tryptophan emission study of cardiac troponin I.

We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility.

Animals↗
PubMed1992

Neutron response of a polyethylene-moderated superheated drop detector.

Monte Carlo neutron transport calculations were performed to obtain the neutron response functions of a superheated drop detector (SDD-100) that is surrounded by polyethylene of various thicknesses. The calculational results suggest that a SDD-100 surrounded by 2.54 cm of polyethylene gives the best dose-equivalent response in a free neutron field. Results were confirmed by a sequence of experiments using various neutron sources. This polyethylene-moderated SDD-100 is ideal for use as an area neutron dose monitor.

Equipment Design↗
PubMed1992

Hepatitis E virus: cDNA cloning and expression.

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus-specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Amino Acid Sequence↗
PubMed1991

Distance distributions and anisotropy decays of troponin C and its complex with troponin I.

We used frequency domain measurements of fluorescence resonance energy transfer to recover the distribution of distances between Met 25 and Cys 98 in rabbit skeletal troponin C. These residues were labeled with dansylaziridine as energy donor and 5-(iodoacetamido)eosin as acceptor and are located on the N- and C-terminal lobes of the two-domain protein, respectively. We developed a procedure to correct for the fraction of the sample that was incompletely labeled with the acceptor independent of chemical data. At pH 7.5 and in the presence of Mg2+, the mean distance was near 15 A with a half-width of the distribution of 15 A; when Mg2+ was replaced by Ca2+, the mean distance increased to 22 A with a decrease in the half-width by 4 A. Similar but less pronounced differences in the mean distance and half-width between samples containing Mg2+ and Ca2+ were also observed with troponin C complexed to troponin I. The results suggest that the conformation of troponin C is altered by Ca2+ binding to the Ca(2+)-specific sites and displacing bound Mg2+ at the Ca2+/Mg2+ sites. This alteration may play an important role in Ca2+ signaling in muscle. At pH 7.5, the anisotropy decays of the donor-labeled troponin C showed two components, with the long rotational correlation time (12 ns) reflecting the overall motion of the protein. When the pH was lowered from 7.5 to 5.2, the mean distribution distance of apotroponin C increased from 22 to 32 A and the half-width decreased by a factor of 2 from 13 to 7 A. The long correlation time of apotroponin C increased to 19 ns at the acidic pH. These results are discussed in terms of a model in which skeletal troponin C is a dimer at low pH and enable comparison of the solution conformation of the protein at neutral pH with a crystal structure obtained at pH 5.2. While the conformation of the monomeric unit of troponin C dimer at pH 5.2 is extended and consistent with the crystal structure, the conformation at neutral pH is likely more compact than the crystal structure predicts.

Animals↗
PubMed1991

The conserved carboxy-terminal domain of Saccharomyces cerevisiae TFIID is sufficient to support normal cell growth.

We have examined the structure-function relationships of TFIID through in vivo complementation tests. A yeast strain was constructed which lacked the chromosomal copy of SPT15, the gene encoding TFIID, and was therefore dependent on a functional plasmid-borne wild-type copy of this gene for viability. By using the plasmid shuffle technique, the plasmid-borne wild-type TFIID gene was replaced with a family of plasmids containing a series of systematically mutated TFIID genes. These various forms of TFIID were expressed from three different promoter contexts of different strengths, and the ability of each mutant form of TFIID to complement our chromosomal TFIID null allele was assessed. We found that the first 61 amino acid residues of TFIID are totally dispensable for vegetative cell growth, since yeast strains containing this deleted form of TFIID grow at wild-type rates. Amino-terminally deleted TFIID was further shown to be able to function normally in vivo by virtue of its ability both to promote accurate transcription initiation from a large number of different genes and to interact efficiently with the Gal4 protein to activate transcription of GAL1 with essentially wild-type kinetics. Any deletion removing sequences from within the conserved carboxy-terminal region of S. cerevisiae TFIID was lethal. Further, the exact sequence of the conserved carboxy-terminal portion of the molecule is critical for function, since of several heterologous TFIID homologs tested, only the highly related Schizosaccharomyces pombe gene could complement our S. cerevisiae TFIID null mutant. Taken together, these data indicate that all important functional domains of TFIID appear to lie in its carboxy-terminal 179 amino acid residues. The significance of these findings regarding TFIID function are discussed.

Amino Acid Sequence↗
PubMed1990

An experimental study of the moderator assembly for a low-energy proton accelerator neutron irradiation facility for BNCT.

An accelerator-based neutron irradiation facility (ANIF), which has been proposed for BNCT, is based on a 2.5-MeV proton beam bombarding a thick lithium target. Neutrons which are emitted from the lithium target are too energetic for BNCT and must be moderated. A calculational study, which was done previously on the moderator assembly for an ANIF, shows that, with an optimized moderator assembly, an ANIF can produce a neutron flux which has quality and intensity sufficient for BNCT. In order to verify our previous calculational study, a lithium target and a non-optimized moderator assembly (a cylindrical tank of D2O) have been constructed and tested at the Ohio State University Van de Graaff proton accelerator. The neutron spectrum was measured for neutrons emerging from the moderator assembly. The measured neutron spectrum agrees reasonably well with that obtained from Monte Carlo calculations, except for neutrons with energies above 100 keV. For those neutrons, the measured spectrum is lower by a factor of two than the calculated one. In addition to the neutron spectrum measurement, the boron-10 absorbed dose was measured on the axis of the neutron field in a 20 cm x 20 cm x 20 cm water phantom, and the result agrees quite well with that obtained from calculation. This experiment confirms that the calculated optimized moderator assembly, consisting of a 22.5-cm thick, 25-cm diameter cylinder of beryllia (BeO) surrounded by a 30-cm thick jacket of alumina (Al2O3), produces an epithermal neutron flux of 3.12 x 10(7) n/cm2-s per mA of protons. For an accelerator delivering 30 mA of 2.5-MeV protons, the irradiation time for a single-session treatment can be as short as 50 minutes. The calculated ratio of absorbed neutron dose to fluence for the optimized moderator assembly is 4.9 x 10(-11) cGy-cm2/n, which is equal to that of a 5-keV neutron beam. Our experimental measurements indicate that the ratio of absorbed neutron dose to fluence may in fact be lower (better) than calculated.

Boron↗
PubMed1990

Cloning of the Schizosaccharomyces pombe TFIID gene reveals a strong conservation of functional domains present in Saccharomyces cerevisiae TFIID.

The gene encoding the Schizosaccharomyces pombe TATA box-binding factor (TFIID) was cloned and sequenced. The gene contains three introns and codes for a polypeptide of 231 amino acids. The cDNA-expressed protein showed both TATA box-binding and basal transcription activities. The carboxy-terminal three-quarters of S. pombe TFIID shares an extraordinary degree of amino acid sequence homology with a corresponding region of Saccharomyces cerevisiae TFIID that has been shown to be necessary and sufficient for TATA box-binding and basal transcription activities. In contrast, the amino-terminal regions of the S. pombe and S. cerevisiae TFIIDs differ markedly in amino acid sequence and composition. Structure and function relationships of TFIID are discussed in light of these data.

Amino Acid Sequence↗
PubMed1990

Eosinophilic gastroenteritis with eosinophilic ascites: report of a case.

Eosinophilic gastroenteritis is a relatively uncommon disease of unknown etiology. Eosinophilic ascites resulting from significant serosal involvement is the rarest clinical subtype. The case reported here is of a 30-year-old male presenting with abdominal pain, diarrhea, and ascites. His personal history included childhood asthma, allergic rhinitis, and recurrent urticaria. The clinical picture was characterized by peripheral eosinophilia and eosinophilic infiltrates of the stomach and small bowel. Computed tomogram (CT) of the abdomen showed generalized thickening of the gastric and small bowel wall. Paracentesis revealed exudative ascites rich in eosinophils. The patient experienced an impressive response to steroid therapy.

Adult↗
PubMed1989

Cloning and structure of a yeast gene encoding a general transcription initiation factor TFIID that binds to the TATA box.

The TATA sequence-binding factor TFIID plays a central role both in promoter activation by RNA polymerase II and other common initiation factors, and in promoter regulation by gene-specific factors. The sequence of yeast TFIID, which seems to be encoded by a single gene, contains interesting structural motifs that are possibly involved in these functions, and is similar to sequences of bacterial sigma factors.

Amino Acid Sequence↗
PubMed1989

Characterization of the ethenoadenosine diphosphate binding site of myosin subfragment 1. Energetics of the equilibrium between two states of nucleotide.S1 and vanadate-induced global conformation changes detected by energy transfer.

The fluorescence decay of 1,N6-ethenoadenosine diphosphate (epsilon ADP) bound to myosin subfragment 1 (S1) was studied as a function of temperature. The decay was biexponential, and the two lifetimes were quenched relative to the single lifetime of free epsilon ADP. The temperature dependence of the fractional intensities of the decay components showed two states of the S1.epsilon ADP complex. At pH 7.5 in 30 mM TES, 60 mM KCl, and 3 mM MgCl2, the equilibrium constant for the conversion of the low-temperature state (S1L.epsilon ADP) to the high-temperature state (S1H.epsilon ADP) was 40 at physiological temperatures, and delta H degrees = 13 kcal.mol-1 and delta S degrees = 49 cal.deg-1.mol-1. At 10 degrees C the equilibrium constant of S1 for epsilon ADP was 5, indicating that S1H.epsilon ADP was the dominant state, and that for the vanadate complex epsilon ADP.Vi was 0.7, suggesting that in S1.epsilon ADP.Vi the dominant state of the S1-nucleotide complex was converted from S1H.epsilon ADP to S1L.epsilon ADP. The single rotational correlation time of bound epsilon ADP at 10 degrees C decreased from 107 ns in S1.epsilon ADP to 74 ns in S1+.epsilon ADP.Vi. Conversion of the binary complex to the ternary vanadate complex resulted in a 3-A decrease in the energy transfer distance between bound epsilon ADP and N-[4-(dimethylamino)-3,5-dinitrophenyl]maleimide attached to SH1 and a decrease of the average distance between bound epsilon ADP and bound Co2+ from 12.6 to 8.3 A.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1989

Purification and characterization of Saccharomyces cerevisiae transcription factor IIIA.

Saccharomyces cerevisiae Transcription Factor IIIA (TFIIIA) has been purified to apparent homogeneity. Two polypeptides copurified with TFIIIA activity. Yeast TFIIIA is a DNA-binding protein which exhibits a high affinity for the internal control region of the homologous 5 S ribosomal RNA gene. Characterization of the yeast protein indicates that it shares most, but not all, of the molecular properties of its Xenopus TFIIIA counterpart.

Animals↗
PubMed1989

Acid-induced dimerization of skeletal troponin C.

We have investigated pH-dependent changes of the properties of troponin C from rabbit skeletal muscle. At pH 7.5 this protein is a monomer and at pH 5.2 it is a dimer. In contrast, bovine cardiac troponin C remains essentially monomeric at pH 5.2. Bovine brain calmodulin is not a dimer, but significantly aggregated at the same acidic pH. The dimerization of skeletal troponin C was demonstrated by low-speed (16,000 rpm) sedimentation equilibrium measurements carried out at 20 degrees C and by polyacrylamide gel electrophoresis under nondenaturing conditions. Dimer formation was significantly inhibited in the ultracentrifuge at rotor speeds of 30,000 and 40,000 rpm at 20 degrees C, and was completely prevented at a rotor speed of 40,000 rpm and 4 degrees C. This temperature and pressure dependence of dimerization strongly suggests that hydrophobic bonding is a major factor in promoting skeletal troponin C association at pH 5.2. The intramolecular distance between Met-25 and Cys-98 of rabbit skeletal troponin C deduced from fluorescence resonance energy transfer measurements increased by a factor of two upon lowering the pH from 7.5 to 5.2, indicating a pH-dependent transition in which the protein changed from a relatively compact conformation to an elongated conformation. The proton-induced increase in the energy transfer distance is related to the acid-induced dimerization of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Purification of a yeast TATA box-binding protein that exhibits human transcription factor IID activity.

By a series of conventional chromatographic procedures we have purified from whole-cell extracts of Saccharomyces cerevisiae yeast transcription factor IID (TFIID), which functionally substitutes for human TFIID in a complementation assay comprised of the adenovirus type 2 major late promoter and HeLa cell-derived RNA polymerase II, transcription factors IIA, IIB, and IIE. Similar to its human counterpart, yeast TFIID also exhibited specific binding to the adenovirus type 2 major late promoter TATA element, as shown by both DNase I footprinting and gel mobility shift assays. NaDodSO4/PAGE analyses showed that a 27-kDa polypeptide coeluted with TFIID complementing activity through each chromatographic step. In agreement with this result and also suggesting that the native protein is a monomer, gel-filtration experiments indicated a molecular mass of 28 kDa for TFIID under nondenaturing conditions. That the 27-kDa polypeptide represented TFIID was further demonstrated by the ability of an HPLC-purified protein to bind specifically after renaturation to the adenovirus type 2 major late promoter TATA sequence.

Antigens, Neoplasm↗
PubMed1988

Distance distributions in proteins recovered by using frequency-domain fluorometry. Applications to troponin I and its complex with troponin C.

We used resonance energy transfer to examine the distribution of distances between two sites on troponin I (TnI). The donor (D) was the single tryptophan residue at site 158 (Trp 158), and the acceptor (A) was cysteine 133 (Cys 133) which was labeled with N-(iodoacetyl)-N'-(1-sulfo-5-naphthyl)ethylenediamine (IE). A distribution of D-A distances results in a distribution of donor decay times, which were resolved by using frequency-domain fluorometry. In the native state we recovered a relatively narrow distribution of D-A distances. The widths of the distance distributions were found to increase progressively and dramatically with increasing concentrations of guanidine hydrochloride. Binding of calcium-free troponin C (TnC) to troponin I did not alter the distance distribution. Addition of Ca2+ to the TnI.TnC complex resulted in a sharper distance distribution and protected against the guanidine hydrochloride induced increase in the width of the distance distribution. Additionally, the same distance distributions were recovered for native and denatured TnI when the Forster distance for energy transfer was decreased by acrylamide quenching. These results demonstrate that distance distributions can be recovered with good accuracy, to the extent of revealing modest changes due to binding of other components. This technique should have widespread applications in studies of protein folding.

Fluorometry↗