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C H Chung

Publications and source records attributed to C H Chung.

At least 109 records · Page 6Linked to original sources
PubMed1997

Purification and characterization of a new ubiquitin C-terminal hydrolase (UCH-1) with isopeptidase activity from chick skeletal muscle.

We have previously shown that chick muscle extracts contain at least 10 different ubiquitin C-terminal hydrolases (UCHs). In the present studies, one of the enzymes, called UCH-1 was partially purified by conventional chromatographic procedures using (125)I-labeled ubiquitin-alphaNH-MHISPPEPESEEEEEHYC as a substrate. The purified enzyme behaved as a 35-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consisted of a single polypeptide chain. It was maximally active at pHs between 8 and 9, but showed little or no activity at pH below 6 and above 11. Like other UCHs, its activity was strongly inhibited by sulfhydryl blocking reagents, such as iodoacetamide, and by ubiquitin-aldehyde. In addition to Ub-PESTc, UCH-1 hydrolyzed ubiquitin-alphaNH-protein extensions, including ubiquitin-alphaNH-carboxyl extension protein of 80 amino acids, ubiquitin-alphaNH-dihydrofolate reductase, and poly-His-tagged di-ubiquitin. This enzyme was also capable of generating free ubiquitin from mono-ubiquitin-epsilonNH-protein conjugates and from branched poly-ubiquitin chains that are ligated to proteins through epsilon NH-isopeptide bonds. These results suggest that UCH-1 may play an important role in the generation of free ubiquitin from ubiquitin-ribosomal protein fusions and linear poly-ubiquitin, as well as in recycling of Ub molecules after degradation of poly-ubiquitinated protein conjugates by the 26S proteasome.

Amino Acid Sequence↗
PubMed1997

A possible role of inwardly rectifying K+ channels in chick myoblast differentiation.

We examined the developmental change of inwardly rectifying K+ channels (IRK) and its possible role in myogenesis. Northern blot analysis revealed an increase in the level of IRK mRNA during myogenesis. Accordingly, IRK current was not detectable in replicating myoblasts but first appeared in aligned myoblasts that were competent for fusion and gradually increased thereafter. The time course change of IRK activity was closely related to the increase in resting membrane potential during myogenesis. Application of 0.5 mM Ba2+ to the bath depolarized the membrane and blocked IRK currents dramatically but not outwardly rectifying K+ currents. Myoblasts devoid of IRK had low resting K+ permeability, whereas myotubes that possess IRK had high resting K+ permeability. In some aligned myoblasts, anomalous hyperpolarization was elicited by increasing extracellular K+ concentration, which may be attributable to the increased conductance of IRK. Noteworthy was the fact that maximal fusion was obtained at this range of K+ concentration. These findings imply that IRK is responsible for the change in the K+ permeability during chick myogenesis, which may provide a larger driving force for Ca2+ influx that is a prerequisite for myoblast fusion.

Animals↗
PubMed1997

Protective effect of tetrandrine on normal human mononuclear cells against ionizing irradiation.

Tetrandrine, an alkaloid isolated from the plant Stephania tetrandra, at low concentration (2 micrograms/ml) was shown to protect normal human mononuclear cells in vitro against damage due to a single high-dose of ionizing irradiation (10 Gy). The cell survival rate increased from 58.3 +/- 2.2% in the irradiated group to 78.0 +/- 2.6% in the tetrandrine-pretreated group, and similarly, the percentage of necrotic cells declined from 20.7 +/- 2.5% to 10.7 +/- 1.9%, respectively. This protective effect of tetrandrine for cell surviving fraction increased in a dose-dependent manner. Tetrandrine was also found to inhibit inflammatory responses induced by irradiation including the release of superoxide (NBT [nitroblue tetrazolium] reduction decreased from 21.3 +/- 2.3% to 10.2 +/- 2.5%) and phagocytic activity (decreased from 80.7 +/- 3.8% to 50.7 +/- 2.3%, the same range level as that of the control group). However, the alkaloid demonstrated no effect on the production of nitric oxide. In terms of cell morphology, only two types were observed-normal or necrotic cells, and there were no characteristics of programmed cell death. These results indicate that tetrandrine possesses radioprotective activity against 10 Gy of ionizing irradiation and could suppress irradiation-induced inflammatory processes.

Alkaloids↗
PubMed1997

Influence of pentobarbital-Na on stimulation-evoked catecholamine secretion in the perfused rat adrenal gland.

OBJECTIVES: The present study was attempted to investigate the effects of pentobarbital-Na, one of the barbiturates which are known to depress excitatory synaptic transmission in the central nervous system at concentrations similar to those required for the induction and maintenance of anesthesia, on catecholamines (CA) secretion evoked by cholinergic stimulation and membrane-depolarization from the isolated perfused rat adrenal gland, and to clarify the mechanism of its action. METHODS: Mature male Sprague-Dawley rats were anesthetized with thiopenal-Na (40 mg/kg, s.c.). The adrenal gland was isolated by the methods of Wakade. A cannula used for perfusion of the adrenal gland was inserted into the distal end of the renal vein. The adrenal gland was carefully removed from the animal and placed on a platform of a leucite chamber. RESULTS: The perfusion of pentobarbital-Na(30-300 uM) into an adrenal vein for 20 min produced relatively dose-dependent inhibition in CA secretion evoked by ACh(5.32 mM), DMPP(100 uM for 1 min), McN-A-343(200 uM for 2 min), Bay-K-8644(10 uM) and high potassium(56 mM), while it did not affect the CA secretion of cyclopiazonic acid(10 uM). Also, in the presence of thiopental-Na (100 uM), CA secretory responses evoked by ACh, DMPP, McN-A-343 and high K+ were markedly depressed. Moreover, in adrenal glands preloaded with ketamine(100 uM for 20 min), which is known to be a dissociative anesthetic, CA secretion evoked by ACh, DMPP, McN-A-343 and high K+ were significantly attenuated. CONCLUSION: Taken together, these experimental results suggest that pentobarbital-Na depresses CA release evoked by both cholinergic stimulation and membrane-depolarization from the isolated rat adrenal medulla and that this inhibitory activity may be due to the result of the direct inhibition of Ca++ influx into the chromaffin cells without any effect on the calcium mobilization from the intracellular store.

Acetylcholine↗
PubMed1997

Site-directed mutagenesis of the Cys residues in ClpA, the ATPase component of protease Ti (ClpAP) in Escherichia coli.

The ATP-dependent casein hydrolysis by protease Ti (ClpAP) has been shown to be inhibited by sulfhydryl blocking agents, such as N-ethylmaleimide (NEM), when preincubated with ClpA but not with ClpP. To define the role of three Cys residues in ClpA, site-directed mutagenesis was performed to substitute each of them with Ser or Ala. None of the mutations showed any effect on the ATPase activity of ClpA or its ability to support the casein degradation by ClpP. However, NEM could no longer block the ability of ClpA/C47S or ClpA/C47A in supporting the ClpP-mediated proteolysis, unlike that of ClpA, ClpA/C203S, or ClpA/C243S. Furthermore, in the presence of NEM, casein could stimulate the ATPase activities of ClpA/C47S and ClpA/C47A and protect from their degradation by ClpP, but not of the other ClpA proteins. These results suggest that the inhibitory effect of NEM is due to prevention of the interaction of ClpA with casein by introduction of a bulky alkyl group to Cys47, but not linked to the catalytic function of the ATPase.

Adenosine Triphosphatases↗
PubMed1996

Poly-L-lysine activates both peptide and ATP hydrolysis by the ATP-dependent HslVU protease in Escherichia coli.

Hs1VU in E. coli is a new type of ATP-dependent protease composed of two heat shock proteins, the HslU ATPase and the HslV peptidase related to certain beta-type subunits of the 20S proteasome. Here we show that the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methylcoumarin by the HslVU protease can be markedly stimulated by poly-L-lysine, that is known to activate the casein-degrading activity of the 20S proteasome. However, poly-L-lysine showed little or no effect on the peptidase activity of HslV itself. Instead, it stimulated the hydrolysis of ATP by HslU several-fold. Histone that could stimulate the ATPase activity of HslU also increased the rate of the ATP-dependent peptide hydrolysis by HslV, although to a much lesser extent than by poly-L-lysine. Thus, the poly-L-lysine-mediated increase in the ATPase activity of HslU appears to be responsible for the dramatic activation of the ATP-dependent peptide hydrolysis by HslV. These results suggest that, in the reconstituted HslVU complex, the peptide hydrolysis by HslV occurs in a tightly coupled process with the cleavage of ATP by HslU.

ATP-Dependent Proteases↗
PubMed1996

Mutational analysis of the ATP-binding site in HslU, the ATPase component of HslVU protease in Escherichia coli.

HslU is the ATPase component of the ATP-dependent HslVU protease in Escherichia coli. To gain an insight into the structure and function of HslU, site-directed mutagenesis was performed to generate a mutation in the ATP-binding site of the ATPase (i.e., to replace the Lys63 with Thr). Unlike the wild-type HslU, the mutant form (referred to as HslU/K63T) could not hydrolyze ATP or support the ATP-dependent hydrolysis of N-carbobenzoxy-Gly-Gly-Leu-7-amido-4-methyl coumarin by HslV. The wild-type HslU (a mixture of monomer and dimer) formed a multimer containing 6-8 subunits in the presence of either ATP or ADP, indicating that ATP-binding, but not its hydrolysis, is required for oligomerization of HslU. However, HslU/K63T remained as a monomer whether or not the adenine nucleotides were present. Furthermore, ATP or ADP could protect HslU, but not HslU/K63T, from degradation by trypsin. These results suggest that the mutation in the ATP-binding site results in prevention of the binding of the adenine nucleotides to HslU and hence in impairment of both oligomerization and ATPase function of HslU.

ATP-Dependent Proteases↗
PubMed1996

Molecular symmetry of the ClpP component of the ATP-dependent Clp protease, an Escherichia coli homolog of 20 S proteasome.

The ClpP component Clp protease from Escherichia coli has been crystallized and examined by X-ray crystallography and self-rotation function calculations. The crystal belongs to the monoclinic space group P2(1) with unit cell dimensions of a = 196.9 A, b = 104.3 A, c = 162.4 A and beta = 98.3 degrees. The X-ray diffraction pattern extends at least to 2.5 A Bragg spacing when exposed to CuK alpha X-rays. Self-rotation function analyses indicate that the ClpP oligomer has 72-point group symmetry. This symmetry suggests that the ClpP oligomer is a tetradecamer, (ClpP)14, consisting of two heptamers, (ClpP)7 stacked on top of each other in a head-to-head fashion. The measurement of crystal density indicates that two independent copies of the ClpP oligomers are present in the asymmetric unit, giving a crystal volume per protein mass (VM) of 2.73 A3/Da and a solvent content of 54.9% (v/v). Self-rotation function calculations are consistent with the presence of two ClpP tetradecamers in the asymmetric unit. The Patterson function suggests that a translation of x = 0.5 and y = 0.5 relates a pair of ClpP oligomers in one asymmetric unit to another pair in the other asymmetric unit. And the two independent tetradecamers in one asymmetric unit are related by a relative rotation of about 18 degrees around the 7-fold axis.

Adenosine Triphosphatases↗
PubMed1996

Purification and characterization of the heat shock proteins HslV and HslU that form a new ATP-dependent protease in Escherichia coli.

The hslVU operon in Escherichia coli encodes two heat shock proteins, HslV, a 19-kDa protein homologous to beta-type subunits of the 20 S proteasomes, and HslU, a 50-kDa protein related to the ATPase ClpX. We have recently shown that HslV and HslU can function together as a novel ATP-dependent protease, the HslVU protease. We have now purified both proteins to apparent homogeneity from extracts of E. coli carrying the hslVU operon on a multicopy plasmid. HslU by itself cleaved ATP, and pure HslV is a weak peptidase degrading certain hydrophobic peptides. HslU dramatically stimulated peptide hydrolysis by HslV when ATP is present. With a 1:4 molar ratio of HslV to HslU, approximately a 200-fold increase in peptide hydrolysis was observed. HslV stimulated the ATPase activity of HslU 2-4-fold, but had little influence on the affinity of HslU to ATP. The nonhydrolyzable ATP analog, beta,gamma-methylene-ATP, did not support peptide hydrolysis. Other nucleotides (CTP, dATP) that were slowly hydrolyzed by HslU allowed some peptide hydrolysis. Therefore, ATP cleavage appears essential for the HslV activity. Upon gel filtration on a Sephacryl S-300 column, HslV behaved as a 250-kDa oligomer (i.e. 12-14 subunits), and HslU behaved as a 100-kDa protein (i.e. a dimer) in the absence of ATP, but as a 450-kDa multimer (8-10 subunits) in its presence. Therefore ATP appears necessary for oligomerization of HslU. Thus the HslVU protease appears to be a two-component protease in which HslV harbors the peptidase activity, while HslU provides an essential ATPase activity.

ATP-Dependent Proteases↗
PubMed1996

HslV-HslU: A novel ATP-dependent protease complex in Escherichia coli related to the eukaryotic proteasome.

We have isolated a new type of ATP-dependent protease from Escherichia coli. It is the product of the heat-shock locus hslVU that encodes two proteins: HslV, a 19-kDa protein similar to proteasome beta subunits, and HslU, a 50-kDa protein related to the ATPase ClpX. In the presence of ATP, the protease hydrolyzes rapidly the fluorogenic peptide Z-Gly-Gly-Leu-AMC and very slowly certain other chymotrypsin substrates. This activity increased 10-fold in E. coli expressing heat-shock proteins constitutively and 100-fold in cells expressing HslV and HslU from a high copy plasmid. Although HslV and HslU could be coimmunoprecipitated from cell extracts of both strains with an anti-HslV antibody, these two components were readily separated by various types of chromatography. ATP stimulated peptidase activity up to 150-fold, whereas other nucleoside triphosphates, a nonhydrolyzable ATP analog, ADP, or AMP had no effect. Peptidase activity was blocked by the anti-HslV antibody and by several types of inhibitors of the eukaryotic proteasome (a threonine protease) but not by inhibitors of other classes of proteases. Unlike eukaryotic proteasomes, the HslVU protease lacked tryptic-like and peptidyl-glutamyl-peptidase activities. Electron micrographs reveal ring-shaped particles similar to en face images of the 20S proteasome or the ClpAP protease. Thus, HslV and HslU appear to form a complex in which ATP hydrolysis by HslU is essential for peptide hydrolysis by the proteasome-like component HslV.

ATP-Dependent Proteases↗
PubMed1996

Involvement of K(Ca) channels and stretch-activated channels in calcium influx, triggering membrane fusion of chick embryonic myoblasts.

Calcium influx is known to be prerequisite for membrane fusion of myoblasts. However, little is known about the channels that are responsible for the entry of calcium into the cells. Here we show that K(Ca) channels and stretch-activated channels are involved in the calcium influx. Upon analysis of single-channel recordings, calcium sensitivity of K(Ca) channels in myoblasts was found to be about sixfold higher than that in myotubes. Their density in myoblasts (1.68 micron(-2)) was also about sixfold higher than that in myotubes (0.27 micron(-2)). In addition, the opening of the calcium-permeable cationic channels in myoblasts was found to increase with membrane stretching and could be blocked by gadolinium. The density of stretch-activated channels was 0.22 micron(-2) for myoblasts, and the relative permeability of calcium to potassium was P(Ca)/P(K) approximately 3.6. The channels could generate inward calcium currents to open K(Ca) channels in physiological solution. Furthermore, the activation of K(Ca) channels by phloretin dramatically hyperpolarized the resting membrane potential of myoblasts and this effect could be reversed upon treatment of tetraethylammonium. While phloretin induced precocious fusion, tetraethylammonium or gadolinium blocked not only the phloretin-induced precocious fusion but also the spontaneous fusion of myoblasts. These results suggest that hyperpolarization generated by reciprocal activation of stretch-activated channels and K(Ca) channels is involved in the calcium influx that triggers myoblast fusion.

Animals↗
PubMed1996

Crystal structure analyses of uncomplexed ecotin in two crystal forms: implications for its function and stability.

Ecotin, a homodimeric protein composed of 142 residue subunits, is a novel serine protease inhibitor present in Escherichia coli. Its thermostability and acid stability, as well as broad specificity toward proteases, make it an interesting protein for structural characterization. Its structure in the uncomplexed state, determined for two different crystalline environments, allows a structural comparison of the free inhibitor with that in complex with trypsin. Although there is no gross structural rearrangement of ecotin when binding trypsin, the loops involved in binding trypsin show relatively large shifts in atomic positions. The inherent flexibility of the loops and the highly nonglobular shape are the two features essential for its inhibitory function. An insight into the understanding of the structural basis of thermostability and acid stability of ecotin is also provided by the present structure.

Amino Acid Sequence↗
PubMed1996

Clinical significance of K-ras oncogene activation in ampullary neoplasms.

AIMS: To investigate the prevalence of K-ras codon 12 point mutations in ampullary neoplasms, to explore their clinical usefulness, and to test whether the detection of these mutations could be used to identify ampullary malignancies at an early stage. METHODS: Forty one tumour specimens from 28 patients with ampullary neoplasms were analysed for activating point mutations in K-ras codon 12 using a sensitive polymerase chain reaction (PCR) based assay. RESULTS: Eleven (39%) of the 28 primary tumours harboured point mutations in K-ras. Mutations were identified in seven (41%) of the 17 carcinomas and four (36%) of the 11 adenomas. Four of the possible six permutations in codon 12 were found in these 11 samples. This spectrum of mutations is different from pancreatic carcinoma but resembles that of colorectal neoplasms. Cytological brush specimens were available in 11 cases, and in all of these specimens, the K-ras status in the primary tumour and brush specimens was identical. CONCLUSIONS: K-ras codon 12 point mutations occur in about 40% of ampullary neoplasms at a relatively early stage in tumorigenesis. The pattern of mutations in these tumours resembles that of the adenoma-carcinoma sequence in the colorectum. These results indicate that ampullary neoplasms can be detected at an early stage by searching for genetic alterations in the K-ras oncogene in cytological brush specimens.

Adenocarcinoma↗
PubMed1996

Protease Ti (Clp), a multi-component ATP-dependent protease in Escherichia coli.

The ATP-dependent protease Ti(Clp) consists of two different multimeric components: ClpA containing ATP-cleaving sites and ClpP, with serine active sites for proteolysis. Here we summarize the most recent results on the structure and function of protease Ti. (1) The clpA gene has dual translational initiation sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa. The abbreviated form of ClpA may play an important role in regulation of the ATP-dependent proteolysis, since it inhibits the ability of the 84-kDa ClpA in supporting the ClpP-mediated protein breakdown and the autodegradation of the 84-kDa ClpA. (2) ClpA contains two highly conserved sequences for ATP-binding: the first site is essential for oligomerization and the second site is responsible for ATP hydrolysis. (3) ATP hydrolysis by ClpA is required not only for assembly of the ClpA/ClpP complex but also for its rapid dissociation.

Adenosine Triphosphatases↗
PubMed1995

Purification and characterization of protease Ci, a cytoplasmic metalloendoprotease in Escherichia coli.

Protease Ci, a cytoplasmic metalloprotease in Escherichia coli, has been purified to apparent homogeneity by conventional chromatographic procedures using 125I-labeled oxidized insulin B-chain as a substrate. The purified enzyme behaves as a 54-kDa protein under both denaturing and nondenaturing conditions, suggesting that it consists of a single polypeptide chain. It is inhibited by metal-chelating agents, including o-phenanthroline and NaCN, but not by inhibitors of serine proteases or thiol-blocking agents. Furthermore, protease Ci was found to contain 1.1 mol of zinc per mol of the enzyme upon analysis by HR ICP mass spectroscopy. Thus, protease Ci must be a zinc metalloprotease. Among the polypeptides tested as substrates, oxidized insulin B-chain and glucagon are most rapidly hydrolyzed. Intact insulin is a much poorer substrate than oxidized insulin B-chain, even though the affinity of the enzyme to intact insulin is approximately 100-fold greater than that to the B-chain. Since unlabeled oxidized insulin A-chain is capable of inhibiting the hydrolysis of 125I-labeled insulin B-chain, it also appears to be a substrate. Protease Ci also degrades lysozyme and lactalbumin, although to a much lesser extent than oxidized insulin B-chain. However, it shows little or no activity against proteins larger than 15 kDa (e.g. ovalbumin and denatured bovine serum albumin). Hydrolysis of oxidized insulin B-chain followed by amino acid composition analyses of the cleavage products reveals that as many as 10 of its 29 peptide bonds are hydrolyzed by protease Ci. This ability to hydrolyze relatively small polypeptides suggests that protease Ci may catalyze the later steps in the pathway for intracellular protein breakdown.

Amino Acid Sequence↗
PubMed1995

The 65-kDa protein derived from the internal translational start site of the clpA gene blocks autodegradation of ClpA by the ATP-dependent protease Ti in Escherichia coli.

The ATP-dependent protease Ti consists of two different components: ClpA containing ATP-cleaving sites and ClpP having serine active sites for proteolysis. The clpA gene has dual translational start sites and therefore encodes two polypeptides with sizes of 84 and 65 kDa (referred to as ClpA84 and ClpA65, respectively). Here we show that ClpA84, but not ClpA65, is degraded in vitro by ClpP in the presence of ATP. The ClpP-mediated hydrolysis of ClpA84 could be prevented by casein, which is an excellent substrate of protease Ti (i.e. ClpA84/ClpP complex). Thus, it appears that free form of ClpA84 competes with casein for the degradation by ClpA/ClpP complex. Furthermore, ClpA65 inhibited the auto-degradation of ClpA84 by the complex. These results suggest that ClpA65 may play an important role in the control of the ClpA84 level and in turn in the regulation of ATP-dependent protein breakdown in E. coli.

Adenosine Triphosphatases↗