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Biomedical subjects

C C Chin

Publications and source records attributed to C C Chin.

At least 19 recordsLinked to original sources
PubMed1992

Method for the detection of glycopeptides at the picomole level in HPLC peptide maps.

Glycopeptide-containing fractions in HPLC peptide maps can be detected by a simple application of the microtiter plate-bound streptavidin-biotinylated glycopeptide-lectin method (M.-C. Shao, 1992, Anal. Biochem., 205, 77-82). To illustrate this application, the glycoproteins, ovalbumin and asialofetuin, reduced and S-alkylated with vinylpyridine, were digested with trypsin-L-1-p-tosylamino-2-phenylethylchloromethyl ketone and the tryptic peptides were fractionated by reverse-phase HPLC, monitoring for absorbance at 230 nm. Aliquots of the HPLC fractions (typically 0.2-0.5% of the total volume) were biotinylated and complexed with streptavidin in the wells of a microtiter plate, allowing the streptavidin-glycopeptide complex to adhere to the plate. Suitable lectins, such as concanavalin A, Datura stramonium agglutinin, and peanut agglutinin, all of which had been coupled to horse radish peroxidase, were added, and after thorough washing, only the wells containing streptavidin-bound glycopeptides retained the complementary lectin and gave a positive peroxidase reaction. Less than 1 pmol of glycopeptide can be detected. The demonstration that the glycopeptide detection could be inhibited either by addition of an excess of the appropriate sugar inhibitor to the different lectins or by digestion of the biotinylated glycopeptides with N-glycosidase F or O-glycosidase shows that the glycopeptide-lectin interaction is the basis for the reaction.

Amino Acid Sequence↗
PubMed1991

N-terminal sequence analysis of N alpha-acetylated proteins after unblocking with N-acylaminoacyl-peptide hydrolase.

The enzyme acylaminoacyl-peptide hydrolase represents an attractive reagent for the removal of acetylamino acids from the N-terminus of proteins prior to sequencing. However, the enzyme will not accept intact proteins as substrates, and a blocked protein must consequently be fragmented to generate a relative short blocked peptide, and all the newly generated amino termini must be blocked with an hydrolase-resistant reagent before the enzyme can be used to specifically unblock the N-terminus. When a number of N-acetylated proteins (enolase, alpha-crystallin, ovalbumin, cytochrome c, parvalbumin, superoxide dismutase, and myelin basic protein) were subjected to fragmentation with proteases or cyanogen bromide, treatment with succinic anhydride and exhaustive extraction with ether, and the resulting salt-free, succinylated peptides were incubated with the hydrolase, the N-terminal sequence was specifically unblocked. An aliquot of the entire peptide mixture was applied to the protein sequencer, and a single sequence, corresponding to the known N-terminal sequence starting at residue 2, was obtained. When another aliquot of the same hydrolase-treated peptide mixture was treated with the enzyme acylase I, the liberated acetylamino acid was cleaved, and the N-terminal amino acid (residue 1) could be identified by amino acid analysis. The amount of sequence information obtained from different proteins with different fragmentation methods varied considerably; in the case of parvalbumin a sequence of 12 residues was obtained, while for myelin basic protein, only 3 residues could be identified; the other proteins yielded from 5- to 9-residue sequences.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylation↗
PubMed1990

The primary structure of rabbit muscle enolase.

The primary amino acid sequence of rabbit muscle enolase has been determined by standard spinning-cup sequencing techniques applied to peptides produced by chemical (cyanogen bromide and mild acid hydrolysis) and enzymatic fragmentation of the enzyme. The 433 amino acid sequence has been compared to other available enolase sequences from eukaryotic and prokaryotic sources, confirming a high degree of conserved sequence identity; the three mammalian muscle sequences (mouse and rat deduced from c-DNA sequences and rabbit) show 94% identity.

Amino Acid Sequence↗
PubMed1989

Amino acid sequence of cytochrome c from Aspergillus niger.

Cytochrome c from Aspergillus niger consists of two forms, a major one (80%) with 111 amino acid residues and a minor one (20%) with 108 residues, missing the three N-terminal residues of the major one. The primary sequence of A. niger cytochrome c was determined by standard spinning-cup Edman degradation of purified peptides and of pairs of peptides, from which the desired sequence was readily deduced by subtraction of common sequencies. Except for the extension and some variability at the N-terminal sequence, the A. niger protein conforms well with other cytochrome c structures.

Amino Acid Sequence↗
PubMed1989

Glycoproteins in Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles.

1. The Rathke's gland secretions of loggerhead (Caretta caretta) and Kemp's ridley (Lepidochelys kempi) sea turtles contain 20 and 10 mg of protein/ml, respectively. The proteins of each species were separated by gel filtration into two major fractions, one (35%) in the excluded volume, and one (50%) with a molecular mass of approximately 55 kDA. 2. The 55 kDa fraction from each species' secretions exhibits a single band on SDS-PAGE (Mr approximately equal to 55,000) and a single amino-terminal sequence. 3. The amino acid compositions of the two 55 kDa proteins are similar, and the first 15 residues of their amino terminus are identical. Both proteins contain glucosamine. 4. Analyses of the amino acid and amino sugar composition of the high molecular weight fractions from the two turtle species also indicate similarities; there are distinct differences between them and their 55 kDa proteins.

Amino Acid Sequence↗
PubMed1989

[Self-concept of school-age children: the norm in Kaohsiung].

This study has two objectives: (1) to establish the norm of three graphical projective tests (Children's Social-Self Test, Children's Attitude Inventory and Children's Self-Concept Inventory), and (2) to explore the influence of sex, order of birth, social economic status of family, and leadership of class that effect self-concept of the school-age children. These subjects are selected from elementary schools in Kaohsiung, twenty-two classes are drawn out from each grade by systemic-random sampling. Then two classes are drawn out from 22 classes at random to arrange retest for reliability. The total subjects are 6207 students. From October 1987 to June 1988, six researchers are divided into three group to conduct the test. Besides establishing the norm, we find results from analysis of data as follow: 1. The reliability and validity of three graphical projective tests are good, they can be used for assessing the self-concept of school-age children. 2. The grade, sex, order of birth, social economic status of family, leadership of class all influence the self-concept of school-age children. 3. The self-concept and the grade has negative correlation. 4. The self-concept and the social economic status of family has positive correlation. 5. The girls have higher self-concept than the boys. 6. The order of first-born or second-born has higher self-concept than the others. 7. The ones who are leaders of class have higher self-concept than the others.

Adolescent↗
PubMed1988

The covalent structure of individual N-linked glycopeptides from ovomucoid and asialofetuin.

In order to explore whether individual N-linked glycans in a given glycoprotein may be processed to different end products and at the same time prepare a number of well characterized glycopeptides as substrates for glycopeptide hydrolases, we have prepared the individual glycopeptides representing the four major glycosylation sites in ovomucoid and the three sites in asialofetuin. The individual glycopeptides were characterized by amino acid sequence determination before and after removal of the glycan by peptide:N-glycanase (amidase), and the liberated glycans were subjected to mass spectrometric analysis. As expected from available sugar analyses of the individual glycans in ovomucoid, no major differences were detected between the four glycosylation sites in this glycoprotein, but a definite trend toward less processed (less extensively branched) species was observed in going from site 1 to 4. In fetuin, for which the glycan pool is known to be made up of about two-thirds triantennary and one-third biantennary structures, the analysis of the three glycopeptides gave triantennary to biantennary ratios of 75/25, 67/33, and 70/30, respectively, demonstrating that the three sites are processed to a very similar, albeit perhaps not identical, extent. All the glycopeptides obtained in these studies, including the CNBr-produced glycopeptide from ovalbumin, were purified by a set series of steps, gel filtration on Sephadex G-50 followed by ion-exchange chromatography on DE52 and/or reverse phase high performance liquid chromatography. Based on the results, these procedures appear to have general application for the preparation of glycopeptides.

Amino Acids↗
PubMed1987

The regulation of glycan processing in glycoproteins. The effect of avidin on individual steps in the processing of biotinylated glycan derivatives.

The effect of the protein matrix on glycan processing by rat liver Golgi enzymes has been evaluated by a direct comparison of substrate----products conversion of a free glycan and of the same glycan linked to a protein. The glycan substrates had the general structure R-glycan where R represented either biotinyl-Asn-GlcNAc2- or 6-(biotinamido)hexanoyl-Asn-Glc-NAc2- and the protein used was avidin; the extension arm in one of the glycan substrates permitted the additional comparison of two avidin-biotin-glycan complexes. By the use of different glycans as substrates, by the presence or absence of donor substrates (UDP-GlcNAc, UDP-Gal, and CMP-sialic acid (Sia) and/or the inhibitor, swainsonine, it was possible to dissect the individual steps involved in the conversion of R-Man6 (or R-Man5) to a biantennary complex glycan, R-Man3-GlcNAc2-Gal2-Sia2 or to the hybrid glycan R-Man5-GlcNAc-Gal-Sia. Using fast atom bombardment-mass spectrometry to identify and quantify the substrates and products of each parallel incubation of free and avidin-bound substrates, the following observations were made. With the substrate without the extension arm, avidin-binding inhibited mannosidase I, GlcNAc transferase I, and the second step of the reaction catalyzed by mannosidase II (R-Man4-GlcNAc----R-Man3-GlcNAc); the second step of the reaction catalyzed by Gal-transferase was also inhibited to a lesser extent. This inhibition was greatly reduced or absent with the substrates with the extension arm and was consequently referred to as the short range effect. A long range effect of avidin binding expressed by both substrates with and without extension arm was observed for Gal-transferase acting in the hybrid glycan pathway (R-Man5-GlcNAc----R-Man5-GlcNAc-Gal) in the presence of swainsonine and also for Sia-transferase in the catalysis of the incorporation of the second Sia residue into the complex product (R-Man3-GlcNA2-Gal2-Sia----R-Man3-GlcNAc2- Gal2-Sia2) and to a lesser extent in the hybrid pathway (R-Man5-GlcNAc-Gal----R-Man5-GlcNAc-Gal-Sia). GlcNAc transferase II did not appear to be affected by avidin. Based on the information available on the biotin-binding site in avidin, it is proposed that the short range effect reflects the masking of the core chitobiose unit in the avidin-glycan complexes in the absence of the extension arm, but not in the presence of the arm, and that the early processing enzymes thus may require a fully exposed chitobiose for full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Alkaloids↗
PubMed1986

Human placental estradiol 17 beta-dehydrogenase: sequence of a histidine-bearing peptide in the catalytic region.

The amino acid sequence of an octapeptide from the catalytic site of human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was established by affinity-labeling techniques. The enzyme was inactivated separately by 12 beta-hydroxy-4-estrene-3,17-dione 12-(bromo[2-14C]acetate) and 3-methoxyestriol 16-(bromo[2-14C]acetate) at pH 6.3. The inactivations, in both cases, followed pseudo-first-order kinetics with half-times for the 12 beta and 16 alpha derivatives being 192 and 68 h, respectively. Both derivatives are known substrates that inactivate in a time-dependent, irreversible manner and that modify cysteine residues to form (carboxymethyl)cysteine and histidine residues to form either N tau- or N pi-(carboxymethyl)histidine. The inactivated enzyme samples were separately reduced, carboxymethylated, and digested with trypsin. The tryptic digests were applied to Sephadex G-50 and the radioactive N tau- and N phi-(carboxymethyl)histidine-bearing peptides identified. The peptides were further purified by cation-exchange chromatography and gel filtration. Final purification was achieved by HPLC prior to sequencing. It was determined that both steroid derivatives modified either of the two histidine residues in the peptide Thr-Asp-Ile-His-Thr-Phe-His-Arg. These histidines are different from a histidine that was previously shown to be alkylated by estrone 3-(bromoacetate) and that was presumed to proximate the A ring of the bound steroid. It is concluded that the two histidine residues identified in the present study proximate the D ring of the steroid as it binds at the active site and may participate in the hydrogen transfer effected by human placental estradiol 17 beta-dehydrogenase.

17-Hydroxysteroid Dehydrogenases↗
PubMed1985

Overproduction and nucleotide sequence of the respiratory D-lactate dehydrogenase of Escherichia coli.

Recombinant DNA plasmids containing the gene for the membrane-bound D-lactate dehydrogenase (D-LDH) of Escherichia coli linked to the promoter PL from lambda were constructed. After induction, the levels of D-LDH were elevated 300-fold over that of the wild type and amounted to 35% of the total cellular protein. The nucleotide sequence of the D-LDH gene was determined and shown to agree with the amino acid composition and the amino-terminal sequence of the purified enzyme. Removal of the amino-terminal formyl-Met from D-LDH was not inhibited in cells which contained these high levels of D-LDH.

Amino Acid Sequence↗
PubMed1983

Human placental estradiol 17 beta-dehydrogenase. Identification of a single histidine residue affinity-labeled by both 3-bromoacetoxyestrone and 12 beta-bromoacetoxy-4-estrene-3,17-dione.

Human placental estradiol 17 beta-dehydrogenase (EC 1.1.1.62) was affinity-labeled at pH 6.3 by 3-bromo[2'-14C]acetoxyestrone and 12 beta-bromo-[2'-14C] acetoxy-4-estrene-3,17-dione (both are substrates) in separate incubations. The affinity-alkylated enzyme samples were then treated separately as described below. Amino acid compositions of both samples revealed radioactive 3-carboxymethylhistidine. Tryptic digests of each sample were prepared, applied to Sephadex G-50, and 3-carboxymethylhistidine-bearing fractions identified. These peptides were further purified by cation exchange chromatography, gel filtration, and paper electrophoresis. The purified, 3-carboxymethylhistidine-bearing peptides labeled by the two steroids had identical electrophoretic mobilities at pH 6.5, 3.5, and 1.9. The amino acid sequence of the radioactive peptide alkylated by 3-bromo[2'-14C]acetoxyesterone was determined as: Leu-Ala-3-[14C]CmHis-Ser-Lys. The smaller quantity of peptide obtained from the inactivation with 12 beta-bromo[2'-14C]acetoxy-4-estrene-3,17-dione precluded the determination of its complete sequence. However, the first 3 residues were found to be Leu-Ala-3-[14C]CmHis and the amino acid composition showed that serine and lysine were also present. It is concluded that the steroid-binding site of human placental estradiol 17 beta-dehydrogenase contains a histidine residue which proximates the upper A-ring region of the steroid as it undergoes the reversible binding step.

17-Hydroxysteroid Dehydrogenases↗
PubMed1983

The amino acid sequence of Escherichia coli cyanase.

The amino acid sequence of the enzyme cyanase (cyanate hydrolase) from Escherichia coli has been determined by automatic Edman degradation of the intact protein and of its component peptides. The primary peptides used in the sequencing were produced by cyanogen bromide cleavage at the methionine residues, yielding 4 peptides plus free homoserine from the NH2-terminal methionine, and by trypsin cleavage at the 7 arginine residues after acetylation of the lysines. Secondary peptides required for overlaps and COOH-terminal sequences were produced by chymotrypsin or clostripain cleavage of some of the larger peptides. The complete sequence of the cyanase subunit consists of 156 amino acid residues (Mr 16,350). Based on the observation that the cysteine-containing peptide is obtained as a disulfide-linked dimer, it is proposed that the covalent structure of cyanase is made up of two subunits linked by a disulfide bond between the single cystine residue in each subunit. The native enzyme (Mr 150,000) then appears to be a complex of four or five such subunit dimers.

Amino Acid Sequence↗
PubMed1983

CNS manifestations of epidemic hemorrhagic fever. An advanced manifestation of disease associated with poor prognosis.

The records of 134 patients with the clinical diagnosis of epidemic hemorrhagic fever (EHF) were evaluated. The conditions of 74 patients could be categorized as "serious" based on hemorrhagic complications and magnitude of proteinuria, BP abnormality, and pyrexia. Forty-six of these patients with a serious condition had one or more CNS abnormalities. Common manifestations included confusion, meningismus, and convulsions. Whereas abnormality in fever could not be related to CNS abnormality, BP disturbance, acidosis, and azotemia were significantly more common among patients with CNS disease. Fourteen percent of the patients in this study died, and all of them had CNS disturbance; mortality among patients with CNS disturbance was 41%. These results suggest that the CNS manifestations of EHF are multifactorial in cause and they identify a subset of patients with an advanced stage of disease and a grave prognosis.

Central Nervous System Diseases↗
PubMed1982

Spatial relationship of steroid and cofactor at the active site of human placental estradiol 17 beta-dehydrogenase.

Two affinity-labeling steroids (2-bromo[2'-14C]acetamidoestrone methyl ether and 16 alpha-bromo[2'-14C]acetoxyestradiol 3-methyl ether) which bear their reagent groups on the A- and D-ring of the molecule, respectively, and which are both substrates, have been used to elucidate spatial relationships of steroid and cofactor as they undergo the reversible binding step at the active site of human placental estradiol 17 beta-dehydrogenase. The 2-derivative alkylates its evolutive cofactor (NADH) in the presence of the enzyme but not in the absence of enzyme. The rate of cofactor alkylation increases with increasing quantities of enzyme and is slowed by estrone which competes for the enzyme-active site. To the contrary, the 16 alpha-derivative does not detectably alkylate its evolutive cofactor (NAD+). The product of cofactor alkylation by 2-bromoacetamidoestrone methyl ether was treated to effect hydrolytic removal of the adenine moiety from the remainder of the cofactor, reduction of the steroid 17-keto group, and crystallization. The final crystalline product has been identified as 2-[2'-6N-adenyl]acetamidoestradiol 3-methyl ether (IUPAC name: N-(3-methoxy-17 beta-hydroxy-1,3,5(10)-estratrien-2-yl)-2-(purin-6-ylamino) acetamide) by ultraviolet, infrared, NMR, and mass spectral analysis.

17-Hydroxysteroid Dehydrogenases↗