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Biomedical subjects

I Y Huang

Publications and source records attributed to I Y Huang.

At least 19 recordsLinked to original sources
PubMed1993

Biosynthesis of lipoic acid: characterization of the lipoic acid auxotrophs Escherichia coli W1485-lip2 and JRG33-lip9.

The abilities of the Escherichia coli lipoic acid auxotrophs W1485-lip2 and JRG33-lip9 to grow on succinate medium in the presence of octanoate, 8-mercaptooctanoate, or 6-mercaptooctanoate have been determined. Both organisms are mutated in lipA. Neither organism can use octanoate or 6-mercaptooctanoate for production of lipoate, but the lip2 allele can use 8-mercaptooctanoate. Chromosomal DNA from the auxotrophs was amplified by PCR using primers derived from the DNA sequence of wild-type lipA and then sequenced. Both mutants contain single G/C to A/T mutations in lipA, resulting in conversion of Ser307 into Phe in W1485-lip2 and Glu195 into Lys in JRG33-lip9. These results support the hypothesis that lipA is involved in the sulfur insertion step(s) of lipoate biosynthesis and indicate that it is possible to selectively block formation of the C8-S bond through suitable mutation in lipA.

Amino Acid Sequence↗
PubMed1992

Study of the changes in collagen of the tunica albuginea in venogenic impotence and Peyronie's disease.

We identified the collagen type and content of the tunica albuginea in Peyronie's disease and venogenic impotence compared with the tunica albuginea from the donor of the renal transplant and patients with penile injury. Type III collagen was detected obviously in Peyronie's plaque and was also present in venogenic impotence. It can be hardly found in normal controls. The ratios of type III to type I collagen were significantly higher in Peyronie's plaque while there was a moderate increase in venogenic impotence. The scarcity of type V collagen was noted in human tunica albuginea. The decreased percentage of glycine and alanine in Peyronie's disease and venogenic impotence implied the abnormal composition of collagen or presence of noncollagen protein. The results suggest the biochemical aberration of the tunica albuginea might interfere with the normal function of the penile drainage system.

Amino Acids↗
PubMed1990

Purification and characterization of hementin, a fibrinogenolytic protease from the leech Haementeria ghilianii.

The fibrinogenolytic enzyme hementin, present in extracts of the posterior salivary glands of the giant leech Haementeria ghilianii, was isolated by ultrafiltration, high-performance ion-exchange chromatography and subsequent reversed-phase liquid chromatography. Approximately 100 micrograms (1 nmol) of hementin, present at less than 0.5% in the crude leech salivary extract, was brought to about 90% purity in three steps. Hementin migrated at an Mr of about 73,000 on non-reducing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and at 82,000 on reducing SDS-PAGE. The amino terminal sequence was determined to be TTLTE-PEPDL. The amino terminal sequences of two inactive proteins that partially coeluted with hementin in the first chromatographic step were also determined.

Amino Acid Sequence↗
PubMed1989

Two structural genes on different chromosomes are required for encoding the major subunit of human red cell glucose-6-phosphate dehydrogenase.

Structural analysis revealed the existence of two types of subunits in human red cell glucose-6-phosphate dehydrogenase. The two subunits have the same COOH region consisting of 479 amino acid residues, but their NH2-terminal regions are different in size and sequence. The minor subunit can be fully encoded by the X-linked G6PD cDNA, but the NH2-terminal region of the major subunit cannot. The cDNA and the gene for the NH2-terminal region of the major subunit were cloned and characterized. Southern blot hybridization indicated that the gene for the NH2-terminal region is on chromosome 6, not on the X chromosome. Northern blot hybridization demonstrated an existence of two separate mRNA components, one for the COOH-terminal region and the other for the NH2-terminal region. Two separate structural genes, the X-linked and chromosome 6-linked genes, must be coresponsible for encoding the single chain subunit. Either cross-translation of two mRNAs, or transpeptidation, or some other mechanism must be involved in the synthesis of human red cell G6PD.

Amino Acid Sequence↗
PubMed1989

[Collagen content and types in oral submucous fibrosis].

Oral submucous fibrosis is a chronic disease of the oral cavity. The basic histological change which occurs is a fibroelastic transformation of the connective tissue in the laminal propria layer associated with epithelial atrophy. The etiology of the disease is uncertain, but there is a close association suggested both geographically and epidemiologically with the habitual chewing of betel nuts. The accumulation of collagen fibers increases with the severity of the disease, and the fibroblasts in the normal mucosa and in the fibrotic mucosa increased their proliferation and collagen synthesis. This can be activated by arecoline, an extract from betel nuts, as described in a recent study. In order to obtain some information about the basic characteristics of the collagen in submucous fibrosis and its correlation with the fibrotic changes, the following study was conducted. In this study, collagen was extracted from the tissues of normal mucosa, normal skin and oral submucous fibrosis with pepsin and disodium hydroxyphosphate. The amino acid compositions of collagen, collagen content, types and their ratios were measured and analyzed. The results indicated that the characteristics of collagen in normal mucosa and skin were similar in content (normal mucosa: 111.8 + 31 micrograms/mg; normal skin: 131.4 + 56.4 micrograms/mg), amino acid compositions, types (I, III, V), and ratios of different types (III/I: normal mucosa: 0.119 + 0.03; normal skin: 0.187 + 0.046, V/I: 0.024 + 0.01; 0.0036 + 0.01). Collagen content in the advanced group with oral submucous fibrosis (221.6 + 58.2 micrograms/mg) was higher than that of the normal mucosa group (111.8 + 31 micrograms/mg) and the moderate group with oral submucous fibrosis (107.1 + 37.8 micrograms/mg) by a significant difference. Put no difference occurred between normal mucosa and moderate group with oral submucous fibrosis. The collagen of normal skin, normal mucosa and oral submucous fibrosis (both the advanced & moderate groups) had similar amino acid compositions, except that the presence of hydroxyproline, proline, and glycine were less in oral submucous fibrosis. The conversion factors for determining the total collagen, done by measuring the concentration of hydroxyproline, were 10.15 for oral submucous fibrosis, 9.21 for normal skin, and 8.52 for normal mucosa. Normal skin, normal mucosa, and oral submucous fibrosis have the same collagen types (I, III, V). The ratios of type III to type I collagen and type V to type I were compared between every two groups and the results showed no significant difference.(ABSTRACT TRUNCATED AT 400 WORDS)

Adolescent↗
PubMed1988

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis.

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.

Amino Acid Sequence↗
PubMed1987

Complete amino acid sequence of staphylococcal enterotoxin A.

The amino acid sequence of staphylococcal enterotoxin A is presented. Staphylococcal enterotoxin A is a single-chain polypeptide which consists of 233 amino acid residues with a molecular weight of 27,078 and has the amino acid composition Cys2, Asp17, Asn19, Thr16, Ser13, Glu15, Gln12, Pro4, Gly15, Ala7, Val13, Met2, Ile10, Leu23, Tyr18, Phe8, His6, Lys24, Arg7, Trp2, with serine as both amino- and carboxyl-terminal amino acids. Automated sequence analysis of intact enterotoxin A, as well as characterization of the peptides obtained from cyanogen bromide treatment and trypsin and chymotrypsin digestion, led to the elucidation of the complete primary structure of this protein. Less structural homology is observed among staphylococcal enterotoxins A, B (Huang, I-Y., and Bergdoll, M. S. (1970) J. Biol. Chem. 245, 3518-3525), and C1 (Schmidt, J. J., and Spero, L. (1983) J. Biol. Chem. 258, 6300-6306) than that seen between enterotoxins B and C1.

Amino Acid Sequence↗
PubMed1987

Prostatic spermine-binding protein. Cloning and nucleotide sequence of cDNA, amino acid sequence, and androgenic control of mRNA level.

cDNA for mRNA of an androgen-dependent spermine-binding protein (SBP) of rat ventral prostate was cloned by inserting cDNA into a dG-tailed expression vector, pUC8, and screening the expression library with anti-SBP antibodies. Hybrid-selected translation using plasmid DNA from positive clones yielded a 34-kDa protein which was immunoprecipitated by affinity-purified anti-SBP antibodies. SBP mRNA is about 1260 bases long as measured by Northern blot hybridization. An amino acid sequence deduced from the nucleotide sequence of the cDNA was identical to an amino acid sequence found in SBP. SBP is extremely rich in acidic residues. Aspartic and glutamic acids, which make up about 33% of the total sequence, comprise 89 of a stretch of 126 amino acids at the carboxyl-terminal end. By dot hybridization analysis, SBP mRNA was not detected in rat liver, kidney, brain, submaxillary gland, or uterus. The prostate levels of SBP mRNA were measured by mRNA translation and dot hybridization. SBP mRNA level decreased to less than 20% of normal 2 days after castration of rats, and this decrease was reversed by 5 alpha-dihydrotestosterone injection into castrated rats.

Amino Acid Sequence↗
PubMed1986

Human glucose-6-phosphate dehydrogenase: primary structure and cDNA cloning.

The X-chromosome-linked glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP+ oxidoreductase, EC 1.1.1.49) of humans and other mammals consists of a subunit with a molecular weight of about 58,000. The enzyme plays a key role in the generation of NADPH, particularly in matured erythrocytes, and the genetic deficiency of the enzyme is associated with chronic and drug- or food-induced hemolytic anemia in humans. The enzyme was purified to homogeneity from human erythrocytes. The complete amino acid sequence of the subunit, consisting of 531 amino acid residues, was determined by automated and manual Edman degradation of tryptic, chymotryptic, thermolytic, and cyanogen bromide peptides obtained from the enzyme. Based on the amino acid sequence data thus obtained, a 41-mer oligonucleotide with unique sequence was prepared. Two cDNA libraries constructed in phage lambda gt11--i.e., a human liver cDNA library and a human hepatoma Li-7 cDNA library--were screened with the synthetic nucleotide probe. Two positive clones, lambda G6PD-19 and lambda G6PD-25, were obtained from the hepatoma library. lambda G6PD-19 contained an insertion of 2.0 kilobase pairs (kbp), and encoded 204 amino acid residues that were completely compatible with the COOH-terminal portion of the enzyme. The insertion of the clone had a 3' noncoding region of 1.36 kbp. The other clone, lambda G6PD-25, had an insertion of 1.8 kbp and encoded 362 amino acid residues of G6PD. Southern blot analysis of DNA samples obtained from cells with and without the human X chromosome indicated that the cDNA hybridizes with a sequence in the X chromosome.

Amino Acid Sequence↗
PubMed1984

Molecular abnormality of an inactive aldehyde dehydrogenase variant commonly found in Orientals.

Usual human livers contain two major aldehyde dehydrogenase [(ALDH) aldehyde:NAD+ oxidoreductase] isozymes--i.e., a cytosolic ALDH1 component and a mitochondrial ALDH2 component--whereas approximately equal to 50% of Orientals are "atypical" and have only the ALDH1 isozyme and are missing the ALDH2 isozyme. We previously demonstrated that atypical livers contain an enzymatically inactive but immunologically crossreactive material (CRM) corresponding to the ALDH2 component. The enzymatically active ALDH2 obtained from a usual liver and the CRM obtained from an atypical liver were reduced, S-carboxymethylated, and digested by trypsin. Separation of their digests by high-performance reverse-phase chromatography and by two-dimensional paper chromatography and electrophoresis revealed that ALDH2 contained a peptide sequence of -Glu-Leu-Gly-Glu-Ala-Gly-Leu-Gln-Ala-Asn-Val-Gln-Val-Lys- and that the glutamine adjacent to lysine was substituted by lysine in CRM. All other tryptic peptides, including eight peptides containing S-carboxymethylcysteine, were common in ALDH2 and CRM. It is concluded that a point mutation in the human ALDH2 locus produced the glutamine leads to lysine substitution and enzyme inactivation.

Aldehyde Dehydrogenase↗
PubMed1983

Amino acid sequence of the active site of human pseudocholinesterase.

The usual E1u and atypical E1a human pseudocholinesterases (acylcholine acylhydrolase, EC 3.1.1.8) were purified to homogeneity. The active-site serine residue was conjugated with diisopropyl fluorophosphate and digested with trypsin. The tryptic peptide containing the active site was isolated by gel filtration followed by two-dimensional paper chromatography and electrophoresis. The amino acid sequence of the active site peptide obtained from the usual E1u enzyme was found to be Gly-Glu-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu. A remarkable structural homology exists between the human and the horse enzymes in their active sites. From the difference in electrophoretic mobility of the active-site peptides obtained from the usual and atypical enzymes, the probable structure of the atypical human enzyme was deduced as Gly-His-Ser-Ala-Gly-Ala-Ser-Ala-Val-Ser-Leu.

Amino Acid Sequence↗
PubMed1982

Prostate alpha-protein. Isolation and characterization of the polypeptide components and cholesterol binding.

alpha-Protein, a major glycoprotein in the cytosol fraction of rat ventral prostate, has a molecular weight of about 50,000 and can be dissociated, by sodium dodecyl sulfate, into two different subunits (A and B). alpha-Protein has three different polypeptide components with apparent molecular weights of 10,000 (I), 14,000 (II), and 15,000 (III). These components were purified to homogeneity and their amino acid compositions were determined. Subunit A is composed of Components I and III, whereas subunit B is composed of Components II and III. Carbohydrate was detectable only on Component III. Component III isolated from subunit A and Component III isolated from subunit B appear to be identical. The purified alpha-protein contains 0.7-1 mol of cholesterol/mol of protein. If cholesterol was removed by acetone, about 1 mol of 5 alpha-dihydrotestosterone or pregnenolone could bind to 1 mol of alpha-protein. In the presence of 2 mM ZnCl2, alpha-protein can form dimers and tetramers. In cell-free systems, alpha-protein can inhibit binding of the androgen-receptor complex to nuclear chromatin and also can promote the release of the complex already bound to chromatin. This effect is due to polypeptide Component I.

Amino Acids↗
PubMed1982

Prostate alpha-protein. Complete amino acid sequence of the component that inhibits nuclear retention of the androgen-receptor complex.

The amino acid sequence of Component I of alpha-protein, a glutamic acid-rich protein, is presented. Component I is a single chain polypeptide which consists of 88 amino acid residues with a molecular weight of 10,191. Component I has the amino acid composition Lys6, His, Arg2, Cys3, Asp5, Asn2, Thr3, Ser4, Glu13, Gln3, Pro3, Gly2, Ala6, Val9, Met4, Ile4, Leu8, Tyr6, Phe3, Trp, with serine and asparagine as NH2(-) and COOH-terminal amino acids, respectively. Automated sequences analysis of the whole protein, as well as characterization of the peptides obtained from trypsin, chymotrypsin, and staphylococcal protease digestion and cyanogen bromide treatment, led to the elucidation of the complete primary structure of this protein.

Amino Acid Sequence↗
PubMed1981

Complete amino acid sequence of mouse liver metallothionein-II.

The complete amino acid sequence of thionein-II, one of the two major mouse liver thionein components, was determined. The main fragmentation of thionein-II, which consists of 61 amino acid residues, was accomplished by digesting the S-[14C]-carboxymethylated protein and the cyanogen bromide-treated oxidized protein with trypsin. The peptides obtained by papain digestion of S-[14C]carboxymethylated thionein-II were used to align the major tryptic peptides. The sequence was determined by a combination of automated and manual Edman degradation techniques. Remarkable structural homology is observed in mouse thionein-I, mouse thionein-II, and thioneins from man and horse.

Amino Acid Sequence↗
PubMed1980

Complete amino acid sequence of human phosphoglycerate kinase. Isolation and amino acid sequence of tryptic peptides.

As a part of the elucidation of the complete amino acid sequence of human phosphoglycerate kinase, 46 tryptic peptides, ranging in length from 1 to 26 residues, were isolated and characterized from the reduced and S-carboxymethylated enzyme. The isolated peptides were subjected to sequence analysis by the modified dansyl-Edman degradation procedure and automated Edman degradation technique. The results, together with the data on cyanogen bromide peptides and two additional tryptic peptides from cyanogen bromide peptides reported in the accompanying paper, established the complete amino acid sequence of human erythrocyte phosphoglycerate kinase.

Amino Acid Sequence↗