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101.
A cDNA encoding human delta-aminolevulinic acid dehydratase (ALA-D; EC 4.2.1.24), the second enzyme in the heme biosynthetic pathway, was isolated from a human liver cDNA expression library. Of the original 17 clones selected with anti-ALA-D antibody, only four expressed anti-ALA-D epitopes as assessed by rescreening with antibody preabsorbed with purified antigen. Subsequent screening of the antibody-positive clones with mixed oligodeoxynucleotide (oligo) probes, synthesized to correspond to human N-terminal and bovine active-site peptide sequences, identified three clones which hybridized only with the oligo probes for the bovine amino acid (aa) sequences. Restriction endonucleases analysis revealed that these three clones contained the same 800-bp cDNA insert. This insert was recloned into bacteriophage M13mp18 and mp19 and sequenced by primer extension. The aa sequence predicted from the partial nucleotide sequence was found to be essentially colinear with the sequences of four bovine ALA-D peptides, totaling 35 non-overlapping aa residues. 相似文献
102.
Human arylsulfatase B: MOPAC cloning, nucleotide sequence of a full-length cDNA, and regions of amino acid identity with arylsulfatases A and C 总被引:13,自引:0,他引:13
cDNAs encoding the human lysosomal hydrolase, arylsulfatase B (ASB; N-acetylgalactosamine-4-sulfatase, EC 3.1.6.1), were isolated from a hepatoma cell cDNA library using an ASB-specific oligonucleotide generated by the MOPAC (mixed oligonucleotide primed amplification of cDNA) technique. To facilitate cDNA cloning, human ASB was purified to apparent homogeneity and a total of 112 amino acid residues were microsequenced from the N-terminus and four internal tryptic peptides of the 47-kDa subunit. Based on the ASB N-terminal amino acid sequence, two oligonucleotide mixtures containing inosines to reduce the mixture complexity were constructed and used as primers to amplify an ASB-specific product from human placental cDNA by the polymerase chain reaction. DNA sequencing of this MOPAC product demonstrated colinearity with 21 N-terminal ASB amino acids. Based on this sequence and on codon usage for the adjacent conserved amino acids in human arylsulfatases A and C, a unique 66-mer was synthesized and used to screen a human hepatoma cell cDNA library. Four putative positive cDNA clones were isolated, and the largest insert (pASB-1) was sequenced in both orientations. The 1834-bp pASB-1 insert had a 1278-bp open reading frame encoding 425 amino acids that was colinear with 85 microsequenced amino acids of the purified enzyme, demonstrating its authenticity. Using the pASB-1 cDNA as a probe, a full-length cDNA clone, pASB-4, was isolated from a human testes library and sequenced in both orientations. pASB-4 had a 2811-bp insert containing a 559-bp 5' untranslated sequence, a 1602-bp open reading frame encoding 533 amino acids (six potential N-glycosylation sites), a 641-bp 3' untranslated sequence, and a 9-bp poly(A) tract. Comparison of the predicted amino acid sequences of arylsulfatases A, B, and C revealed regions of identity, particularly in their N-termini. 相似文献
103.
Assignment of the structural gene encoding human aspartylglucosaminidase to the long arm of chromosome 4 (4q21----4qter) 总被引:3,自引:0,他引:3
The structural gene for the human lysosomal enzyme aspartylglucosaminidase (AGA) has been assigned to chromosome 4 using somatic cell hybridization techniques. The human monomeric enzyme was detected in Chinese hamster-human cell hybrids by a thermal denaturation assay that selectively inactivated the Chinese hamster isozyme, while the thermostable human enzyme retained activity. Twenty informative hybrid clones, derived from seven independent fusions, were analyzed for the presence of human AGA activity and their human chromosomal constitutions. Without exception, the presence of human AGA in these hybrids was correlated with the presence of human chromosome 4. All other human chromosomes were excluded by discordant segregation of the human enzyme and other chromosomes. Two hybrid clones, with interspecific Chinese hamster-human chromosome translocations involving the long arm of human chromosome 4, permitted the assignment of human AGA to the region 4q21----4qter. 相似文献
104.
Nonparametric regression in the presence of measurement error 总被引:4,自引:0,他引:4
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106.
Uroporphyrinogen III synthase (URO-synthase) catalyzes the cyclization and D-ring isomerization of hydroxymethylbilane (HMB) to uroporphyrinogen (URO'gen) III, the cyclic tetrapyrrole and physiologic precursor of heme, chlorophyl, and corrin. The deficient activity of human URO-synthase results in the autosomal recessive cutaneous disorder, congenital erythropoietic porphyria. Mapping of the structural determinants that specify catalysis and, potentially, protein-protein interactions is lacking. To map the active site and assess the enzyme's possible interaction in a complex with hydroxymethylbilane-synthase (HMB-synthase) and/or uroporphyrinogen-decarboxylase (URO-decarboxylase) by NMR, an efficient expression and purification procedure was developed for these cytosolic enzymes of heme biosynthesis that enabled preparation of special isotopically-labeled protein samples for NMR characterization. Using an 800 MHz instrument, assignment of the URO-synthase backbone (13)C(alpha) (100%), (1)H(alpha) (99.6%), and nonproline (1)H(N) and (15)N resonances (94%) was achieved as well as 85% of the side-chain (13)C and (1)H resonances. NMR analyses of URO-synthase titrated with competitive inhibitors N(D)-methyl-1-formylbilane (NMF-bilane) or URO'gen III, revealed resonance perturbations of specific residues lining the cleft between the two major domains of URO synthase that mapped the enzyme's active site. In silico docking of the URO-synthase crystal structure with NMF-bilane and URO'gen III was consistent with the perturbation results and provided a 3D model of the enzyme-inhibitor complex. The absence of chemical shift changes in the (15)N spectrum of URO-synthase mixed with the homogeneous HMB-synthase holoenzyme or URO-decarboxylase precluded occurrence of a stable cytosolic enzyme complex. 相似文献
107.
Synthesis of a fluorescent derivative of glucosyl ceramide for the sensitive determination of glucocerebrosidase activity 总被引:7,自引:0,他引:7
A fluorescent derivative of glucosyl ceramide was synthesized by covalently linking a fluorescent fatty acid, 12-[N-methyl-N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)] aminododecanoic acid to the amino group of sphingosyl-1-O-beta-D-glucoside, glucosyl sphingosine. For hydrolysis by glucocerebrosidase, this substrate was dispersed in mixed micelles with Triton X-100 and sodium taurocholate or in unilamellar liposomes with phosphatidylcholine and the negatively charged lipid, dicetylphosphate. In either micellar or liposomal dispersions of the fluorescent substrate, reaction rates were linear with time and protein concentration, and saturation kinetics were observed. The rate of hydrolysis of this fluorescent substrate was equal to that obtained with radiolabeled glucosyl ceramide. The fluorescent glucosyl ceramide was used to determine glucocerebrosidase activity in extracts of human leukocytes, cultured skin fibroblasts, and various tissues as well as in partially purified splenic and placental glucocerebrosidase preparations. This fluorescent derivative of the natural substrate was not hydrolyzed by aryl beta-glucosidase(s), thereby facilitating the specific and reliable diagnosis of heterozygotes and homozygotes with Gaucher disease. 相似文献
108.
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110.
Adel Shalata Maria?C. Ramirez Robert?J. Desnick Nolan Priedigkeit Christoph Buettner Claudia Lindtner Mohammed Mahroum Muhammad Abdul-Ghani Feng Dong Nazik Arar Olga Camacho-Vanegas Rui Zhang Sandra?C. Camacho Ying Chen Mwafaq Ibdah Ralph DeFronzo Virginia Gillespie Kevin Kelley Brian?D. Dynlacht Sehyun Kim Marc?J. Glucksman Zvi?U. Borochowitz John?A. Martignetti 《American journal of human genetics》2013,93(6):1061-1071
Obesity is a major public health concern, and complementary research strategies have been directed toward the identification of the underlying causative gene mutations that affect the normal pathways and networks that regulate energy balance. Here, we describe an autosomal-recessive morbid-obesity syndrome and identify the disease-causing gene defect. The average body mass index of affected family members was 48.7 (range = 36.7–61.0), and all had features of the metabolic syndrome. Homozygosity mapping localized the disease locus to a region in 3q29; we designated this region the morbid obesity 1 (MO1) locus. Sequence analysis identified a homozygous nonsense mutation in CEP19, the gene encoding the ciliary protein CEP19, in all affected family members. CEP19 is highly conserved in vertebrates and invertebrates, is expressed in multiple tissues, and localizes to the centrosome and primary cilia. Homozygous Cep19-knockout mice were morbidly obese, hyperphagic, glucose intolerant, and insulin resistant. Thus, loss of the ciliary protein CEP19 in humans and mice causes morbid obesity and defines a target for investigating the molecular pathogenesis of this disease and potential treatments for obesity and malnutrition. 相似文献