Analysis And Microsequence Of Human Erythrocyte Pyrimidine5'-Nucleotidase Protein And Construction Of CDNA Library

Human erythrocyte pyrimidine 5'-nucleotidase (P5'N) deficiency may results in hereditary nonspheroeytie hemolytic anemia(HNSHA). The incidence rate of P5'N deficiency is the third in the hereditary erythrocyte enzyme disease. Up to date over 70 cases of P5' N deficiency from 35 families have been described. Two types of human red cell P5' N isoenzymes, termed P5' N- Ⅰ and P5' N-Ⅱ, are identified on the basis of their different molecular properties and substrate speeificities. The deficiency of P5'N- Ⅰ is associated to a hemolytic nonspherocytic anemia , while the active of P5'N-Ⅱ is normal. The pathogenesis of P5'N deficiency is still not clear. To further study the P5' N biochemical characters and it' s pathogenesis in HNSHA, we evaluated the properties and microsequence of P5' N-Ⅰ , investigated the family of a subject with P5' N-Ⅰ deficiency and constructed a correspondence cDNA library.Method:Ⅰ .Construction of specific affinity chromatography in order topurificate human erythrocyte P5' N- ⅠⅡ. Analysis and microsequence of human erythrocyte P5' N-Ⅰ protein1.Analysis of pure P5' N- Ⅰ protein by ESI-Mass Spectrometry2.Microsequencing of pure P5' N- Ⅰ protein by direct to detect and transplant to PVDF membrane or fragment hydrolysised by trypsin and collected by HPLC.3.Analysis of the sequence by bioinformatics in order to search homology fragment.4.Analysis of amino acid conformation by acid hydrolysis the enzyme protein.Ⅲ.Analysis of the enzyme active and enzyme protein quantity, observed the form of peripheral blood erythrocyte in the family of a subject with P5' N- Ⅰ deficiency.Ⅳ.Construction of the cDNA library of normal human which mRNA obtained from CD34+ cell of his peripheral blood mobilizated by G-CSF and3GM-CSF.Result:Ⅰ Cross-linking rate is 40%. Pure P5' N-Ⅰ protein show as a single band in SDS-PAGE gel.Ⅱ.Analysis and microsequence of human erythrocyte P5'N-Ⅰ protein1.The molecular weight of P5' N-Ⅰ was detected as 26952. 5, while there also existed two fragment of 55476 and 110938. 2.There were 10 amino acid showed by the microsequecing. The sequence is Ile-Glu-Gly-Pro-Thr-Ile-Arg-GlN-Ile-Glu. 3.The homologous sequence has not found compared with the ten-amino-acid sequence in Genebank by blast procedure. 4. The amino acid component of P5' N-Ⅰ include 18 species and 243 pieces amino acid residues per subunit.Ⅲ.A heterozygote whose enzyme active was less than 50% of normal value and enzyme content less than 50% of contrast was found in the family investigation.Ⅳ.The volume of cDNA was 2. 8x10～6 pfu/ug. The rate of full-length in insert was 77. 5%, the average length was 1235bp.Conclusion:1.P5' N-Ⅰ , which produce a marked effect in physiological status of normal human erythrocyte, may be as homologous tetramer. The enzyme may be an allosteric enzyme.2.The 10-aa peptide sequence obtained by microsequencing can be designed as the probe for screening purpose gene. The compoent of amino acid may apply an important imformation in definition of it' s protein primary stucture.3.In addition to assay of the enzyme active , the determination of the P5' N-Ⅰ protein content may be a useful method in detection of the heterozygote.4.The volume , integrity and the rate of full-length of the cDNA library constructed by normal human peripheral blood CD34～+ cell had4been achived the standard which can use to screen the goal gene.