{"title":"两种尿苷-胞苷激酶亚型在神经母细胞瘤细胞系中的纯化、活性和表达水平","authors":"R. Meinsma, A. V. van Kuilenburg","doi":"10.1080/15257770.2015.1124998","DOIUrl":null,"url":null,"abstract":"ABSTRACT Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme. The UCK1 antibody showed no cross reactivity against UCK2. Our data showed that UCK1 and 2 are both expressed in several neuroblastoma cell lines, including four MYCN single copy cell lines and five MYCN amplified cell lines, with the exception that UCK1 was not expressed in SJNB8. These results indicate that UCK2 in neuroblastoma might be used as a selective target for chemotherapy using UCK2-dependent pyrimidine analogues.","PeriodicalId":19306,"journal":{"name":"Nucleosides, Nucleotides and Nucleic Acids","volume":"49 1","pages":"613 - 618"},"PeriodicalIF":0.0000,"publicationDate":"2016-12-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"8","resultStr":"{\"title\":\"Purification, activity, and expression levels of two uridine-cytidine kinase isoforms in neuroblastoma cell lines\",\"authors\":\"R. Meinsma, A. V. van Kuilenburg\",\"doi\":\"10.1080/15257770.2015.1124998\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"ABSTRACT Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme. The UCK1 antibody showed no cross reactivity against UCK2. Our data showed that UCK1 and 2 are both expressed in several neuroblastoma cell lines, including four MYCN single copy cell lines and five MYCN amplified cell lines, with the exception that UCK1 was not expressed in SJNB8. These results indicate that UCK2 in neuroblastoma might be used as a selective target for chemotherapy using UCK2-dependent pyrimidine analogues.\",\"PeriodicalId\":19306,\"journal\":{\"name\":\"Nucleosides, Nucleotides and Nucleic Acids\",\"volume\":\"49 1\",\"pages\":\"613 - 618\"},\"PeriodicalIF\":0.0000,\"publicationDate\":\"2016-12-01\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"8\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Nucleosides, Nucleotides and Nucleic Acids\",\"FirstCategoryId\":\"1085\",\"ListUrlMain\":\"https://doi.org/10.1080/15257770.2015.1124998\",\"RegionNum\":0,\"RegionCategory\":null,\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"\",\"JCRName\":\"\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Nucleosides, Nucleotides and Nucleic Acids","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1080/15257770.2015.1124998","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
Purification, activity, and expression levels of two uridine-cytidine kinase isoforms in neuroblastoma cell lines
ABSTRACT Uridine-cytidine kinase (UCK) catalyzes the phosphorylation of uridine, cytidine, and several pyrimidine ribonucleoside analogs. We overexpressed and purified the two known isoforms of human UCK in Escherichia coli, produced a specific antibody against UCK1 and characterized the kinetic properties of UCK1 and 2. The Vmax of purified recombinant UCK2 was 22- and 8-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK1 enzyme. The Km of UCK1 was 39- and 40-fold higher with uridine and cytidine, respectively, compared to those observed for the purified recombinant UCK2 enzyme. The UCK1 antibody showed no cross reactivity against UCK2. Our data showed that UCK1 and 2 are both expressed in several neuroblastoma cell lines, including four MYCN single copy cell lines and five MYCN amplified cell lines, with the exception that UCK1 was not expressed in SJNB8. These results indicate that UCK2 in neuroblastoma might be used as a selective target for chemotherapy using UCK2-dependent pyrimidine analogues.
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