Abstract
Guanylate kinase catalyzes the phosphorylation of either GMP to GDP or dGMP to dGDP and is an essential enzyme in nucleotide metabolism pathways. Despite its involvement in antiviral drug activation in humans and in mouse model systems and as a target for chemotherapy, the human and mouse primary structures have never been elucidated. Full-length cDNA clones encoding enzymatically active guanylate kinase were isolated from mouse B-cell lymphoma and human peripheral blood lymphocyte cDNA libraries. Multiple tissue Northern blots demonstrated an mRNA species of approximately 1 kilobase for both mice and humans in all tissue types examined. The mouse cDNA is predicted to encode a 198-amino acid protein with a molecular mass of 21,904 daltons. The human cDNA is predicted to encode a 197-amino acid protein with a molecular mass of 21,696 daltons. These proteins share 88% sequence identity with each other and 52-54% identity with the yeast guanylate kinase. Molecular modeling using the yeast diffraction coordinates indicates a high degree of conservation within the active site and maintenance of the overall structural integrity, despite a lack of similarity along the periphery of the enzyme.
Highlights
As with other enzymes involved in nucleotide metabolism, guanylate kinase is a target for cancer chemotherapy and is inhibited by the potent antitumor drug, 6-thioguanine [3,4,5]
We describe the isolation, expression, and functional analysis of both human and mouse guanylate kinases in an effort to aid improvements in antiviral and tumor chemotherapeutic approaches through molecular modeling and rational drug design
This is in close agreement with the bovine gmk that encodes a 198-amino acid protein with a calculated molecular mass of 22,051 daltons [1]
Summary
Materials—Restriction enzymes, deoxynucleoside triphosphates, and random-primed DNA labeling kits were purchased from Boehringer Mannheim. Vector Constructions—The NcoI/BamHI fragment from pUC118: hgmk was gel purified and ligated to NcoI/BamHI-digested pET23d and used to transform BL21(DE3) tkϪ cells. The resulting plasmid was sequenced and designated pETHT Both human and mouse guanylate kinase cDNAs were cloned as NcoI/BamHI fragments isolated from pET23d:hgmk or pET23d:mgmk into the NcoI/BamHI sites of pETHT. The resulting 7-kilobase plasmid was designated pREP8⌬7 Both guanylate kinase cDNAs were cloned into the HindIII (blunt)/ BamHI sites of pREP8⌬7 as NcoI(blunt)/BamHI fragments. A small amount (8000 cell eq) was removed and subjected to polyacrylamide gel electrophoresis, followed by immunoblot analysis using a 1:5000 dilution of either preimmune serum or anti-MGMK serum These lysates were used for determination of guanylate kinase activity. The change in A340 was measured over time with cell lysates as the source of GMK enzyme on a Hewlett-Packard model 8452A diode array spectrophotometer (Hewlett-Packard, Fullerton, CA)
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