GDP-mannose pyrophosphorylase. Purification to homogeneity, properties, and utilization to prepare photoaffinity analogs.
Pig liver GDP-mannose pyrophosphorylase was purified 5,000-fold to apparent homogeneity using standard techniques. The native enzyme showed a single band on gels of about 450 kDa and two subunits of 43 and 37 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 37-kDa (beta-) subunit had only methionine at its amino terminus and a surprisingly hydrophobic sequence: Met-Lys-Ala-Leu-Ile-Leu-Val-Gly-Gly-Tyr-Gly-Thr-Arg-Leu- Arg-Pro-Leu-Thr-Leu-Ser-Ile-Pro-Lys. The 43-kDa (alpha-) subunit was blocked at the amino terminus, but a 29-kDa CNBr fragment had the following sequence: Leu-Asp-Ala-His-Arg-His-Arg-Pro-His-Pro- Phe-Leu-Leu-. Substrate specificity studies done in the direction of formation of nucleoside triphosphate and sugar-1-P indicated that the enzyme was most effective with GDP-glucose as substrate (100%) followed by IDP-mannose (72%) and then GDP-mannose (61%). That GDP-mannose and GDP-glucose activities were indeed catalyzed by the same enzyme was indicated by the following. (i) Various studies indicated that the enzyme was homogeneous. (ii) A staining procedure for production of GTP stained the same single band on native gels when either GDP-mannose or GDP-glucose was the substrate. (iii). GDP-mannose inhibited the utilization of GDP-glucose by the enzyme, and vice versa. When 8-azido-[32P]GTP was incubated with native enzyme and exposed to UV light, both the 43-kDa and the 37-kDa subunits became labeled, although the 37-kDa subunit reacted more strongly. On the other hand, 8-azido-GDP-[32P]mannose only photolabeled the 43-kDa band. Most importantly, the purified enzyme can be utilized to produce 8-azido-[32P]GDP mannose or 8-azido-[32P]GDP glucose.
Szumilo
T
T
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205-7199.
Drake
R R
RR
York
J L
JL
Elbein
A D
AD
D000345:Affinity Labels / Q000138:chemical synthesis*; D000595:Amino Acid Sequence; D000818:Animals; D001386:Azides / Q000138:chemical synthesis*; D002250:Carbon Radioisotopes; D002845:Chromatography; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D006155:Guanosine Diphosphate Mannose / Q000031:analogs & derivatives* / Q000138:chemical synthesis; D006156:Guanosine Diphosphate Sugars / Q000138:chemical synthesis*; D006160:Guanosine Triphosphate / Q000031:analogs & derivatives*; D006882:Hydroxyapatites; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011487:Protein Conformation; D013379:Substrate Specificity; D013552:Swine
C021961:8-azidoguanosine triphosphate; D000345:Affinity Labels; D001386:Azides; D002250:Carbon Radioisotopes; D006156:Guanosine Diphosphate Sugars; D006882:Hydroxyapatites; D046911:Macromolecular Substances; D010761:Phosphorus Radioisotopes; C082643:8-azidoguanosine diphosphate-mannose; C082645:8-azidoguanosine diphosphate-glucose; D006155:Guanosine Diphosphate Mannose; D006160:Guanosine Triphosphate; D017886:Durapatite; D009713:Nucleotidyltransferases; C026917:mannose 1-phosphate guanylyltransferase