GDP-mannose pyrophosphorylase. Purification to homogeneity, properties, and utilization to prepare photoaffinity analogs.

T Szumilo,R.R Drake,J.L York,A.D Elbein

doi:10.1016/s0021-9258(17)46796-2

T Szumilo, R.R Drake + Show 2 more

Open Access

https://doi.org/10.1016/s0021-9258(17)46796-2

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Abstract

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.

Highlights

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 43-kDa (a-) subunit was blocked at the amino terminus, but a 29-kDa CNBr fragment had the following sequence: Leu-Asp-Ala
We describe the 5,000-fold purification to apparent homogeneity of the pig liver GDP-mannose pyrophosphorylase and the properties of the purified protein

Summary

PROCEDURES

Mmol) were obtained from ICN. [U-‘4C]Mannose l-phosphate was obtained by hydrolysis of GDP-r”Clmannose with snake venom phosphodie&&e. The incubation mixtures contained the following components in afinal volume of 50 gl: 5 mM sodium pyrophosphate, 100 KM GDP-[U-'4C]mannose (5.8 Cilmol), 100 mM Tris-HCI buffer, pH 7.5, 4 mM MgCI2,and various amounts of the enzyme at different steps in the purification. In thiscase,the assay mixture contained the following components in a final volume of I ml: 50 mM Tris-HC1 buffer, pH 7.5, 5 mM MgCI,, 0.1 mM ADP, 1mM glucose, 0.5 mM NADP, 1 mM inorganic pyrophosphate, a 0.2 mM concentration of GDP-mannose or GDP-glucose,and 2.5 units each of nucleoside diphosphate kinase, hexokinase, and glucose-6-P dehydrogenase. Incubation mixtures contained the following components in a final volume of 60 &I: 125 mM TrisHCl buffer, pH 7.5, 4 mM MgCl,, 10 mM GTP, 2 mM [U-'4C-sugar-lP (1.34 Ci/mmol), 1 unit of inorganic pyrophosphatase, and various amounts of the pyrophosphorylase, a t various stages of purification. Units are expressed as pmol of mannose 1-phosphateformed/min

Blue Sepharose

RESULTS

C GDP-Glc