Purification of the major protein-tyrosine-phosphatases of human placenta.

N K Tonks,C D Diltz,E H Fischer

doi:10.1016/s0021-9258(18)68702-2

N K Tonks, C D Diltz + Show 1 more

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https://doi.org/10.1016/s0021-9258(18)68702-2

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Abstract

This report describes the purification of the major protein-tyrosine-phosphatases from human placenta. Enzyme activity was followed with a novel artificial substrate, namely reduced, carboxamidomethylated, and maleylated lysozyme, phosphorylated on tyrosine by a partially purified preparation of insulin and epidermal growth factor receptor kinases, also from human placenta. The key step in the purification of the protein-tyrosine-phosphatases was affinity chromatography on a column of thiophosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme-Sepharose. Purification was carried out separately from both the soluble and particulate fractions. Whereas multiple and distinct enzyme forms were obtained from each of these, little difference could be detected between the behavior of the "soluble" enzyme subtypes and their "particulate" counterparts. The major subtypes were purified to apparent homogeneity with an approximately 23,000-fold enrichment and 10% yield from the soluble fraction and a 4,300-fold enrichment and 13% yield from the particulate fraction. Both samples migrated as bands of 35 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and had specific activities of approximately 45,000 nmol of Pi released min-1 mg-1, at least 2-3-fold higher than that of the type 1 and 2A serine/threonine phosphatases. The level of protein-tyrosine-phosphatases in the soluble fraction of human placenta (2,000 units/g of protein) was approximately the same as protein-serine/threonine-phosphatases 1 and 2A in skeletal muscle.

Highlights

We introduce a novel substrate which can be conveniently used in routine assays of PTPase activity: RCM lysozyme
Affinity columns of phosphorylated substrate coupled to Sepharose were ineffective because the ligand was rapidly dephosphorylated, and enzyme activity aprather than thegradient elution currentlyused, may speed up this stepwithout sacrificing yield or enrichment
The a c f ~ v e fractions were combined a5 Indicated and dialyzed against buffer E

Summary

DISCUSSION

The study of PTPases to date habseen limited by the lack of any characterized physiological substrates that may be prepared in sufficient quantities for routine use. The alternative has been to use artificial substrates such as casein and histone, as well as proteins that are npoht osphorylated until they have been chemically modified so as to expose their. A total of 6.5 kg of placental tissue was used in this preparation. Material was processed through steps 1and 2 in small batches of < 1kg and pooled. Yield and purification are expressed relative to thecombined pool obtained at the endof step 2. PTPase activity was measured in thepresence of 5 mM EDTA throughout.

Affi-Gel blue

RESULTS

E IO a a 0 ta 30