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

TLDR

The study aims to purify the major protein‑tyrosine‑phosphatases from human placenta. The phosphatases were isolated using a novel artificial substrate—thiophosphorylated, reduced, carboxamidomethylated, maleylated lysozyme—phosphorylated by placenta‑derived kinases, followed by affinity chromatography on lysozyme‑Sepharose, with separate purification of soluble and particulate fractions. The purification yielded two major 35‑kDa phosphatase subtypes with ~45,000 nmol Pi min⁻¹ mg⁻¹ activity, 2–3 fold higher than serine/threonine phosphatases, and the placenta fraction contained ~2,000 units g⁻¹ protein, comparable to skeletal muscle serine/threonine phosphatases.

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 and 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 fraction and a 4,300-fold enrichment and 13% yield from the 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 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.