In Vitro Enzymatic Assays of Protein Tyrosine Phosphatase 1B

Abstract

Many hormone or growth factor receptors signal via the activation of protein-tyrosine kinases and phosphatases. Alteration of the phosphorylation state of tyrosine residues in certain proteins can directly regulate enzyme activity or cause formation of protein complexes necessary for transducing intracellular signals. Genetic and biochemical evidence also implicates protein-tyrosine phosphatases in several disease processes, including negative regulation of insulin receptor signaling at the level of the insulin receptor and perhaps in signaling at the IRS-1 level. The expression of protein tyrosine phosphatase-1B (PTP1B) is elevated in muscle and adipose tissue in insulin-resistant states both in man and rodents suggesting that PTP1B may play a role in the insulin-resistant state associated with diabetes and obesity. As described in this unit, PTP1B activity can be determined with the small molecule substrate, p-nitrophenyl phosphate (pNPP), in which the cleavage of the phosphate results in production of p-nitrophenol (pNP) and an increase in absorbance at 405 nm. Alternatively, PTP1B activity can be measured as described using model phosphotyrosyl-containing peptide substrates in which the release of free phosphate from the peptide is determined using a malachite green colorimetric assay.

Connecting Quorum Sensing, c-di-GMP, Pel Polysaccharide, and Biofilm Formation in Pseudomonas aeruginosa through Tyrosine Phosphatase TpbA (PA3885)

“…tpbA encodes a 218 aa protein that has the conserved domain for a protein tyrosine phosphatase [28] , [29] since it has the C(X) 5 R(S/T) motif beginning at aa 132 ( C KHGNN RT ). To confirm it is a tyrosine phosphatase, we purified TpbA by adding a polyhistidine tag at either the N-terminus (TpbA-nHis) or the C-terminus (TpbA-cHis) (note only the C-terminus fusion protein was active).…”

“…6A ). The purified TpbA protein had phosphatase activity with p -nitrophenyl phosphate (pNPP) that is often used as a general phosphatase substrate [29] ( Fig. 6B ).…”

Ag(I)‐Catalyzed/Acid‐Mediated Cascade Cyclization of ortho‐Alkynylaryl‐1,3‐dicarbonyls to Access Arylnaphthalenelactones and Furanonaphthol Libraries via Aryl‐Disengagement

“…The results were shown in Table 6. Notably, 3 c , 3 j , and 3 o showed approximately four times stronger inhibitory activities towards PTP1B enzyme than compound 9 j and more than double potency of ursolic acid, [9] a known inhibitor of PTP1B. The results suggested that this class of compounds could be developed as potential PTP1B inhibitors.…”

“…In this work, an ingenious method was reported for the construction of arylnaphthenelactone 3 using ortho ‐alkynylarylketones 8 as the starting material. Substrate 8 could be prepared from inexpensive and readily available dimethyl malonate 7 which could be easily functionalized at R 3 to increase the diversity of the desired products [7] . Previously, our research group reported the synthesis of naphthofurans 5 and 6 using ortho ‐alkynylarylketones 4 as the starting material [5d] .…”

Polyhydroxy p-Terphenyls from a Mangrove Endophytic Fungus Aspergillus candidus LDJ-5

“…The enzyme activity was estimated by measuring the absorbance at 405 nm with appropriate corrections. Each experiment was performed in triplicate, and IC 50 data were derived from three independent experiments [34].…”