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Purification and characterization of the human protein tyrosine phosphatase, PTP mu, from a baculovirus expression
1Cold Spring Harbor Labs, NY 11724-2208.
Abstract:
The receptor like PTPase, PTP mu, displays structural similarity in its extracellular segment to members of the immunoglobulin superfamily of cell adhesion molecules. The full length form of PTP mu (200 kD) and a construct expressing only the intracellular PTPase domain-containing segment (80 kD) were expressed in the baculovirus/Sf9 cell system, purified and characterized. Full length PTP mu was membrane associated while the truncated form was recovered in the soluble fraction. PTP mu preferentially dephosphorylated a reduced carboxamidomethylated and maleylated derivative of lysozyme (RCML) over other tyrosine phosphorylated substrates such as myelin basic protein (MBP) or the synthetic peptide EDNDYINASL. The enzymatic properties of the soluble, truncated form of the enzyme were examined in detail. The pH optimum was 7.5. It dephosphorylated RCML with a Km of 400 nM and a Vmax of 725 nmol/min/mg. This form of the enzyme was 2 fold more active than full length PTP mu. Trypsinization of the full length form inhibited activity. Vanadate and molybdate, potent tyrosine phosphatase inhibitors, abolished activity of the enzyme. Zn++ and Mn++ ions, polylysine, poly-glu/tyr, and spermine were also inhibitory.
Insights
Protein tyrosine phosphatase mu (PTP mu) dephosphorylates specific substrates, with activity influenced by pH and inhibitors. Characterizing PTP mu provides insights into its enzymatic function and regulation.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Adhesion
Background:
- Receptor-like protein tyrosine phosphatase mu (PTP mu) shares structural similarities with immunoglobulin superfamily cell adhesion molecules.
- PTP mu plays a role in cellular processes, necessitating a detailed understanding of its enzymatic activity.
Purpose of the Study:
- To express, purify, and characterize both full-length and truncated forms of PTP mu.
- To investigate the substrate specificity and enzymatic properties of PTP mu, particularly its soluble intracellular domain.
Main Methods:
- Expression of full-length and truncated PTP mu using the baculovirus/Sf9 cell system.
- Purification and characterization of recombinant PTP mu variants.
- Enzymatic assays to determine substrate preference, pH optimum, kinetics (Km, Vmax), and inhibition by various agents.
Main Results:
- Full-length PTP mu is membrane-associated, while the truncated form is soluble.
- PTP mu preferentially dephosphorylates a modified lysozyme derivative (RCML) over other tyrosine phosphorylated substrates.
- The soluble PTP mu domain exhibits optimal activity at pH 7.5 with specific kinetic parameters (Km=400 nM, Vmax=725 nmol/min/mg) and is more active than the full-length form.
- Enzyme activity is inhibited by trypsinization, vanadate, molybdate, certain metal ions (Zn++, Mn++), polycations, and poly-glu/tyr.
Conclusions:
- The intracellular domain of PTP mu possesses distinct enzymatic properties and substrate specificity.
- Enzymatic activity of PTP mu is modulated by pH, specific substrates, and various inhibitory molecules, including metal ions and known phosphatase inhibitors.