Catalytic domains of the LAR and CD45 protein tyrosine phosphatases from Escherichia coli expression systems: Purification and characterization for specificity and mechanism

Journal Article · Tue Jan 14 00:00:00 EST 1992 · Biochemistry; (United States)

DOI:https://doi.org/10.1021/bi00116a019· OSTI ID:5688173

Cho, Hyeongjin; Ramer, S E; Itoh, Michiyasu; Saito, Haruo; Walsh, C T ^[1]; Kitas, E; Bannwarth, W; Burn, P ^[2]

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1. Harvard Medical School, Boston, MA (United States)
2. Hoffmann-La Roche, Basel (Switzerland)

The cytoplasmic domains of two human transmembrane protein tyrosine phosphatases (PTPases), LAR and CD45, have been expressed in Escherichia coli, purified to near-homogeneity, and compared for catalytic efficiency toward several phosphotyrosine-containing peptide substrates. A 615-residue LAR fragment (LAR-D1D2) containing both tandemly repeated PTPase domains shows almost identical specific activity and high catalytic efficiency as the 40-kDa single-domain LAR-D1 fragment, consistent with a single functional active site in the 70-kDa LAR-D1D2 enzyme. A 90-kDa fragment of the human leukocyte CD45 PTPase, containing two similar tandemly repeated PTPase domains, shows parallel specificity to LAR-D1 and LAR-D1D2 with a high k{sub cat}/K{sub M} value for a phosphotyrosyl undecapeptide. Sufficient purified LAR-D1 and LAR-D1D2 PTPases were available to demonstrate enzymatic exchange of {sup 18}O from {sup 18}O{sub 4} inorganic phosphate into H{sub 2} {sup 16}O at rates of {approximately}1 {times} 10{sup {minus}2} s{sup {minus}1}. The oxygen-18 exchange probably proceeds via a phosphoenzyme intermediate. Brief incubation of all three PTPase fragments with a ({sup 32}P)phosphotyrosyl peptide substrate prior to quench with SDS sample buffer and gel electrophoresis led to autoradiographic detection of {sup 32}P-labeled enzymes. Pulse/chase studies on the LAR {sup 32}P-enzyme showed turnover of the labeled phosphoryl group.

• OSTI ID: 5688173
• Journal Information: Biochemistry; (United States), Vol. 31:1; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
MEMBRANE PROTEINS
AUTORADIOGRAPHY
OXYGEN 16
ISOTOPIC EXCHANGE
PHOSPHATASES
BIOCHEMICAL REACTION KINETICS
ESCHERICHIA COLI
MAN
PHOSPHORUS 32
TYROSINASE
ANIMALS
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
DAYS LIVING RADIOISOTOPES
ENZYMES
ESTERASES
EVEN-EVEN NUCLEI
HYDROLASES
HYDROXYLASES
ISOTOPES
KINETICS
LIGHT NUCLEI
MAMMALS
MICROORGANISMS
NUCLEI
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
OXYGEN ISOTOPES
PHOSPHORUS ISOTOPES
PRIMATES
PROTEINS
RADIOISOTOPES
REACTION KINETICS
STABLE ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques