A molecular biology-based approach to resolve the subunit orientation of lipoprotein lipase.
The subunit orientation of a dimeric enzyme influences the mechanism of action and function. To determine the subunit arrangement of lipoprotein lipase (LPL), a molecular biology-based approach was initiated. An eight amino acid linker region was engineered between two LPL monomers and expressed in COS-7 cells. The resultant tandem-repeat molecule (LPLTR) was lipolytically active and had kinetic parameters, salt inhibition, cofactor-dependent activity, heparin-binding characteristics, and a functional unit size very similar to the expressed native human enzyme. By these criteria, LPLTR was the functional equivalent of native LPL. Considering the length of the linker peptide (no more than 24 A), monomers in the tethered molecule were restricted to a head-to-tail subunit arrangement. Since LPLTR demonstrated native enzyme-like properties while constrained to this subunit arrangement, these results provide the first compelling evidence that native LPL monomers are arranged in a head-to-tail subunit orientation within the active dimer. Thus, LPL function in physiology, lipolysis, and binding to cell-surface components must now be addressed with this subunit orientation in mind. The utility of the tandem-repeat approach to resolve the subunit arrangement of an obligate dimer has been demonstrated with LPL and could be generalized for use with other oligomeric enzymes.
Wong
H
H
Lipid Research Laboratory, West Los Angeles Veterans Affairs Medical Center, Los Angeles, CA 90073, USA. wongh@ucla.edu
Yang
D
D
Hill
J S
JS
Davis
R C
RC
Nikazy
J
J
Schotz
M C
MC
D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D017931:DNA Primers; D019281:Dimerization; D006801:Humans; D008071:Lipoprotein Lipase / Q000096:biosynthesis / Q000737:chemistry* / Q000302:isolation & purification; D046911:Macromolecular Substances; D008967:Molecular Biology / Q000379:methods; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000302:isolation & purification; D014162:Transfection
D017931:DNA Primers; D046911:Macromolecular Substances; D011994:Recombinant Proteins; D008071:Lipoprotein Lipase