Proenzyme of Manduca sexta phenol oxidase: purification, activation, substrate specificity of the active enzyme, and molecular cloning.
Phenol oxidase (PO) was isolated as a proenzyme (pro-phenol oxidase, pro-PO) from the hemolymph of Manduca sexta larvae and purified to homogeneity. Pro-PO exhibits a M(r) of 130,000 on gel filtration and two bands with an apparent M(r) of approximately 100,000 on SDS/PAGE, as well as size-exclusion HPLC. Activation of pro-PO was achieved either by specific proteolysis by a cuticular protease or by the detergent cetylpyridinium chloride at a concentration below the critical micellar concentration. A cDNA clone for M. sexta pro-PO was obtained from a larval hemocyte cDNA library. The clone encodes a polypeptide of approximately 80,000 Da that contains two copper-binding sites and shows high sequence similarity to POs, hemocyanins, and storage proteins of arthropods. The M. Sexta pro-PO, together with other arthropod pro-POs, contains a short stretch of amino acids with sequence similarity to the thiol ester region of alpha-macroglobulins and complement proteins C3 and C4.
Hall
M
M
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Scott
T
T
Sugumaran
M
M
Söderhäll
K
K
Law
J H
JH
D000595:Amino Acid Sequence; D000818:Animals; D001181:Arthropods / Q000201:enzymology / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D018402:Manduca / Q000201:enzymology* / Q000235:genetics; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017931:DNA Primers; D004792:Enzyme Precursors; D011994:Recombinant Proteins; D014442:Monophenol Monooxygenase