Lipase activities of p37, the major envelope protein of vaccinia virus.
p37, the major protein of the extracellular enveloped form of vaccinia virus, is involved in the biogenesis of the viral double membrane and in egress of virus from the cell. p37 was expressed as a glutathione S-transferase fusion protein and was purified to homogeneity by silver staining using glutathione-agarose, Sephacryl S-200, and DEAE-cellulose chromatography. Incubation of p37 with phosphatidylcholine labeled in the fatty acyl side chains resulted in the production of multiple lipid products that were identified by thin layer chromatography and mass spectrometry as diacylglycerol, free fatty acid, monoacylglycerol, and lysophosphatidylcholine. Lipid-metabolizing activities colocalized with p37-containing fractions throughout the chromatographic steps. p37 also metabolized phosphatidylethanolamine efficiently, but it had less activity toward phosphatidylinositol and little or no activity toward phosphatidylserine. The purified enzyme also metabolized triacylglycerol to diacylglycerol but was inactive toward sn-1, 2-diacylglycerol. p37 was also expressed in insect cells as a poly-His fusion protein; cell lysates and partially purified proteins also generated products expected from phospholipase C and A activities. Thus, p37 is a broad specificity lipase with phospholipase C, phospholipase A, and triacylglycerol lipase activities.
Baek
S H
SH
Department of Biochemistry, Emory University Medical School, Atlanta, Georgia 30322, USA.
Kwak
J Y
JY
Lee
S H
SH
Lee
T
T
Ryu
S H
SH
Uhlinger
D J
DJ
Lambeth
J D
JD
D000595:Amino Acid Sequence; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D002855:Chromatography, Thin Layer; D005982:Glutathione Transferase / Q000378:metabolism; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014280:Triglycerides / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism*; D014616:Vaccinia virus / Q000201:enzymology*; D014759:Viral Envelope Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D008565:Membrane Proteins; D011993:Recombinant Fusion Proteins; D014280:Triglycerides; D014759:Viral Envelope Proteins; C084759:p37 protein, Vaccinia virus; D005982:Glutathione Transferase; D010741:Phospholipases A; D010738:Type C Phospholipases