Characterization of rat adipose tissue lipoprotein lipase using a monospecific antibody.
An antibody to a highly pure enzyme preparation was developed to facilitate detailed studies of rat adipose tissue lipoprotein lipase regulation. Lipoprotein lipase was purified by heparin-Sepharose affinity chromatography followed by preparative isoelectric focusing. The enzyme migrated as a single broad band on SDS disc gel and two-dimensional gel electrophoresis with an apparent molecular mass of 67 000 and 62 000 Da, respectively. The amino acid composition of the purified rat enzyme was virtually identical to that of bovine milk. A major protein component with no lipase activity co-eluted with the enzyme from the affinity column, but was separated by the isoelectric focusing step. The molecular mass was slightly lower (58 000 Da) but the amino acid composition of this protein was similar to that of the enzyme. An antibody raised against the purified rat enzyme was highly potent and was effective in inhibiting rat heart lipoprotein lipase, but not the salt-resistant hepatic lipase. Analysis of crude acetone-ether adipose tissue preparation on SDS slab polyacrylamide gel coupled to Western blotting revealed five protein bands = (62 000, 56 000, 41 700, 22 500, 20 000 Da). Similarly, following affinity purification by immunoadsorption, the purified antibody reacted with five equivalent protein bands. Fluorescent concanavalin A binding data indicated that the 56 kDa band is a glycosylated form of lipoprotein lipase. Pretreatment of adipose tissue with proteinase inhibitors revealed that the lower molecular mass proteins (41 700 and 20 000 Da) were degradation products of lipoprotein lipase, and the 22 500 Da band could be accounted for by non-specific binding.
N A
NA
Vydelingum
F
F
AlQuadan
A H
AH
Kissebah
J
J
Etienne
D000273:Adipose Tissue / Q000201:enzymology*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D000911:Antibodies, Monoclonal; D002241:Carbohydrates / Q000032:analysis; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D007525:Isoelectric Focusing; D008071:Lipoprotein Lipase / Q000032:analysis* / Q000276:immunology; D008297:Male; D008892:Milk / Q000201:enzymology; D008970:Molecular Weight; D051381:Rats; D011919:Rats, Inbred Strains
D000596:Amino Acids; D000911:Antibodies, Monoclonal; D002241:Carbohydrates; D008071:Lipoprotein Lipase