Isolation and Characterization of Fatty Acid Binding Protein From Lactating Bovine Mammary Gland

Abstract

An intracellular protein which binds fatty acids had been found in a wide range of organisms and tissues. Its presence had not been documented in mammary tissue. The purpose of this study was to determine whether fatty acid binding protein (FABP) exists in bovine mammary tissue and if so, to isolate and characterize it. Mammary tissue was obtained at slaughter from five cows from the University dairy herd which were producing 5-25 kg milk/day. The tissue was homogenized and centrifuged to obtain the cytosolic and microsomal fractions. FABP was isolated from the cytosolic fraction by gel filtration on a Sephadex G-75 column followed by ion exchange chromatography on DEAE Sephadex A-25. FABP was eluted from the ion exchange column by .1 M NaCl. Molecular weight of FABP was estimated to be 12,000 by SDS polyacrylamide gel electrophoresis (PAGE). Two or three bands were seen on nondenaturing PAGE, two of which bound fatty acids. Activity of FABP was retained after incubation at 60 C for 15 min. FABP bound long chain fatty acids and their CoA derivatives but did not bind medium or short chain fatty acids. The affinity constant (Ka) of FABP for oleate was estimated to be 2 micromolar. The isolated FABP contained endogenous fatty acids, both covalently and noncovalently associated, which were primarily oleate, palmitate and stearate. The isoelectric point as determined by isoelectric focusing was about 5.6. Neither FABP nor albumin affected the activities of microsomal phosphatidic acid phosphatase, fatty acid:CoA ligase, or diacylglycerol acyltransferase. Fatty acid binding protein was not detected in skim milk or skim colostrum. This study demonstrated that mammary gland does possess a FABP which has properties similar to that found in other tissues.