Isolation and characterization of human breast milk lipoamidase.
The mean lipoamidase activity in human breast milk was found to be 0.073 nmol/min per mg (S.D. = 0.028, range = 0.020-0.123, n = 44). The mean lipoamidase activity is approximately 3-fold higher in milk than that in serum (0.023 nmol/min per mg, S.D. = 0.016, range = 0.001-0.059, n = 32). Lipoamidase was purified to 4400-fold by a four-step procedure from 330 ml of human breast milk. The purified enzyme was identified as a single band (Mr = 135,000) by sodium dodecyl sulfate/polyacrylamide electrophoresis. Analysis by Edman degradation indicated that the N-terminal amino acid was glycine. These results strongly suggest that milk lipoamidase is composed of a single polypeptide chain. The enzyme is considered to be a glycoprotein since it reacted positively to periodate-Schiff (PAS) staining. The isoelectric point of the enzyme was 4.2. After treatment of lipoamidase with sialidase, its position on isoelectric focusing gel moved from pH 4.2 to 4.6. This is strongly indicative that lipoamidase contains sialic acid residues. The optimum pH for the enzyme activity is 7.0. The Michaelis constant (KM) for lipoyl p-aminobenzoate is calculated as 25 microM. The enzyme activity was completely lost by heating 60 degrees C for 5 min. The effects of thiol-reactive agents, such as 2-mercaptoethanol (ME) and p-chloromercuribenzoate, were not significant. However, the enzyme activity was completely inhibited by 50 microM diisopropylfluorophosphate. Thus, this enzyme seemed to contain an essential serine residue in the active site.
Hayakawa
K
K
National Children's Medical Research Center, Division of Metabolism, Tokyo, Japan.
Oizumi
J
J
D000581:Amidohydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D004355:Drug Stability; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing; D007526:Isoelectric Point; D007531:Isoflurophate / Q000494:pharmacology; D007700:Kinetics; D008895:Milk, Human / Q000201:enzymology*; D008970:Molecular Weight; D009439:Neuraminidase / Q000494:pharmacology; D010503:Periodic Acid-Schiff Reaction; D013379:Substrate Specificity
D006023:Glycoproteins; D007531:Isoflurophate; D009439:Neuraminidase; D000581:Amidohydrolases; C023940:lipoamidase