Hydrolysis of Vitamin A Acetate by Unspecific Carboxylesterases from Liver and Kidney

Abstract

Vitamin A acetate is hydrolysed by highly purified carboxylesterases (EC 3.1.1.1) from liver and kidney of several species (pig, ox, man), whereas long-chain fatty acid esters of vitamin A are not split. [7,8]. The substrate specifity of these carboxylesterases is remarkably low. Observations of Frimmer and Gries [9] gave first indications that vitamin A acetatc is hydrolysed by pig liver esterase. In the present communication this reaction has been characterized in more detail by experiments on the enzymatic hydrolysis of vitamin A acetate in vitro by highly purified carboxyl-esterases of different origin. MATERIALS AND METHODS MaterialAll organic solvents not designated as analytical grade were distilled prior to use. All other reagents were of analytical grade.Vitamin A stearate and -oleate were kindly supplied by Prof. Dr. 0. Wiss, Hoffman la Roche, Basel/Switzerland. Nicrosomes and EnzymesPig liver microsomes a,nd carboxylesterases from pig liver, pig kidney and ox liver were isolated according to methods reporhed previously [3,5,6]. The analogous enzyme from human liver is purified a t present in our laboratory [lo].Determinations o/ Enzyme Activity For assaying the activity of the various carboxyesterase preparations, acetanilide [3,5] and p-nitrophenylacetate [Ill were used as substrates. ProteinProtein was determined by a biuret method [12); in the case of microsomes a turbidity correction was made by addition of KCN [13]. Substrate SolutionsVitamin A Acetate (10 m M ) . 16.43 mg crystalline vitamin A acetate + 5.5 mg hydroquinone were dissolved by slight warming in 1 ml of methanol and diluted to 5 ml with 0.1 M Tris-C1, pH 8

Hydrolysis of retinyl esters by non-specific carboxylesterases from rat liver endoplasmic reticulum

“…The specific activities of the rat liver esterases for the hydrolysis of retinyl acetate are about 10-fold lower than those reported by Bertram & Krisch [3] for pig liver esterase. It is not clear whether their pig liver esterase preparation really lacked retinyl palmitate hydrolase activity or whether their measurement was not sensitive enough.…”

“…Several attempts have been undertaken to ascertain such functions from their possible activity on physiological ester-type lipids. In 1969, Bertram & Krisch [3] demonstrated that vitamin A acetate is a substrate for purified pig, ox and human liver esterases. As a result, the Nomenclature Committee of the International Union of Biochemistry deleted the entry EC 3.1.1.12 (vitamin A esterase) [4].…”

“…It was therefore thought worthwhile to determine whether one of these esterases selectively hydrolyses retinyl esters or whether this is a function of all esterases of this type. Beside this, the question of whether these esterases only [3] act on retinyl acetate or also on the palmitate needs to be reinvestigated. The latter substrate could indicate a physiological function, since this ester is the main storage form of vitamin A in the liver.…”

The morphology of release of vitamin A‐containing lipid droplets by hepatocytes in rat liver

“…In addition, purified carboxylesterase (EC 3.1.1.1) from rat serum has been reported to be very sensitive to DFP but not to PCMB or eserine (Hashinotsume et al, 1978). Furthermore, highly purified carboxylesterases (EC 3.1.1.1) from liver and kidney of several species hydrolyze vitamin A acetate, but not vitamin A palmitate, stearate, and oleate in vitro (Bertram and Krisch, 1969), regardless of the storage in the liver of these long-chain fatty acid esters of vitamin A. Highly purified carboxylesterases are inhbited by 10 p,M E 600 but not by eserine or PCMB (Bertram and Krisch, 1969).…”

“…Furthermore, highly purified carboxylesterases (EC 3.1.1.1) from liver and kidney of several species hydrolyze vitamin A acetate, but not vitamin A palmitate, stearate, and oleate in vitro (Bertram and Krisch, 1969), regardless of the storage in the liver of these long-chain fatty acid esters of vitamin A. Highly purified carboxylesterases are inhbited by 10 p,M E 600 but not by eserine or PCMB (Bertram and Krisch, 1969). Lipid droplets in mouse hepatocytes have been demonstrated to be positive for esterase activity, as revealed by using 8-actoxy-quinoline as a substrate.…”