Abstract
Acid trehalase was purified from the yeast suc2 deletion mutant. After hydrophobic interaction chromatography, the enzyme could be purified to a single band or peak by a further step of either polyacrylamide gel electrophoresis, gel filtration, or isoelectric focusing. An apparent molecular mass of 218,000 Da was calculated from gel filtration. Polyacrylamide gel electrophoresis of the purified enzyme in the presence of sodium dodecyl sulfate suggested a molecular mass of 216,000 Da. Endoglycosidase H digestion of the purified enzyme resulted after sodium dodecyl sulfate gel electrophoresis in one distinct band at 41,000 Da, representing the mannose-free protein moiety of acid trehalase. The carbohydrate content of the enzyme was 86%. Amino acid analysis indicated 354 residues/molecule of enzyme including 9 cysteine moieties and only 1 methionine. The isoelectric point of the enzyme was estimated by gel electrofocusing to be approximately 4.7. The catalytic activity showed a maximum at pH 4.5. The activity of the enzyme was not inhibited by 10 mM each of HgCl2, EDTA, iodoacetic acid, phenanthrolinium chloride or phenylmethylsulfonyl fluoride. There was no activation by divalent metal ions. The acid trehalase exhibited an apparent Km for trehalose of 4.7 +/- 0.1 mM and a Vmax of 99 mumol of trehalose min-1 X mg-1 at 37 degrees C and pH 4.5. The acid trehalase is located in the vacuoles. The rabbit antiserum raised against acid trehalase exhibited strong cross-reaction with purified invertase. These cross-reactions were removed by affinity chromatography using invertase coupled to CNBr-activated Sepharose 4B. Precipitation of acid trehalase activity was observed with the purified antiserum.
Highlights
Talization of trehalase and its substrate yineast [4,5]
One unit of acid trehalase is defined as theamount of enzyme that catalyzes the hydrolysis of 1pmol of trehalose/min at 37 "C and pH
As separation of invertase from acid trehalase was found to be extremely difficult andtime consuming, trehalase was purified from the yeast suc2 deletion mutant, which contains no external invertase
Summary
A nonreducing disaccharide (a-D-glucopyranosyl(l+ 1)-a-D-glucopyranoside)was first isolated by Wiggers in 1832 [1].Since it hasbeen found in a wide variety of organisms including yeast, fungi, bacteria, plants,insects, and other invertebrates. The large pools of trehalose remain constant in cells starved by incubation in water even though these cells contain high activities of trehalase, the trehalose-hydrolyzing enzyme, first described in yeast by Fischer [3]. Such observations led to thepostulation of an intracellular compartmen-. Acid trehalase was assayed by incubating 30 pl of extract with 270 pl of 200 mM sodium citrate, pH 4.5, containing 1 mM EDTA and Purification of Acid Trehalase from the Yeast suc Mutant (All Operations Were Performedat 4 "C). Precipitated proteinwas removed by centrifugation at 10,000 X g for 45 min
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