Trehalase and the transport of glucose in the mammalian kidney and intestine.

Abstract

The enzyme trehalase, which hydrolytically splits trehalose (1-a-D-glucopyranosyl-a-D-glucopyranoside) to two glucose moieties, has been reported from yeast, fungi and other plants, and several phyla of invertebrate animals; it is ubiquitously found in insects.' Trehalose is the maj or blood sugar of insects.2-4 The "fat body" rapidly converts glucose and glycogen to trehalose, which is released to the hemolymph and is used to support a variety of energy-requiring functions. Trehalase is especially active in the insect gut,5-7 although the insect may never ingest trehalose. The enzyme is also prevalent in flight muscle,8-10 where the precipitous hydrolysis of the disaccharide at the initiation of flight is the rate-limiting reaction in glycolysis.10 These findings, plus others, led us to suggest that in insects trehalose is a vehicle of carbohydrate transport and that trehalase functions in the mechanism of sugar transport."In an investigation of digestive disaccharidases in rat intestine, Dahlqvist and Brun"2 have shown that maltose, sucrose, lactose, and trehalose are hydrolyzed. An intestinal trehalase, specific for trehalose, has been purified 20-fold.'3 The presence of trehalase in the mammalian intestine, which assumes added significance in light of our suggested role for the enzyme, prompted us to look for trehalase in other mammalian tissues and to see if trehalase may function in glucose transport in higher animals. In this communication we report a highly active trehalase in kidney as well as in intestine. The specific localization of the enzyme, plus the discovery in kidney and jejunum of the enzymes which in sequence lead to the synthesis of trehalose from glucose, support the hypothesis that the disaccharide has a role in the resorption of glucose by the kidney and the transport of the hexose across the intestinal mucosa. '4 Methods.-Trehalase was determined by either of two procedures, depending on the activity of the preparation. With more active preparations, the enzyme was assayed directly. The reaction mixture contained 0.5 Mmole of NADP, 0.5 Mmole of ATP, 12 Mmoles of trehalose, 5 /Amoles of MgCl2, 6 Mimoles of potassium phosphate, adjusted to pH 6.3, excesses of glucose-6-P dehydrogenase and hexokinase, and trehalase in a total volume of 0.62 ml. With less active or turbid preparations, tissue samples were incubated for 10 or 30 min in a reaction containing 12 mM trehalose and 10 mM potassium phosphate, pH 6.3, in a volume of 1.0 ml. The reaction was stopped with the addition of 0.1 ml of 8% perchloric acid. Glucose in the neutralized extract was determined as described previously. 10

Isolation and Biochemical Characterization of Brush Borders From Rabbit Kidney

“…Other disaccharidases and a-glucosidase activities not present in the kidney were reported previously (11) .…”

“…To be noted from Several enzymes, including hexokinase, phosphoglucomutase, and UDPG pyrophosphorylase, which were shown to participate in the synthesis of trehalose from glucose in yeast and insects, were found previously in the renal cortex (I1) . Hexokinase, which was predominantly membrane bound (11), was now found to be associated, in part, with the mitochondrial fraction . The specific activity of the enzyme in fraction P 4 was four-to fivefold that in the homogenate .…”

“…Maltase, isomaltase, sucrase, lactase, and cellobiase were assayed similarly, except that 20 µmoles of the appropriate substrate were used . UDPG pyrophosphorylase, phosphoglucomutase, and hexokinase were measured as previously reported (11) .…”

“…addition, the disaccharidase, trehalase, previously found uniquely in the particulate fraction of rabbit renal tubules (11,12) and postulated to have a role in the transport of glucose (11,13), is now localized exclusively in the brush border . The use of this disaccharidase, as well as maltase, for "marker" enzymes for rabbit renal brushborder preparations is suggested .…”

A novel trehalase inhibitor, trehalostatin, produced by Amycolatopsis trehalostatica HG-25.

“…Trehalose, found in hemolymphs of insects, is used to support various energy-requiring functions, such as insect flight.1>2) The other biological functions of trehalose or trehaiase are also reported for germination of ascospores in fungi3~6) and glucose transport in mammalian kidney or intestine. 7) In the course of our screening of specific and potent inhibitors of trehalase, we have succeeded in isolating a microorganism producing a potent and specific inhibitor of trehalase. As described below, the inhibitor was different from the reported trehalase inhibitors, such as proteinaceous inhibitor,8'9) S-GI (deoxynojirimycin), 10) validamycins,11 '12) and validoxylamine.13) Therefore, the trehalase inhibitor was named "trehalostatin" for its specific inhibitory spectrum.…”