Rat Submaxillary Gland

Biochem. J. (1980) 187, 269-272 269 Printed in Great Britain Substrate Stabilization of the Palmitoyl-Coenzyme A Hydrolase Activity of Rat Submaxillary Gland Thomas E. KNAUER,* John J. GURECKIt and Gleness R. KNAUER* *Department ofMedicine, Medical College of Virginia, Richmond, VA 23298, and tDepartment of Pharmacology-Physiology, University ofPittsburgh, School ofDental Medicine, Pittsburgh, PA 15261, U.S.A. (Received 2 January 1980) The long-chain acyl-CoA hydrolase (EC 3.1.2.2) activity of rat submaxillary salivary gland, found in the postmicrosomal supernatant fraction, has a pH optimum of 7.4. This hydrolase activity was found to be extremely labile, but inclusion of glycerol or the substrate palmitoyl-CoA in the preparations markedly stabilized the activity. Gel- filtration studies revealed multiple forms of the hydrolase, a lower-molecular-weight species of approx. 45000 and a higher-molecular-weight species of approx.- 130000 observed when glycerol (20%, v/v) or palmitoyl-CoA (10puM) were included in the eluting buffer. This phenomenon is similar to that observed with the palmitoyl-CoA hydrolase of rat brain, except that there is no evidence that the higher-molecular-weight species of the hydrolase of submaxillary gland is generated by substrate-induced dimerization of the lower-molecular-weight species. The submaxillary salivary gland of the rat is a 0.25 M-sucrose/0. lOM-potassium phosphate (pH 7.4) metabolically active secretory organ that utilizes with a Polytron ST1O homogenizer (Brinkman fatty acids both as precursors for complex-lipid Instruments, Westbury, NY, U.S.A.). The homo- synthesis and as sources of energy (Pritchard, 1970, genates were centrifuged for 5min at 700g (ra, 1972; Pritchard et al., 1971; Horak & Pritchard, 4.13 cm). Particulate and supernatant fractions were 1971). The lipid composition of rat submaxillary prepared by centrifuging the 700g supernatant for gland, which is approx. 5-6% lipid by wet weight, 60 min at 105OOOg (ra. 5.95 cm). Except as noted, includes all of the usual phospholipids, neutral lipids, all operations were carried out at 0-40C. cholesterol and its esters (Gilbertson et al., 1975). Our radioassay for palmitoyl-CoA hydrolase has Pritchard (1972) has presented evidence that the been described in detail previously (Knauer, 1979). increased energy demand of the submaxillary gland Briefly, 20-70,ug of submaxillary gland protein and during adrenaline-stimulated secretion is met by 88nmol of palmitoyl-CoA (containing 0.05#uCi of accelerated oxidation of fatty acids. Although no [1-14Clpalmitoyl-CoA) were incubated together at details were reported, Pritchard et al. (1971) noted 370C in a final volume of 1.Oml of 0.10M-potas- that palmitoyl-CoA was readily hydrolysed in vitro sium phosphate/lmM-EDTA (pH7.4). After 5- during incubations with subcellular fractions pre- 60min the incubations were extracted twice with pared from submaxillary gland. During our initial chloroform/methanol/acetic acid (50: 50: 1, by vol.), work we observed that the palmitoyl-CoA hydrolase the combined chloroform extracts washed with activity of submaxillary gland was extremely labile, water, dried under N2 and the residue was re- but could be stabilized by the addition of glycerol or dissolved in n-hexane. Portions of the n-hexane were palmitoyl-CoA to the preparations. This suggested taken for radioassay by liquid-scintillation spectro- that palmitoyl-CoA might have an effect on the metry or for analysis by chromatography on thin hydrolase similar to that observed with the hydro- layers of silica gel G with a developing solvent of lase of rat brain (Knauer, 1979). n-hexane/diethyl ether/acetic acid (70:30: 1, by vol.). Experimental The sources of the chemicals and supplies used in the present study were as previously reported Submaxillary glands were removed from groups (Knauer, 1979). Columns of Sephadex G-200 of four or more adult rats under light diethyl ether were calibrated by using pure proteins obtained from anaesthesia and chilled in ice-cold 0.9% NaCl. Boehringer Mannheim, Indianapolis, IN, U.S.A. Homogenates (10 or 20%, w/v) were prepared in Columns of Sephadex G-25 were used for desalting Vol. 187 0306-3275/80/040269-04$01.50/1 © 1980 The Biochemical Society 270 T. E. KNAUER, J. J. GURECKI AND G. R. KNAUER protein samples. All gel-filtration procedures were calibrated columns of Sephadex G-200 and eluted performed at 2-40C. Protein was determined by the with 0.lOM-potassium phosphate/imm EDTA method of Lowry et al. (1951), with crystalline (pH 7.4) (Fig. la), the major peak of hydrolase bovine serum albumin as the standard. Radioactive activity, preceded by a broad shoulder, eluted in a samples were counted in a Beckman LS250 liquid- region consistent with a molecular weight of approx. scintillation spectrometer with external standardiza- 45000. An additional peak of activity in the void tion for determining counting efficiencies. volume of the column probably represents the long-chain acyl-CoA thioesterase activity of the Results and Discussion fatty acid synthetase complex (Kumar, 1975; Lornitzo et al., 1975). Recovery of activity was low, Activity ofsubmaxillary gland hydrolase in vitro approximately 5-10% of that applied, and was not Under the conditions of the radioassay, the increased by including dithiothreitol (3mM) in the hydrolysis of palmitoyl-CoA was linearly dependent eluting buffer. on protein concentration and incubation time up to at least 25% hydrolysis of the substrate. The optimal pH for hydrolysis was approx. 7.4. When particulate and soluble fractions prepared from cell-free homogenates of submaxillary gland were assayed for hydrolase activity, over 82% of the activity was found in the 105 OOOg supernatant fraction. This fraction was used as the source of palmitoyl-CoA hydrolase during the remainder ofthese studies. 8 j 6 I--. Stabilization of hydrolase activity by glycerol and 0 palmitoyl-CoA 4 *g- The hydrolase was found to be sensitive to 2 Z manipulation and considerable activity was lost after 20o .*2 wcn dilution, dialysis or column desalting. For example, 0 only 40 ± 4% (mean ± S.E.M., n = 5) of the original 00 0 hydrolase activity remained 3 h after 50-fold dilution 12 vc of the 105 000g supernatant (5-7 mg of protein/ml) 10 in 0.1OM-potassium-phosphate, pH7.4. However, 10. similar dilutions retained full potency for 3 h when glycerol (20%, v/v) was included in the buffer. 6 E The hydrolase activity was found to be heat-labile and was completely inactivated by exposure to 600C 2 for 10min. However, during studies concerning the effect of temperature on the assay of the hydrolase 140 220 300 380 460 540 in vitro, we observed that in the standard incubation Elution volume (ml) system a substantial portion of the original hydro- Fig. 1. Gel filtration of the 105 000g supernatant frac- lase activity remained even after 30min at 600C. To tion ofsubmaxillary gland determine if the presence of the substrate stabilized (a) A portion (6ml) of the 105OOg supernatant the hydrolase activity against heat inactivation, we prepared from a 20% (w/v) homogenate of sub- heated of maxillary gland was applied to a column (2.6cm x portions submaxillary gland protein at 98cm) of Sephadex G-200 equilibrated and eluted 600C for 10min with and without added palmitoyl- by ascending flow (14 ml/h) with 0. 10M-potassium CoA (401M). Subsequent radioassays revealed that phosphate/1.0mM-EDTA (pH 7.4). Column frac- the preparations heated with palmitoyl-CoA retained tions were assayed for hydrolase activity by the 63 ± 5% (mean + S.E.M., n = 3) of the original standard method described in the text. One unit of activity, whereas preparations heated without pal- enzyme activity will hydrolyse 1 nmol of palmitoyl- mitoyl-CoA had virtually no hydrolase activity CoA/min. Recovery of hydrolase activity for this (2%). column was 5% ofthe applied activity. (b) A portion (7 ml) of the 105 000g supernatant prepared from a Gel-filtration studies 10% (w/v) homogenate of submaxillary gland was Gel-filtration adjusted to 20% (v/v) glycerol and applied to a studies revealed multiple forms of column (2.6 cm x 96 cm) of Sephadex G-200 equilib- the palmitoyl-CoA hydrolase activity of sub- rated and eluted by ascending flow (13 ml/h) with maxillary gland. (Column profiles are representative potassium phosphate/EDTA buffer containing 20% of several experiments.) When portions of the (v/v) glycerol. Recovery of hydrolase activity from 105 OOOg supernatant fraction were applied to this column was 22% ofthe applied activity 1980 RAPID PAPERS 271 In an attempt to increase the recovery of activity glycerol columns (Fig. lb) were treated similarly, no during gel filtration, we added glycerol (20%, v/v) to hydrolase activity was recovered from the Sephadex the eluting buffer. Two major peaks of activity G-200 column. Thus for either brain (Knauer, 1979) emerged from the columns eluted with glycerol or submaxillary gland, removal of glycerol results in buffer (Fig. lb). The recovery of hydrolase activity a 95-100% loss of hydrolase activity, whereas a was increased to 20-25% of that applied, with the substantial portion (>65%) of their original activities bulk of this increased activity appearing as a new remains after excess (non-protein bound) palmitoyl- peak between the void volume and the peak CoA has been removed by column desalting. corresponding to a molecular weight of approx. Since a higher-molecular-weight form of the 45000. Similarly, including palmitoyl-CoA (10puM) hydrolase of rat brain is generated from a lower- in the eluting buffer promoted the appearance of an molecular-weight form by exposure to palmitoyl- additional peak of hydrolase activity between the CoA (Knauer, 1979), we examined the possibility void volume and the peak of mol.wt.

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