Biochem. J. (1980) 187, 269-272 269 Printed in Great Britain

Substrate Stabilization of the Palmitoyl-Coenzyme A 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 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 homo- genates 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- 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. 45 000 (Fig. 2). that by exposing the 45 000-mol.wt. species of Recoveries of hydrolase activity were also sub- submaxillary gland hydrolase to palmitoyl-CoA or stantially increased (65-80%). A portion of the glycerol, the 130000-mol.wt. species could be pooled column fractions from this large peak was generated. However, all such attempts were un- desalted by using Sephadex G-25 to remove excess successful. In addition we could find no evidence of a (non-protein bound) palmitoyl-CoA, concentrated, precursor using activated-thiol-Sepharose covalent applied to a column of Sephadex G-200 and eluted chromatography as described previously (Knauer, with buffer that did not contain palmitoyl-CoA 1979). There is a clear difference between the (insert, Fig. 2). A single symmetrical peak of activity thiol-binding characteristics of the brain hydrolase was eluted from the column in a region consistent in the absence and presence of palmitoyl-CoA, with a molecular weight of 130000. However, when which represents a conversion of the non-binding portions of the corresponding peak of activity from form into the thiol-binding form of the hydrolase. As is the case with the brain system, exposure of preparations of submaxillary gland hydrolase to palmitoyl-CoA increased the recovery of activity as the thiol-binding form of the hydrolase. This is consistent with the fact that the substrate both stabilizes a 130000-mol.wt. form of u generates and 2.0 we 0 the hydrolase. However, for submaxillary gland -002-. could not detect any conversion of a non-binding 1.6 0 form of the hydrolase into a thiol-binding form by 1.2 24 c c exposure to palmitoyl-CoA. For either submaxillary gland or brain a higher- ° 0.8 16 °-= molecular-weight form of the enzyme of approx. 0E 0.4 130000 is found to be generated and stabilized by 8 136 the presence of glycerol or palmitoyl-CoA. In the O 50 100 150 200 25 case of the brain hydrolase, this higher-molecular- Elution volume (ml) weight form appears to be a dimer comprising subunits of approx. 70000mol.wt. On the other Fig. 2. Gel filtration of the palmitoyl-CoA hydrolase of rat submaxillary gland hand, the higher- and lower-molecular-weight A portion (5 ml) of the 105 OOOg supernatant pre- species of submaxillary gland hydrolase are prob- pared from a 10% (w/v) homogenate of sub- ably unrelated, since neither species could be maxillary gland was adjusted to lOM-palmitoyl- generated from the other. Furthermore, a peak of CoA and applied to a column (2.6cm x 36cm) of hydrolase activity corresponding to a mol.wt. of Sephadex G-200 equilibrated and eluted by ascending 45000 was always eluted from Sephadex G-200 flow (9ml/h) with 0.10M-potassium phosphate/ columns regardless of the presence of absence of lmM-EDTA (pH7.4), containing 10M-palmitoyl- glycerol or palmitoyl-CoA, and this peak always CoA. Column fractions were assayed for hydrolase represented 5-10%Y of the applied activity whether activity by the standard method described in the the total recovery was 5, 20 or 70% of the applied text. One unit of enzyme activity will hydrolyse 1 1 nmol of palmitoyl-CoA/min. After desalting to activity (see Figs. and 2). remove non-protein bound palmitoyl-CoA, a portion The palmitoyl-CoA of rat sub- of the pooled fractions from the major peak of maxillary gland and brain exhibit many common hydrolase activity was applied to the same Sephadex features. As examples, the hydrolases of both G-200 column equilibrated and eluted by ascending tissues: (1) have the same subcellular distribution flow (9ml/h) with potassium phosphate/EDTA and pH optimum; (2) are inhibited by albumin buffer containing no palmitoyl-CoA (insert). (approx. 1 mg/ml or greater) included in the assay in Vol. 187 272 T. E. KNAUER, J. J. GURECKI AND G. R. KNAUER vitro; (3) are extremely sensitive to thiol-blocking portion of the overall lipid metabolism of this gland reagents such as HgCl2, p-hydroxymercuribenzoate may be involved in the production of energy required and 5,5'-dithiobis-(2-nitrobenzoic acid); (4) are for its secretory function. In spite of these differ- unstable in dilute solution, but are stabilized by the ences creation and stabilization of a higher-mole- inclusion of glycerol or palmitoyl-CoA in the buffer; cular-weight form of palmitoyl-CoA hydrolase by (5) are not (product) inhibited by palmitate or the substrate palmitoyl-CoA is a phenomenon coenzyme A in vitro; (6) are not affected by common to both rat brain and submaxillary gland. exposure to phenylmethanesulphonyl fluoride, sug- gesting that these hydrolases are distinct from This work was supported by research grants DE-04372 general non-specific , which fall in the and DE-05000 from the National Institute of Dental category of 'serine esterases'. Furthermore the pal- Research, Bethesda, MD, U.S.A. mitoyl-CoA hydrolase activities of neither rat brain (Knauer, 1979) nor submaxillary gland can be attributed to triacylglycerol activities in these References tissues. The submaxillary gland of the neonatal (3- Gilbertson, J. R., Gelman, R. A. & Schmutz, J. A. (1975) day-old) rat is devoid of lipase activity (Hamosh & Arch. Oral Biol. 20, 527-530 Scow, 1973). We examined submaxillary glands Hamosh, M. & Scow, R. 0. (1973) J. Clin. Invest. 52, from neonatal rats and found the specific activity of 88-95 the hydrolase to be approx. 120% that for adult Horak, H. & Pritchard, E. T. (1971) Biochim. Biophys. glands. This rules out the possibility that the Acta 253, 12-23 hydrolase activity of submaxillary gland is merely an Knauer, T. E. (1979) Biochem. J. 179, 515-523 expression of the triacylglycerol lipase activity of the Kumar, S. (1975)J. Biol. Chem. 250, 5150-5158 gland. Lornitzo, F. A., Qureshi, A. A. & Porter, J. W. (1975) J. Biol. Chem. 250, 4520-4529 Brain and submaxillary gland are quite dissimilar Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, with respect to lipid metabolism. Brain has a R. J. (195 1)J. Biol. Chem. 193, 265-275 relatively high lipid content with most metabolic Pritchard, E. T. (1970)Arch. Oral Biol. 15, 879-891 activity directed toward synthesis for membrane Pritchard, E. T. (1972) FEBS Lett. 23, 314-316 maintenance. On the other hand, submaxillary gland Pritchard, E. T., Horak, H. & Yamada, J. A. (197 1) Arch. contains considerably less lipid and a substantial Oral Biol. 16, 915-928

1980