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Partial glyceridase activity of a protein-kinase activatable triglyceride lipase from rat adipose tissue
65
BIOCHIMICA ET BIOPHYSICA ACTA BBA
56051
PARTIAL
GLYCERIDASE
ACTIVATABLE
RENU
A. HELLER
TRIGLYCERIDE
and DANIEL
Department of Medicine, 92037 (U.S.A.) (Received
December
ACTIVITY
OF A PROTEIN-KINASE
LIPASE FROM RAT ADIPOSE TISSUE
STEINBERG
School of Medicine,
University of Califonzia,
San Diego, La Jolla, Calif.
zgth, 1971)
SUMMARY I. A preparation of hormone-sensitive triglyceride lipase purified approximately Ioo-fold from a high-speed supernatant fraction of rat adipose tissue homogenates also hydrolyzed monoolein and diolein emulsions. The rate of hydrolysis of these lower glycerides was about 5 times that for hydrolysis of triolein. 2. Triglyceride lipase activity was increased by 70% after incubation with protein kinase, cyclic AMP and ATP-Mge+; monoglyceride lipase activity in the same preparations, on the other hand, was not altered by this activating system. Diglyceride lipase activity was slightly increased but the mean activation was only 15% above control values. 3. NaCl (I M) inhibited monoglyceride lipase activity more than 50% but did not inhibit triglyceride lipase activity. Isopropanol (4%) inhibited triglyceridase activity by 70% but did not inhibit monoglyceride lipase activity. Triglyceride lipase was inhibited by 40 and 90% when the substrate was prepared in I and 5 mM taurodeoxycholate, respectively, whereas monoglyceride lipase activity was actually enhanced relative to activity measured using suspensions stabilized by gum arabic. 4. Heating the enzyme preparation at 48 “C for 45 min caused 40% inactivation of monoglyceride lipase activity without altering triglyceride lipase activity. 5. The optimum for triglyceride lipase activity was at pH 6.8, whereas the optimum for monoglyceride lipase activity was at pH 7.6-7.8. 6. These data are compatible with the presence of a separate monoglyceride lipase not subject to kinase-mediated activation and having rather distinct properties from triglyceride lipase. However, until physical resolution of the two activities can be demonstrated this conclusion must remain tentative.
INTRODUCTION
Crude extracts of rat adipose tissue contain: (I) hormone-sensitive triglyceride lipasel-a; (2) lipoprotein lipase4; (3) a high level of activity against monoglycerides Abbreviations: EGTA, ethyleneglycol-his-(aminoethyl cyclic 3’: f-adenosine monophosphate.
ether)-N,N’-tetraacetic
Biocham. Biophys.
acid; cyclic AMP,
Acta, 270
(1972)
65-73
66
R. A. HELLER,
D. STEINBERG
(monoglyceridase)1-3; (4) a high level of activity against diglycerides (diglyceridase)av3 and (5) activity against short chain glyceryl esters (esterase)6. None of these activities has been prepared in a pure form and it is not known whether each activity is referable to a single enzyme protein or to more than one. Nor is it known to what extent there is overlap, i.e. to what extent assays with a given substrate measure the activity of more than one enzyme or class of enzymes. The properties of the first three (hormone-sensitive lipase, lipoprotein lipase and monoglyceridase*) and their different responses to nutritional and hormone influences strongly suggest that they represent distinct enzymes but that has not been firmly established. Recent work in this laboratory has yielded a partial purification of hormone-sensitive lipase (loo-fold from the 78oooxg supernatant fraction of a crude homogenate)6g7. The present studies were undertaken to determine the extent to which activity against lower glycerides accompanies the triglyceride lipase activity during purification and to compare the properties of the former and the latter, particularly in the preparation purified IOOfold with respect to hormone-sensitive triglyceride lipase. A preliminary report of these findings has appeared elsewhere8. MATERIALS AND METHODS
Preparation
of hormone-sensitive
lipase
The enzyme was prepared from epididymal fat pads of fed Sprague-Dawley rats (150-200 g). The tissue was first incubated for 4 h in Krebs-Ringer bicarbonate buffer, pH 7.4 containing 4% bovine serum albumin. This preincubation has been shown to reduce the tissue content of lipoprotein lipase to very low levels, thereby reducing the possibility of contamination of hormone-sensitive lipase preparations with that enzyme. Homogenization of the tissue and purification of the enzyme were carried out as reported previously’. Briefly, the procedure involves homogenization of the fat pads in sucrose 0.25 M and IO-~ M EDTA. The homogenate is subjected to ultracentrifugation for I h at 78000 xg followed by an isoelectric precipitation of the soluble supernatant fraction at pH 5.2. The precipitate is redissolved in 0.02 M Tris buffer (pH 7.4) containing EDTA (IO-~ M) and dithiothreitol (IO-~ M), adjusted to density 1.12 with sucrose, and centrifuged for 48 h at 105000xg. The top fraction (dc1.12) is adjusted to density 1.06 and centrifuged for 48 h at 105000 xg. The sedimented enzyme (d 1.06-d 1.12) is subjected to gel f&ration on 4% agarose. Lipase activity emerging in the void peak is subjected to a final centrifugation for I h at 87000 xg to remove membrane-like material emerging with the lipase in the void volume. Unless otherwise noted, the results in this paper were obtained with lipase preparations carried through this procedure representing approximately a roe-fold purification from the original 78 000 x g supernatant . Lipase assays
Triolein was obtained from the Sigma Chemical Co., St. Louis, MO. and glyceryl [carboxy-14C]trioleate, spec.. act. 15.2 mCi/mM, was obtained from the International Chemical and Nuclear Corporation, Irvine, Calif. [%]Triolein was diluted with un* The term monoglyceridase is used here in a strictly operational sense to refer to enzyme activity assayed using monoolein as substrate under the conditions specified. Diglyceridase and triglyceridase are used similarly to refer to activity assayed using diolein or triolein as substrate. Biockim. Biophys. Acta, 270 (1972)
65-73
GLYCERIDASEACTIVITYOFTRIGLYCERIDELIPASE
67
labelled triolein to give a final spec. act. of 0.083 &i/pmole. Diolein and monoolein were obtained from the Hormel Institute, Austin, Minn. Monoolein (50 mCi/mmole) and diolein (IOO mCi/mM) both carboxyJ*C-labelled (>gg.5% pure) were purchased from Dhom Products Ltd, North Hollywood, Calif. [W]Diolein and [llC]monoolein were both diluted with unlabelled diolein and monoolein, respectively, to give a final spec. act. of 0.025&i/,umole. Triolein emulsions were prepared by sonication with 5 yO gum arabic as previously described6 and diolein emulsions were prepared in the same way. In our early studies monoolein suspensions were also prepared in gum arabic but prolonged sonication (30 s) was required to obtain homogeneous preparations and this caused significant hydrolysis and high blank values (up to 20 y0 of experimental values). Homogeneous preparations were obtained by mixing the monoolein with IO mM taurodeoxycholate, pH 7.0, on a Vortex mixer. Such preparations, used in all but the preliminary studies, were clear or only slightly opalescent, stable and yielded consistent results. Monoglyceride lipase activity assayed with gum arabic suspension tended to be lower but the best gum arabic preparations yielded activities equal to those obtained with taurodeoxycholate preparations. Triglyceride lipase assay was performed as described previously6 in a final assay volume of 0.2 ml, containing 1.2 ,umoles triolein, 5 mg bovine serum albumin and 50 pmoles phosphate buffer, pH 6.8 Lipolysis was allowed to proceed for I h at 23 “C. Diglyceride lipase and monoglyceride lipase assays were carried out using the same procedure but substituting 2 ,umoles of diolein or 2 pmoles of monoolein and incubating for only 30 min in the case of diolein and 15 min in the case of monoolein. Substrate concentration activity curves showed that the substrate concentrations used were saturating. For all three substrates the assay remained linear over the time intervals used. Using the labelled substrates, zero times values and values after incubation without enzyme were usually less than 1% and always less than 5% of experimental values. When studying enzyme activation by cyclic AMP and protein kinase, activation was carried out at 23 “C for 15 min in 0.4 ml of a mixture containing 2 .IO--~ M Trischloride buffer, pH 7.4; EGTA, I .IO-~M; dithiothreitol, 2 *IO-~M; ATP, I .IO-&M; magnesium acetate, 5 .IO-~M; cyclic AMP, I .10-eM; theophylline, I .IO-~M; and rabbit muscle protein kinase, IO ,ugper assay. The activated enzyme was assayed after the addition of 0.1 ml of an assay mixture containing substrate (triolein 1.2 pmoles, diolein 2 ,umoles, or monoolein 2 pmoles); 0.1 M phosphate buffer, pH 6.8; bovine serum albumin, 5 mg; and EDTA, 5 .IO-"M. Previous studies have shown that the high final concentration of EDTA during assay (I.10-sM) effectively stops further activation by protein kinase$. This was checked under the present conditions using purified enzyme; after kinase activation the time course for triolein or diolein hydrolysis in the assay was linear for 90 min and then fell off slightly. Assays were terminated by adding 5 ml of Dole’s extraction medium” and the free fatty acids were isolated from the heptane phase using the Kelley resin methodll as modified by Huttunen et al.@. In some experiments the net free fatty acids in the Dole extract was measured using the method of Novak’*.
B&him.
Biophys.
Acta.
270 (1972) 65-73
68
R. A. HELLER, D. STEINBERG
RESULTS
Lipase activity against different szlbstrates Activity against all three substrates (triolein, diolein and monoolein) was assayed at each successive step during purification. Results of a representative experiment are shown in Table I. The 78000 xg supernatant fraction showed a ratio of triglyceridase to monoglyceridase of I to 15 (range I : 20 to I : 15 in four experiments). The diglyceridase activity in the crude supernatant was usually one-half that of monoglyceridase. TABLE I RATIOOF TRIGLYCERIDE SUCCESSIVE
STAGES
IN
LIPASE
TO
DIGLYCERIDE
AND
MONOGLYCERIDE
LIPASE
ACTIVITIES
AT
PURIFICATION
Lipase assays were carried out under conditions described in Materials and Methods.Three separate experiments were done to study the relative activities at different stages of purification. Shown below are absolute activities from one representative experiment; ranges for relative activities are given in the text under Results. Fraction
Enzyme activity (pequiv/ml per h) Triglyceride
Diglyceride
lipase
lipase
Monoglyceride lipase
Monoglyceride lipase activity/ Triglyceride lipase activity
78000 xg supernatant
0.10
0.73
1.56
15.6
pH d < I.5.2 I 2precipitate fraction Final preparation
4.90 4.42 0.29
22.7 26.7 I .05
35.9 39.4 I .40
::; 4.8
During purification, lower glyceridase activity decreased relative to triglyceridase activity, indicating some resolution of monoglyceride and diglyceride lipase from the triglyceride lipase. However, the final preparation still contained considerable lower glyceridase activity, the triglyceridase to monoglyceridase ratio being I to 5 (range I : 7 to I : 5 in five experiments). The diglyceridase activity of the final preparation was approximately equal to the monoglyceridase activity. Difleerentiul activation of triglyceride, diglyceride and monoglyceride lipase The purified triglyceride lipase has been shown to be activated by cyclic AMPdependent protein kinase; little or no activation was observed in the absence of any one of the key components of the system (cyclic AMP, protein kinase, ATP, Mga+)ls. Using a similar system the activation of triglyceride lipase, diglyceride lipase and monoglyceride lipase was determined at various steps in the purification. As shown in Table II, the triglyceride lipase activity was enhanced in all fractions by the addition of cyclic AMP, ATP-Mg2+ and protein kinase. Control samples contained cyclic AMP and ATP-Mga+ but no protein kinase. In contrast, there was no significant enhancement of diglyceride lipase or monoglyceride lipase activity. The final purified preparation was also tested for the activation of the three lipases using a control containing protein kinase but no cyclic AMP (Table III). Again, while triglyceride lipase was activated by 50%, the monoglyceride lipase activity remained essentially unchanged. This was true whether the monoolein was prepared in gum arabic suspension or in taurodeoxycholate. Diglyceride lipase activity occasionally showed a slight activation ; Biochim. Biophys. Acfa, 270 (1972) 65-73
GLYCERIDASE
ACTIVITY
OF TRIGLYCERIDE
LIPASE
69
TABLE II PROTEINKINASE-DEPENDENT
ACTIVATION
OF
LIPASE
AT
DIFFERENT
STAGES
OF
PURIFICATION
Activation of the enzyme and the lipase assayswere performedas describedunderMaterialsand Methods. Absolute levels of enzyme activities were in the same range as those shown in Table I. Activation is expressed relative to lipase activity in presence of cofactors, including cyclic AMP, but without addition of protein kinase. Data represent mean + SE. in 3 to 12 determinations.
% Activafion
Subs&ate
Triolein Diolein Monoolein
TABLE
with cyclic AMP,
78oooxg supenzatant fraction
PH 5.2 precipitate
32 % I4 I4 f 9.5 6 + 3.4
45 f 4 3 f I.7 -3 & 2.1
protein
kinase and ATP-MgB+
dcI.12 fraction
44 f 5 f
in:
Final preparation
70 5 S I5 f 4.8
I4 2.5
I5 rt 10
4 zt I.8
III
CYCLIC AMP-DEPENDENT ACTIVATION OF TRIGLYCERIDE, LIPASE IN PURIFIED PREPARATION
DIGLYCERIDE
AND
MONOGLYCERIDE
Activation of the enzyme and lipase assays were carried out under conditions described in Materials and Methods. Activation is expressed relative to lipase activity in presence of ATP-Mg’+and protein kinase but without addition of cyclic AMP. Three separate experiments were carried out and data shown below represent the average of two separate incubations (activation and assay) from one experiment.
Substrate
Lipase
activity
(,uequiv free fatty
With A TP-Mg=+ and protein kinase
Triolein Diolein Monoolein
acids/ml
per h)
yO Change
With A TP-Mg*+, protein kinase and cyclic-d MP
0.702
1.05
2.63 2.61
2.93 2.51
+5o +II 0
however, this was not seen consistently and was always much less than the activation of triglyceride lipase in the same preparation. Diferential efects of additions to the assay system on triglyceride and monoglyceride lipase activities As shown in Fig. I, NaCl over a concentration range of 0.1 M to 1.0 M in the assay mixture had little effect on triglyceride lipase activity. In contrast, monoglyceride lipase activity was inhibited progressively with increasing NaCl concentration. At I M NaCl monoglyceride lipase activity was reduced by more than 50%. Isopropanol has been shown previously to inhibit triglyceride lipase activity in crude homogenates of adipose tissue to a greater extent than monoglyceride lipase activity’. As shown in Fig. 2, similar effects were seen with this purified preparation. At a final isopropanol concentration of 4%, triglyceride lipase was inhibited by more than 70% while monoglyceride lipase was inhibited less than 20%. Preparation of substrates by sonication in taurodeoxycholate to give final concentrations of I and 5 mM in the assay mixture had strikingly different effects as shown in Table IV. Triglyceridase activity was markedly inhibited whereas monoglyceridase activity was enhanced relative to activity assayed using gum arabic suspensions.
Biochim. Bioplrys. Acta,
270 (1972) 65-73
R. A. HELLER,
D. STEINBERG
TRIOLEIN
I
I
I
0
0.5
10
N&l
CONCENTRATION
0
2
4
ISOPROPANOL
(Ml
a
6
10
CONCENTRATION
12 (%I
Fig. I. Effect of increasing NaCl concentrations in the assay mixture on triglyceride and monoglyceride lipase activities of the purified enzyme preparation. Lipase activities are expressed as a percentage of activity obtained in control assays not containing NaCl. Fig. z. Differential inhibition of triglyceride and monoglyceride lipase activities in the purified preparation by isopropanol added to the assay mixture to give the final concentrations indicated. Lipase activity is expressed as the percentage of activity obtained in control assays not containing isopropanol. TABLE
IV
DIFFERENTIAL LIPASE
EFFECT
OF TAURODEOXYCHOLATE
ON
TRIGLYCERIDE
LIPASE
AND
MONOGLYCERIDE
ACTIVITIES
The substrates were sonicated in gum arabic or in taurodeoxycholate (pH 7.0) to give the indicated concentrations in the final assay mixture. Lipase assays were carried out under conditions described in Materials and Methods. Two separate experiments were carried out. Data shown below represent the average of two separate incubations from one experiment. Substrate
Triolein Monoolein
Gum arabic
Taurodeoxycholate (1 mM)
0/ODifference
Tauvodeoxycholate (5 mM)
0/ODifference
(o.~WXJ 0.742 3. I 2 1
0.434 5.352
-42 +7I
0.073 4.606
-91 +47
D$$%rentialintrinsic pro$erties (heat lability and pH optimum) The purified enzyme was preincubated for 45 min at different temperatures and then assayed at 23 “C in the usual way. As shown in Fig. 3, monoglyceridase activity was more temperature sensitive than triglyceridase activity. Preincubation at 48 “C, for example, did not effect triglyceridase activity but reduced monoglyceridase activity by 40%. Similar results were observed in three other experiments. Preincubation of the enzyme at 60 “C resulted in a complete loss of both enzymatic activities. Assays performed over a pH range of 5.2 to 9.0 in phosphate buffer (pH 5.2-7.4) and Tris-chloride (pH 6.8-9) showed a rather broad optimum for triglyceride lipase. The peak was at 6.8 in agreement with previous results’. Monoglyceridase activity showed a much sharper optimum between pH 7.6 and 7.8 with a shoulder of activity over the range from 6.2 to 7.4 (Fig. 4). Biochim.
Biophys.
Acta, 270 (1972) 65-73
GLYCERIDASE
ACTIVITY OF TRSGLYCERIDE LIPASE
0 PHOSPHATE BUFFER l
TRISHCI
BUFFER
I
P---j
MONOOLEIN
i cl--
0
8
16
24
;2
TEMPERATURE
40
46
56
I
4
I
I
I
5
6
7
8
9
(*C)
PH
Fig. 3. Heat stability of triglyceride and monoglyceride lipase activities in the purified enzyme preparation. The enzyme preparation was incubated for 45 min at o, 40. 45, 48, 52 and 55 “C and then assayed for the two activities at 23 “C. Lipase activity of the fraction incubated at o “C was taken as equal to IOO, and the activity of the fractions incubated at the higher temperatures were expressed as a percentage of that value. Fig. 4. The pH activity curve for triglyceride and monoglyceride lipase activities of the purified enzyme preparation. Sodium phosphate buffer, ooz M, was used for the pH range of 5.2-7.4 and
Tris-HCl, 0.02 M, for the pH range 6.8-9.0. DISCUSSION
These results provide additional evidence that adipose tissue contains lower glyceridase activity distinct from its hormone-sensitive triglyceridase activity. During purification of the latter there was a marked decrease in the relative activity of both diglyceridase and monoglyceridase accompanying it (Table I). Preliminary experiments utilizing agarose gel electrophoresis show that monoglyceridase activity can be resolved into as many as four bands, two with little or no accompanying triglyceride lipase activity (R. A. Heller and D. Steinberg, unpublished results). Under the assay conditions used, activity against lower glycerides was considerably greater than that against triglyceride in the crude supernatant fraction and at every step in purification. It is recognized that comparisons of enzyme activities against insoluble substrates pose special and difficult problems. Activity is importantly influenced by the method of suspending the substrate and different methods may be required for optimal results with different substrates. This was the case in the present studies, for example, with the use of taurodeoxycholate for preparing glyceride emulsions (Table IV). Not knowing how the substrates are made available in the intact cell we cannot be certain that results obtained in the artificial systems fairly reflect the relative activities i9t viva. All that can be said is that using saturating levels of substrate emulsions in a given mode of presentation lower glyceridase activity exceeded triglyceridase activity. The excess-of lower glyceridase activity is consonant with the proposition that Biochim.
Biophys.
Acta, 270 (1972) 65-73
72
R. A. HELLER, D. STEINBERG
the rate-limiting step in the mobilization of stored triglycerides is the hydrolysis of the first ester bonds. The lower glyceride content of rat adipose tissue under basal conditions is very low. No increase was detected when free fatty acid mobilization was stimulated by epinephrine ilz vitro 14. However, Scow16reported increases during maximal adrenocorticotropic hormone stimulation of perfused parametrial adipose tissue. If lower glyceridase activity can under some circumstances become rate-limiting it would be important to know if it is responsive to hormone treatment. Earlier studies showed that pretreatment of epididymal fat pads with lipolytic hormones did not affect diglyceridase or monoglyceridase activity in whole homogenates or in etherextracted homogenates, while triglyceridase activity was increased IOO to 200%2. An apparent activation of monoglyceride lipase was reported by Gorin and Shafirle but this occurred only in tissue previously incubated with dinitrophenol to reduce initial monoglyceridase activity. Now that the probable mechanism of activation of hormonesensitive triglyceride lipase has been characterized %17it was possible in the present studies to compare effects of the protein kinase-cyclic AMP system on the various glyceridase activities in single fractions at different stages of purification. Triglyceridase activity was consistently enhanced but there was little or no enhancement of either diglyceridase or monoglyceridase activity (Table II and III). In crude fractions, which appear to contain several monoglyceride lipase activities, activation of only one of them might be overlooked, masked by the large background of non-activatable enzyme. We were particularly interested in whether the lower glyceridase activity associated with the hormone-sensitive lipase might be activatable. However, even in the most highly purified preparation there was no protein kinase activation of lower glyceridase. The possibility that lower glyceridases are activated by other hormone-mediated mechanisms is not ruled out by these studies but is made less likely by the studies of hormone-treated intact tissue mentioned above. Even though purified, approx. roe-fold from the 78000 xg soluble supernatant fraction, the final preparation of hormone-sensitive lipase still contained considerable lower glyceridase activity. This preparation converts triolein to free fatty acids and glycerol without significant accumulation of lower glycerides (R. A. Heller and D. Steinberg, unpublished data). It was of interest to try to determine whether a single enzyme was responsible for overall triglyceride breakdown or whether the preparation included one or more distinct lower glyceridases. The latter remains a real possibility since the final preparation appears to be a very high molecular weight (approx. 5 .IO~), lipid-rich particle that might represent a multi-enzyme complex’. To this end a number of comparisons were made of the properties of monoglyceridase and triglyceridase in the roe-fold purified preparation. The difference in response to protein kinase has already been discussed. Other differences found included differemes in pH optimum, thermal stability, inhibition by high concentrations of NaCl, by isopropanol and by taurodeoxycholate. The latter three differences could easily reflect effects on the physical state of the substrates rather than direct effects on the enzyme. Even the difference in activatability does not necessarily require postulation of two distinct enzyme proteins. One can visualize an enzyme configuration such that monoglyceride hydrolysis proceeds optimally even in the non-activated state; protein kinase treatment might favorably alter configuration so as to increase V for triglyceride hydrolysis without altering V for monoglyceride hydrolysis. The differences in heat lability and pH activity curves seem more likely to reflect different intrinsic enzyme properties. Putting all Biochim.
Biophys.
Acta,
270
(1972)
65-73
GLYCERIDASE ACTIVITY OF TRIGLYCERIDE
73
LIPASE
the data together, it seems probable that more than one enzyme is involved but until the activities can be physically resolved that conclusion must remain tentative. ACKNOWLEDGEMENT
We would like to thank Miss Alegria A. Aquino for her excellent technical assistance. This work was supported by Training Grant HL-o58gg-or and HL-12373 and HL-14197, from the National Heart and Lung Institute. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16
17
M. Vaughan, J. E. Berger and D. Steinberg, J. Biol. Chem., z3g (1964) 401. 0. Strand, M. Vaughan and D. Steinberg, J. Lipid Res., 5 (1964) 554. E. Gorin and E. Shafrir, Biochim. Biophys. Ada, 84 (1964) 24. E. D. Kom and T. W. Quigley, J. Biol. Chem., 226 (1957) 883. Y. Biale, E. Gorin and E. Shafrir, Biochim. Biophys. Acta, 152 (1968) 28. J. K. Huttunen, J. Ellingboe, R. C. Pittman and D. Steinberg, Biochim. Bioehys. Ada, 218 (1970) 333. J. K. Huttunen, A. A. Aquino and D. Steinberg, Biochim. Biophys. Acta, 224 (1970) 295. R. Heller and D. Steinberg, Fed. Pvoc., 30 (1971) logo Abs. J. K. Huttunen and D. Steinberg, Biochim. Biophys. Acta, 239 (1971) 411. V. P. Dole, J. Clin. Invest., 35 (1956) 160. T. F. Kelley, J. Lipid Res., g (1968) 799. M. Novak, J. Lipid Res., 6 (1965) 431. J. K. Huttunen, D, Steinberg and S. E. Mayer,Biochem. Biophys. Res. Commun., 41 (1970) 1350. M. Vaughan and D. Steinberg, J. Lipid Res., 4 (1963) 193. R. 0. Scow, in A. E. Renola and G. F. Cahill, Handbook of Physiology, Section 5: Adipose Tissue, Am. Physiol. Sot., Washington, D. C., 1965, p. 437. E. Gorin and E. Shafrir, Biochim. Biophys. Acta, 137 (1967) 189. J. D. Corbin, E. M. Reimann, D. A. Walsh and E. G. Krebs, J. Biol. Chem., 245 (1970) 4849. Biochim.
Biophys.
Acta,
270 (1972) 65-73
BIOCHIMICA ET BIOPHYSICA ACTA BBA
56051
PARTIAL
GLYCERIDASE
ACTIVATABLE
RENU
A. HELLER
TRIGLYCERIDE
and DANIEL
Department of Medicine, 92037 (U.S.A.) (Received
December
ACTIVITY
OF A PROTEIN-KINASE
LIPASE FROM RAT ADIPOSE TISSUE
STEINBERG
School of Medicine,
University of Califonzia,
San Diego, La Jolla, Calif.
zgth, 1971)
SUMMARY I. A preparation of hormone-sensitive triglyceride lipase purified approximately Ioo-fold from a high-speed supernatant fraction of rat adipose tissue homogenates also hydrolyzed monoolein and diolein emulsions. The rate of hydrolysis of these lower glycerides was about 5 times that for hydrolysis of triolein. 2. Triglyceride lipase activity was increased by 70% after incubation with protein kinase, cyclic AMP and ATP-Mge+; monoglyceride lipase activity in the same preparations, on the other hand, was not altered by this activating system. Diglyceride lipase activity was slightly increased but the mean activation was only 15% above control values. 3. NaCl (I M) inhibited monoglyceride lipase activity more than 50% but did not inhibit triglyceride lipase activity. Isopropanol (4%) inhibited triglyceridase activity by 70% but did not inhibit monoglyceride lipase activity. Triglyceride lipase was inhibited by 40 and 90% when the substrate was prepared in I and 5 mM taurodeoxycholate, respectively, whereas monoglyceride lipase activity was actually enhanced relative to activity measured using suspensions stabilized by gum arabic. 4. Heating the enzyme preparation at 48 “C for 45 min caused 40% inactivation of monoglyceride lipase activity without altering triglyceride lipase activity. 5. The optimum for triglyceride lipase activity was at pH 6.8, whereas the optimum for monoglyceride lipase activity was at pH 7.6-7.8. 6. These data are compatible with the presence of a separate monoglyceride lipase not subject to kinase-mediated activation and having rather distinct properties from triglyceride lipase. However, until physical resolution of the two activities can be demonstrated this conclusion must remain tentative.
INTRODUCTION
Crude extracts of rat adipose tissue contain: (I) hormone-sensitive triglyceride lipasel-a; (2) lipoprotein lipase4; (3) a high level of activity against monoglycerides Abbreviations: EGTA, ethyleneglycol-his-(aminoethyl cyclic 3’: f-adenosine monophosphate.
ether)-N,N’-tetraacetic
Biocham. Biophys.
acid; cyclic AMP,
Acta, 270
(1972)
65-73
66
R. A. HELLER,
D. STEINBERG
(monoglyceridase)1-3; (4) a high level of activity against diglycerides (diglyceridase)av3 and (5) activity against short chain glyceryl esters (esterase)6. None of these activities has been prepared in a pure form and it is not known whether each activity is referable to a single enzyme protein or to more than one. Nor is it known to what extent there is overlap, i.e. to what extent assays with a given substrate measure the activity of more than one enzyme or class of enzymes. The properties of the first three (hormone-sensitive lipase, lipoprotein lipase and monoglyceridase*) and their different responses to nutritional and hormone influences strongly suggest that they represent distinct enzymes but that has not been firmly established. Recent work in this laboratory has yielded a partial purification of hormone-sensitive lipase (loo-fold from the 78oooxg supernatant fraction of a crude homogenate)6g7. The present studies were undertaken to determine the extent to which activity against lower glycerides accompanies the triglyceride lipase activity during purification and to compare the properties of the former and the latter, particularly in the preparation purified IOOfold with respect to hormone-sensitive triglyceride lipase. A preliminary report of these findings has appeared elsewhere8. MATERIALS AND METHODS
Preparation
of hormone-sensitive
lipase
The enzyme was prepared from epididymal fat pads of fed Sprague-Dawley rats (150-200 g). The tissue was first incubated for 4 h in Krebs-Ringer bicarbonate buffer, pH 7.4 containing 4% bovine serum albumin. This preincubation has been shown to reduce the tissue content of lipoprotein lipase to very low levels, thereby reducing the possibility of contamination of hormone-sensitive lipase preparations with that enzyme. Homogenization of the tissue and purification of the enzyme were carried out as reported previously’. Briefly, the procedure involves homogenization of the fat pads in sucrose 0.25 M and IO-~ M EDTA. The homogenate is subjected to ultracentrifugation for I h at 78000 xg followed by an isoelectric precipitation of the soluble supernatant fraction at pH 5.2. The precipitate is redissolved in 0.02 M Tris buffer (pH 7.4) containing EDTA (IO-~ M) and dithiothreitol (IO-~ M), adjusted to density 1.12 with sucrose, and centrifuged for 48 h at 105000xg. The top fraction (dc1.12) is adjusted to density 1.06 and centrifuged for 48 h at 105000 xg. The sedimented enzyme (d 1.06-d 1.12) is subjected to gel f&ration on 4% agarose. Lipase activity emerging in the void peak is subjected to a final centrifugation for I h at 87000 xg to remove membrane-like material emerging with the lipase in the void volume. Unless otherwise noted, the results in this paper were obtained with lipase preparations carried through this procedure representing approximately a roe-fold purification from the original 78 000 x g supernatant . Lipase assays
Triolein was obtained from the Sigma Chemical Co., St. Louis, MO. and glyceryl [carboxy-14C]trioleate, spec.. act. 15.2 mCi/mM, was obtained from the International Chemical and Nuclear Corporation, Irvine, Calif. [%]Triolein was diluted with un* The term monoglyceridase is used here in a strictly operational sense to refer to enzyme activity assayed using monoolein as substrate under the conditions specified. Diglyceridase and triglyceridase are used similarly to refer to activity assayed using diolein or triolein as substrate. Biockim. Biophys. Acta, 270 (1972)
65-73
GLYCERIDASEACTIVITYOFTRIGLYCERIDELIPASE
67
labelled triolein to give a final spec. act. of 0.083 &i/pmole. Diolein and monoolein were obtained from the Hormel Institute, Austin, Minn. Monoolein (50 mCi/mmole) and diolein (IOO mCi/mM) both carboxyJ*C-labelled (>gg.5% pure) were purchased from Dhom Products Ltd, North Hollywood, Calif. [W]Diolein and [llC]monoolein were both diluted with unlabelled diolein and monoolein, respectively, to give a final spec. act. of 0.025&i/,umole. Triolein emulsions were prepared by sonication with 5 yO gum arabic as previously described6 and diolein emulsions were prepared in the same way. In our early studies monoolein suspensions were also prepared in gum arabic but prolonged sonication (30 s) was required to obtain homogeneous preparations and this caused significant hydrolysis and high blank values (up to 20 y0 of experimental values). Homogeneous preparations were obtained by mixing the monoolein with IO mM taurodeoxycholate, pH 7.0, on a Vortex mixer. Such preparations, used in all but the preliminary studies, were clear or only slightly opalescent, stable and yielded consistent results. Monoglyceride lipase activity assayed with gum arabic suspension tended to be lower but the best gum arabic preparations yielded activities equal to those obtained with taurodeoxycholate preparations. Triglyceride lipase assay was performed as described previously6 in a final assay volume of 0.2 ml, containing 1.2 ,umoles triolein, 5 mg bovine serum albumin and 50 pmoles phosphate buffer, pH 6.8 Lipolysis was allowed to proceed for I h at 23 “C. Diglyceride lipase and monoglyceride lipase assays were carried out using the same procedure but substituting 2 ,umoles of diolein or 2 pmoles of monoolein and incubating for only 30 min in the case of diolein and 15 min in the case of monoolein. Substrate concentration activity curves showed that the substrate concentrations used were saturating. For all three substrates the assay remained linear over the time intervals used. Using the labelled substrates, zero times values and values after incubation without enzyme were usually less than 1% and always less than 5% of experimental values. When studying enzyme activation by cyclic AMP and protein kinase, activation was carried out at 23 “C for 15 min in 0.4 ml of a mixture containing 2 .IO--~ M Trischloride buffer, pH 7.4; EGTA, I .IO-~M; dithiothreitol, 2 *IO-~M; ATP, I .IO-&M; magnesium acetate, 5 .IO-~M; cyclic AMP, I .10-eM; theophylline, I .IO-~M; and rabbit muscle protein kinase, IO ,ugper assay. The activated enzyme was assayed after the addition of 0.1 ml of an assay mixture containing substrate (triolein 1.2 pmoles, diolein 2 ,umoles, or monoolein 2 pmoles); 0.1 M phosphate buffer, pH 6.8; bovine serum albumin, 5 mg; and EDTA, 5 .IO-"M. Previous studies have shown that the high final concentration of EDTA during assay (I.10-sM) effectively stops further activation by protein kinase$. This was checked under the present conditions using purified enzyme; after kinase activation the time course for triolein or diolein hydrolysis in the assay was linear for 90 min and then fell off slightly. Assays were terminated by adding 5 ml of Dole’s extraction medium” and the free fatty acids were isolated from the heptane phase using the Kelley resin methodll as modified by Huttunen et al.@. In some experiments the net free fatty acids in the Dole extract was measured using the method of Novak’*.
B&him.
Biophys.
Acta.
270 (1972) 65-73
68
R. A. HELLER, D. STEINBERG
RESULTS
Lipase activity against different szlbstrates Activity against all three substrates (triolein, diolein and monoolein) was assayed at each successive step during purification. Results of a representative experiment are shown in Table I. The 78000 xg supernatant fraction showed a ratio of triglyceridase to monoglyceridase of I to 15 (range I : 20 to I : 15 in four experiments). The diglyceridase activity in the crude supernatant was usually one-half that of monoglyceridase. TABLE I RATIOOF TRIGLYCERIDE SUCCESSIVE
STAGES
IN
LIPASE
TO
DIGLYCERIDE
AND
MONOGLYCERIDE
LIPASE
ACTIVITIES
AT
PURIFICATION
Lipase assays were carried out under conditions described in Materials and Methods.Three separate experiments were done to study the relative activities at different stages of purification. Shown below are absolute activities from one representative experiment; ranges for relative activities are given in the text under Results. Fraction
Enzyme activity (pequiv/ml per h) Triglyceride
Diglyceride
lipase
lipase
Monoglyceride lipase
Monoglyceride lipase activity/ Triglyceride lipase activity
78000 xg supernatant
0.10
0.73
1.56
15.6
pH d < I.5.2 I 2precipitate fraction Final preparation
4.90 4.42 0.29
22.7 26.7 I .05
35.9 39.4 I .40
::; 4.8
During purification, lower glyceridase activity decreased relative to triglyceridase activity, indicating some resolution of monoglyceride and diglyceride lipase from the triglyceride lipase. However, the final preparation still contained considerable lower glyceridase activity, the triglyceridase to monoglyceridase ratio being I to 5 (range I : 7 to I : 5 in five experiments). The diglyceridase activity of the final preparation was approximately equal to the monoglyceridase activity. Difleerentiul activation of triglyceride, diglyceride and monoglyceride lipase The purified triglyceride lipase has been shown to be activated by cyclic AMPdependent protein kinase; little or no activation was observed in the absence of any one of the key components of the system (cyclic AMP, protein kinase, ATP, Mga+)ls. Using a similar system the activation of triglyceride lipase, diglyceride lipase and monoglyceride lipase was determined at various steps in the purification. As shown in Table II, the triglyceride lipase activity was enhanced in all fractions by the addition of cyclic AMP, ATP-Mg2+ and protein kinase. Control samples contained cyclic AMP and ATP-Mga+ but no protein kinase. In contrast, there was no significant enhancement of diglyceride lipase or monoglyceride lipase activity. The final purified preparation was also tested for the activation of the three lipases using a control containing protein kinase but no cyclic AMP (Table III). Again, while triglyceride lipase was activated by 50%, the monoglyceride lipase activity remained essentially unchanged. This was true whether the monoolein was prepared in gum arabic suspension or in taurodeoxycholate. Diglyceride lipase activity occasionally showed a slight activation ; Biochim. Biophys. Acfa, 270 (1972) 65-73
GLYCERIDASE
ACTIVITY
OF TRIGLYCERIDE
LIPASE
69
TABLE II PROTEINKINASE-DEPENDENT
ACTIVATION
OF
LIPASE
AT
DIFFERENT
STAGES
OF
PURIFICATION
Activation of the enzyme and the lipase assayswere performedas describedunderMaterialsand Methods. Absolute levels of enzyme activities were in the same range as those shown in Table I. Activation is expressed relative to lipase activity in presence of cofactors, including cyclic AMP, but without addition of protein kinase. Data represent mean + SE. in 3 to 12 determinations.
% Activafion
Subs&ate
Triolein Diolein Monoolein
TABLE
with cyclic AMP,
78oooxg supenzatant fraction
PH 5.2 precipitate
32 % I4 I4 f 9.5 6 + 3.4
45 f 4 3 f I.7 -3 & 2.1
protein
kinase and ATP-MgB+
dcI.12 fraction
44 f 5 f
in:
Final preparation
70 5 S I5 f 4.8
I4 2.5
I5 rt 10
4 zt I.8
III
CYCLIC AMP-DEPENDENT ACTIVATION OF TRIGLYCERIDE, LIPASE IN PURIFIED PREPARATION
DIGLYCERIDE
AND
MONOGLYCERIDE
Activation of the enzyme and lipase assays were carried out under conditions described in Materials and Methods. Activation is expressed relative to lipase activity in presence of ATP-Mg’+and protein kinase but without addition of cyclic AMP. Three separate experiments were carried out and data shown below represent the average of two separate incubations (activation and assay) from one experiment.
Substrate
Lipase
activity
(,uequiv free fatty
With A TP-Mg=+ and protein kinase
Triolein Diolein Monoolein
acids/ml
per h)
yO Change
With A TP-Mg*+, protein kinase and cyclic-d MP
0.702
1.05
2.63 2.61
2.93 2.51
+5o +II 0
however, this was not seen consistently and was always much less than the activation of triglyceride lipase in the same preparation. Diferential efects of additions to the assay system on triglyceride and monoglyceride lipase activities As shown in Fig. I, NaCl over a concentration range of 0.1 M to 1.0 M in the assay mixture had little effect on triglyceride lipase activity. In contrast, monoglyceride lipase activity was inhibited progressively with increasing NaCl concentration. At I M NaCl monoglyceride lipase activity was reduced by more than 50%. Isopropanol has been shown previously to inhibit triglyceride lipase activity in crude homogenates of adipose tissue to a greater extent than monoglyceride lipase activity’. As shown in Fig. 2, similar effects were seen with this purified preparation. At a final isopropanol concentration of 4%, triglyceride lipase was inhibited by more than 70% while monoglyceride lipase was inhibited less than 20%. Preparation of substrates by sonication in taurodeoxycholate to give final concentrations of I and 5 mM in the assay mixture had strikingly different effects as shown in Table IV. Triglyceridase activity was markedly inhibited whereas monoglyceridase activity was enhanced relative to activity assayed using gum arabic suspensions.
Biochim. Bioplrys. Acta,
270 (1972) 65-73
R. A. HELLER,
D. STEINBERG
TRIOLEIN
I
I
I
0
0.5
10
N&l
CONCENTRATION
0
2
4
ISOPROPANOL
(Ml
a
6
10
CONCENTRATION
12 (%I
Fig. I. Effect of increasing NaCl concentrations in the assay mixture on triglyceride and monoglyceride lipase activities of the purified enzyme preparation. Lipase activities are expressed as a percentage of activity obtained in control assays not containing NaCl. Fig. z. Differential inhibition of triglyceride and monoglyceride lipase activities in the purified preparation by isopropanol added to the assay mixture to give the final concentrations indicated. Lipase activity is expressed as the percentage of activity obtained in control assays not containing isopropanol. TABLE
IV
DIFFERENTIAL LIPASE
EFFECT
OF TAURODEOXYCHOLATE
ON
TRIGLYCERIDE
LIPASE
AND
MONOGLYCERIDE
ACTIVITIES
The substrates were sonicated in gum arabic or in taurodeoxycholate (pH 7.0) to give the indicated concentrations in the final assay mixture. Lipase assays were carried out under conditions described in Materials and Methods. Two separate experiments were carried out. Data shown below represent the average of two separate incubations from one experiment. Substrate
Triolein Monoolein
Gum arabic
Taurodeoxycholate (1 mM)
0/ODifference
Tauvodeoxycholate (5 mM)
0/ODifference
(o.~WXJ 0.742 3. I 2 1
0.434 5.352
-42 +7I
0.073 4.606
-91 +47
D$$%rentialintrinsic pro$erties (heat lability and pH optimum) The purified enzyme was preincubated for 45 min at different temperatures and then assayed at 23 “C in the usual way. As shown in Fig. 3, monoglyceridase activity was more temperature sensitive than triglyceridase activity. Preincubation at 48 “C, for example, did not effect triglyceridase activity but reduced monoglyceridase activity by 40%. Similar results were observed in three other experiments. Preincubation of the enzyme at 60 “C resulted in a complete loss of both enzymatic activities. Assays performed over a pH range of 5.2 to 9.0 in phosphate buffer (pH 5.2-7.4) and Tris-chloride (pH 6.8-9) showed a rather broad optimum for triglyceride lipase. The peak was at 6.8 in agreement with previous results’. Monoglyceridase activity showed a much sharper optimum between pH 7.6 and 7.8 with a shoulder of activity over the range from 6.2 to 7.4 (Fig. 4). Biochim.
Biophys.
Acta, 270 (1972) 65-73
GLYCERIDASE
ACTIVITY OF TRSGLYCERIDE LIPASE
0 PHOSPHATE BUFFER l
TRISHCI
BUFFER
I
P---j
MONOOLEIN
i cl--
0
8
16
24
;2
TEMPERATURE
40
46
56
I
4
I
I
I
5
6
7
8
9
(*C)
PH
Fig. 3. Heat stability of triglyceride and monoglyceride lipase activities in the purified enzyme preparation. The enzyme preparation was incubated for 45 min at o, 40. 45, 48, 52 and 55 “C and then assayed for the two activities at 23 “C. Lipase activity of the fraction incubated at o “C was taken as equal to IOO, and the activity of the fractions incubated at the higher temperatures were expressed as a percentage of that value. Fig. 4. The pH activity curve for triglyceride and monoglyceride lipase activities of the purified enzyme preparation. Sodium phosphate buffer, ooz M, was used for the pH range of 5.2-7.4 and
Tris-HCl, 0.02 M, for the pH range 6.8-9.0. DISCUSSION
These results provide additional evidence that adipose tissue contains lower glyceridase activity distinct from its hormone-sensitive triglyceridase activity. During purification of the latter there was a marked decrease in the relative activity of both diglyceridase and monoglyceridase accompanying it (Table I). Preliminary experiments utilizing agarose gel electrophoresis show that monoglyceridase activity can be resolved into as many as four bands, two with little or no accompanying triglyceride lipase activity (R. A. Heller and D. Steinberg, unpublished results). Under the assay conditions used, activity against lower glycerides was considerably greater than that against triglyceride in the crude supernatant fraction and at every step in purification. It is recognized that comparisons of enzyme activities against insoluble substrates pose special and difficult problems. Activity is importantly influenced by the method of suspending the substrate and different methods may be required for optimal results with different substrates. This was the case in the present studies, for example, with the use of taurodeoxycholate for preparing glyceride emulsions (Table IV). Not knowing how the substrates are made available in the intact cell we cannot be certain that results obtained in the artificial systems fairly reflect the relative activities i9t viva. All that can be said is that using saturating levels of substrate emulsions in a given mode of presentation lower glyceridase activity exceeded triglyceridase activity. The excess-of lower glyceridase activity is consonant with the proposition that Biochim.
Biophys.
Acta, 270 (1972) 65-73
72
R. A. HELLER, D. STEINBERG
the rate-limiting step in the mobilization of stored triglycerides is the hydrolysis of the first ester bonds. The lower glyceride content of rat adipose tissue under basal conditions is very low. No increase was detected when free fatty acid mobilization was stimulated by epinephrine ilz vitro 14. However, Scow16reported increases during maximal adrenocorticotropic hormone stimulation of perfused parametrial adipose tissue. If lower glyceridase activity can under some circumstances become rate-limiting it would be important to know if it is responsive to hormone treatment. Earlier studies showed that pretreatment of epididymal fat pads with lipolytic hormones did not affect diglyceridase or monoglyceridase activity in whole homogenates or in etherextracted homogenates, while triglyceridase activity was increased IOO to 200%2. An apparent activation of monoglyceride lipase was reported by Gorin and Shafirle but this occurred only in tissue previously incubated with dinitrophenol to reduce initial monoglyceridase activity. Now that the probable mechanism of activation of hormonesensitive triglyceride lipase has been characterized %17it was possible in the present studies to compare effects of the protein kinase-cyclic AMP system on the various glyceridase activities in single fractions at different stages of purification. Triglyceridase activity was consistently enhanced but there was little or no enhancement of either diglyceridase or monoglyceridase activity (Table II and III). In crude fractions, which appear to contain several monoglyceride lipase activities, activation of only one of them might be overlooked, masked by the large background of non-activatable enzyme. We were particularly interested in whether the lower glyceridase activity associated with the hormone-sensitive lipase might be activatable. However, even in the most highly purified preparation there was no protein kinase activation of lower glyceridase. The possibility that lower glyceridases are activated by other hormone-mediated mechanisms is not ruled out by these studies but is made less likely by the studies of hormone-treated intact tissue mentioned above. Even though purified, approx. roe-fold from the 78000 xg soluble supernatant fraction, the final preparation of hormone-sensitive lipase still contained considerable lower glyceridase activity. This preparation converts triolein to free fatty acids and glycerol without significant accumulation of lower glycerides (R. A. Heller and D. Steinberg, unpublished data). It was of interest to try to determine whether a single enzyme was responsible for overall triglyceride breakdown or whether the preparation included one or more distinct lower glyceridases. The latter remains a real possibility since the final preparation appears to be a very high molecular weight (approx. 5 .IO~), lipid-rich particle that might represent a multi-enzyme complex’. To this end a number of comparisons were made of the properties of monoglyceridase and triglyceridase in the roe-fold purified preparation. The difference in response to protein kinase has already been discussed. Other differences found included differemes in pH optimum, thermal stability, inhibition by high concentrations of NaCl, by isopropanol and by taurodeoxycholate. The latter three differences could easily reflect effects on the physical state of the substrates rather than direct effects on the enzyme. Even the difference in activatability does not necessarily require postulation of two distinct enzyme proteins. One can visualize an enzyme configuration such that monoglyceride hydrolysis proceeds optimally even in the non-activated state; protein kinase treatment might favorably alter configuration so as to increase V for triglyceride hydrolysis without altering V for monoglyceride hydrolysis. The differences in heat lability and pH activity curves seem more likely to reflect different intrinsic enzyme properties. Putting all Biochim.
Biophys.
Acta,
270
(1972)
65-73
GLYCERIDASE ACTIVITY OF TRIGLYCERIDE
73
LIPASE
the data together, it seems probable that more than one enzyme is involved but until the activities can be physically resolved that conclusion must remain tentative. ACKNOWLEDGEMENT
We would like to thank Miss Alegria A. Aquino for her excellent technical assistance. This work was supported by Training Grant HL-o58gg-or and HL-12373 and HL-14197, from the National Heart and Lung Institute. REFERENCES I 2 3 4 5 6 7 8 9 IO II 12 13 14 15 16
17
M. Vaughan, J. E. Berger and D. Steinberg, J. Biol. Chem., z3g (1964) 401. 0. Strand, M. Vaughan and D. Steinberg, J. Lipid Res., 5 (1964) 554. E. Gorin and E. Shafrir, Biochim. Biophys. Ada, 84 (1964) 24. E. D. Kom and T. W. Quigley, J. Biol. Chem., 226 (1957) 883. Y. Biale, E. Gorin and E. Shafrir, Biochim. Biophys. Acta, 152 (1968) 28. J. K. Huttunen, J. Ellingboe, R. C. Pittman and D. Steinberg, Biochim. Bioehys. Ada, 218 (1970) 333. J. K. Huttunen, A. A. Aquino and D. Steinberg, Biochim. Biophys. Acta, 224 (1970) 295. R. Heller and D. Steinberg, Fed. Pvoc., 30 (1971) logo Abs. J. K. Huttunen and D. Steinberg, Biochim. Biophys. Acta, 239 (1971) 411. V. P. Dole, J. Clin. Invest., 35 (1956) 160. T. F. Kelley, J. Lipid Res., g (1968) 799. M. Novak, J. Lipid Res., 6 (1965) 431. J. K. Huttunen, D, Steinberg and S. E. Mayer,Biochem. Biophys. Res. Commun., 41 (1970) 1350. M. Vaughan and D. Steinberg, J. Lipid Res., 4 (1963) 193. R. 0. Scow, in A. E. Renola and G. F. Cahill, Handbook of Physiology, Section 5: Adipose Tissue, Am. Physiol. Sot., Washington, D. C., 1965, p. 437. E. Gorin and E. Shafrir, Biochim. Biophys. Acta, 137 (1967) 189. J. D. Corbin, E. M. Reimann, D. A. Walsh and E. G. Krebs, J. Biol. Chem., 245 (1970) 4849. Biochim.
Biophys.
Acta,
270 (1972) 65-73