Inhibitors of Lipase Activities in Soybean and Other Oil Seeds'Received for publication July 20, 1984
SHUE-MEI WANG AND ANTHONY H.C. HUANG*Biology Department, University ofSouth Carolina, Columbia, South Carolina 29208
ABSTRACT
In the cotyledon extracts of seedlings of many oil seeds, includingsoybean, sunflower, cucumber, and peanut, the in vitro lipase activitywas too low to account for the observed in vivo lipolysis. The low in vitrolipase activity was due to the presence of lipase inhibitors in the extracts.The inhibitors from soybean were characterized based on their effects onthe hydrolysis of trilinolein by corn, pancreatic, and Rhizopus lipases.The inhibitors were not dialyzable and unaltered by RNase and ,-galactosidase treatment. However, they were sensitive to heafing andprotease digestion. The inhibitory effect of the inhibitors was expressedirrespective of the sequence of the addition of lipase, substrate, andinhibitors to the assay medium. The inhibitory effect was equally ex-pressed when the inhibitors were added either before or after the lipasereaction had been in progress. The inhibitory effect of the inhibitors wasindependent of the amount of lipase present in the assay, but wasdependent on the amount of substrate added. High substrate concentra-tion eliminated totally the inhibitory effect of the inhibitors. Most of theinhibitors were recovered in the soluble fraction in subcellular fractiona-tion. They were present in the 24S and not in the 7S, and IIS (storageproteins) protein fraction. There was a gradual decrease of the inhibitorsin the cotyledons in the postgerminative growth. We suggest that theinhibitors are proteins which bind to the surface of the substrate micelles.The binding prevents the normal functioning of lipase which acts on theinterfacial area between the aqueous medium and the micelle surface.
In the postgerminative growth of seeds, the reserve triacylglyc-erols are mobilized to support the growth of the embryonic axis.The mobilization is via a long gluconeogenic pathway (1, 7), theinitial step ofwhich is the hydrolysis oftriacylglycerols to glyceroland fatty acids in the presence of lipase (EC 3.1.1.3). In severalseed species, including castor bean, corn, cotton, mustard, andrape, the lipase is known to be associated with the membrane ofthe storage lipid bodies (6, 17). However, in several other seedspecies, in vitro lipase activity in the lipid bodies or crude extractswas undetectable or too low to account for the in vivo rate oflipolysis (6, 8, 16, and results to be reported in this paper).Apparently, the enzyme was inactivated soon after tissue ho-mogenization or its activity was somehow inhibited or not as-sayed at suitable conditions.
Seeds generally contain proteins and, depending on the plantspecies, starch and/or triacylglycerols as food reserve for germi-nation. In the mobilization of these three major reserves ingermination, they are hydrolyzed initially by specific proteases,amylases, and lipases. Seeds ofsome species are known to containinhibitors of proteases (9, 10) and amylases (5, 14), whosephysiological functions are still unclear. Inhibitors of lipases inseeds are relatively unknown. There are reports of lipase inhibi-
' Supported by National Science Foundation PCM-8304556.
tors in extracts of soybean (18), but their mechanism of actionis not understood.
In our studies on seed lipases, we detected little or no lipaseactivities in the extracts of several major seed species. We foundthat inhibitory factors of lipase activities were present in theextracts. In this paper, we report the general characteristics ofthese factors and their mechanism of action.
MATERIALS AND METHODS
Plant Materials. Soybean seeds (Glycine max L. Merr. cvCoker 237) from Coker Pedigreed Seed Co. were soaked in waterfor 1 h and allowed to germinate on moist tissue papers at 26°Cin darkness. Cotyledons collected from ungerminated seeds and4-, 6-, and 7-d-old seedlings were used. Seeds of cucumber(Cucumis sativus L.), rape (Brassica napus L. cv Tower), mustard(Brassica juncea L.), glandless cotton (Gossypium hirsutum L.),sunflower (Helianthus annus L.), and corn (Zea mays L. cv Mo17) were soaked in running water for 24 h and allowed togerminate on moist paper towels at 26°C in darkness. Peanut(Arachis hypogaea L. cv Red Valencia) seeds were germinateddirectly on moist paper towels at 26°C in darkness. The cotyle-dons of 4-d-old cucumber, rape, mustard, and sunflower seed-lings, cotyledons of 5-d-old peanut seedlings, and scutella of 6-d-old corn seedlings were used for lipase and inhibitor prepara-tions.Assay of Lipase Activity. Trilinolein from Nu Chek Prep Inc.
(Elysian, MN) was used as the substrate. An emulsion of 25 mMtrilinolein in 5% Gum Arabic was prepared by sonication at highspeed with a Bronwill Biosonik sonicator (VWR Scientific, SanFrancisco) for 30 s. Lipolytic activity was measured in a l-mlreaction mixture containing 0.1 M Tris-HCI buffer (pH 7.5), 5mM trilinolein (from 0.2 ml of the above emulsion), 5 mm DTT,and lipase preparation (1 1). In the assay of pancreatic lipase, 0.2M NaCl and 5 mM CaCl2 were also present in the reactionmixture. In the assay of lipase activities in various plant extractsat different pH values, 0.1 M Na-succinate (pH 4.5), imidazole-HCI (pH 6.0), and Na-glycine (pH 9.0) were used instead of Tris-HCI (pH 7.5) in the assay mixture. The reaction was carried outat 34°C in a shaker-water bath. Aliquots of the reaction mixturewere sampled at time intervals. The amount of fatty acids re-leased was determined using 1,5-diphenylcarbazide (15). Theinitial rate of lipolytic activity was expressed as nmol fatty acidreleased per min.
Preparation of Inhibitors. All operations were performed at4°C. Cotyledons of 6-d-old soybean seedlings were chopped intosmall pieces with a razor blade in a Petri dish and ground gentlyin a mortar and pestle. The grinding medium contained I mMEDTA, 10 mm KCI, 1 mM MgCI2, 2 mm DTT, 0.6 M sucrose,and 0.15 M Tricine adjusted to pH 7.5 with KOH. The homog-enate was filtered through a Nitex cloth of pore size of 20 x 20,m. The filtrate was used directly as the crude extract in theassays of lipase activities or subjected to further fractionation. Itwas centrifuged at 10,000g for 20 min and the pellet was resus-pended in grinding medium. The supernatant was recentrifuged
at 1 50,000g for 2 h. The resulting supernatant was used directlyor dialyzed against 0.1 M Tris-HCI buffer (pH 7.5). The inhibitorpreparations ofother seeds were obtained by the same procedure.Assay of Inhibitor Activity. Fifty to 800 ,ug proteins of the
inhibitors (in crude extract, undialyzed, or dialyzed 150,000gsupernatant, as specified in "Results") were added to a lipaseassay mixture as described above but without lipase, making avolume of 0.9 ml. The mixture was incubated at 24°C for 5 min.Lipase preparation in 100 ul of grinding medium (pH 7.5) wasadded to initiate the lipolytic reaction. The lipolytic activity wasmonitored as described above.Enzymes. Ribonuclease (type I-A), (3-galactosidase (grade IV),
Protease V8, pancreatic lipase (type VI-S), and Rhizopus lipase(type XI) were purchased from Sigma Chemical Co. Pronase wasobtained from CalBiochem Co. Lipid bodies were isolated fromthe scutella of 6-d-old corn seedlings (12). The lipids in theisolated lipid bodies suspended in grinding medium were ex-tracted with diethyl ether. After removal ofthe ether, the aqueousmedium contained the lipid body membrane fragments andsolubilized lipase. It was centrifuged at 150,000g for 2 h. Thesupernatant contained the solubilized lipase, and was retained.This corn lipase preparation, the commercially prepared pan-creatic lipase, and the commercially prepared Rhizopus lipasewere used as lipases for inhibitor studies.Enzyme Hydrolysis of the Inhibitor Fraction. Pronase (1250
jLg/ml), Protease V8 (500 ,ug/ml), RNase A (1250 ,ug/ml), and (3-galactosidase (500 MAg/ml) were dissolved in 0.1 M Na-boratebuffer (pH 7.5) containing 10 mM MgCl2, 5 mM CaC12, and 1mM DTT. Each enzyme solution (100 1l) was mixed with 400 u1of dialyzed 150,000g soybean supernatant containing 6.0 mgprotein. The preparation was dialyzed against 0.1 M Na-boratebuffer (pH 7.5) in a dialyzing bag (Spectra/Por 2, MWCO 12,000,from Fisher Scientific Corp.) at 24°C for 6 h. The preparationinside the dialyzing bag was collected and adjusted to 1.50 mlwith 0.1 M Na-borate buffer (pH 7.5). The inhibitory activity ofthe preparation was measured as described and the results arepresented in Table III. In the study of the time course of pronasedigestion, pronase (10 mg/ml) was dissolved in 0.1 M Tris-HCIbuffer (pH 7.5), and incubated at 34C for 2 h to remove thepossible existence of DNase and RNase by pronase proteolysisofthe nucleases (13). The pretreated pronase solution was dilutedwith buffer, and 0.3 ml containing 180 gg protein was mixedwith the dialyzed 1 50,000g soybean supernatant (2.7 ml contain-ing 18 mg protein). The mixture was incubated at 24C. At timeintervals, 0.5 ml of the mixture was pipetted into a dialyzing bag(Spectra/Por 3, MWCO 3500, from Fisher Scientific Corp.) anddialyzed against 0.1 M Tris-HCI (pH 7.5) at 4°C for 8 h to removeshort peptides produced by proteolytic activity. The preparationinside the dialyzing bag was collected and adjusted to 0.75 mlwith 0.1 M Tris-HCI (pH 7.5). On the basis of an equal volume,the protein content of the preparation and its inhibitory activityon pancreatic lipase were determined.Ammonium Sulfate Fractionation. The 1 50,000g supernatant
of soybean was fractionated by adding solid (NH4)2SO4 to give a40% saturation. It was centrifuged at 10,000g for 20 min, andthe pellet was collected. The procedure was repeated to yield thepellets of 60% and 80% saturation, and the supernatant of 80%saturation. The pellets were resuspended in grinding medium.All the preparations were dialyzed against 0.1 M Tris-HCl (pH7.5) to remove the (NH4)2SO4.
Preparation of IIS, 7S, and 2-4S Proteins from SoybeanSeeds. Cotyledons of ungerminated soybean seeds were groundin I M NaCl and 35 mm Na-phosphate buffer (pH 7.5) (19). Thehomogenate was filtered through a Nitex cloth of 20 x 20 Mumpore size, and the filtrate was centrifuged at 10,000g for 30 min.The supernatant between the lipid layer and the pellet was
5% to 30% [w/w] sucrose in grinding medium). The gradientwas centrifuged at 27,000 rpm for 24 h in a Beckman L2-65Bultracentrifuge with SW 28 rotor. The gradient was fractionated,and the fractions were assayed for protein content. The proteinpeaks corresponding to the 24S and 7-1IS proteins were col-lected. The two protein preparations were dialyzed against 0.1 MTris-HCl (pH 7.5) containing 1 M NaCl. The dialyzed preparationwas then concentrated in a ultrafiltration cell with a AmiconYM 10 filter (Amicon Co.).
Protein Determination. The protein contents were determinedby the Coomassie Blue method using bovine albumin (fractionV) as the standard (4).
RESULTS
Occurrence of Lipase Inhibitors in Oil Seeds. The total lipaseactivities in the crude extracts of the storage tissues of seedlingsofdiverse oil seed species were examined. The measured activitieswere compared with the in vivo rate of lipolysis as estimated bythe assumption that the storage triacylglycerols are consumedwithin a period of 7 to 10 d. The lipase activities were relativelyhigh in corn, cotton, mustard, and rape (Table I). These activitiesare higher than (more than 10-fold) those required to accountfor the in vivo rate of lipolysis in germination. On the contrary,the lipase activities in the total extract of peanut, sunflower,cucumber, and soybean were comparatively low and, except inpeanut, they are insufficient to account for the in vivo rate oflipolysis.The oil seeds of the latter group were examined for their
possession of lipase inhibitors in the 150,000g supernatants ofthe storage tissues. These supernatants were tested for theirinhibitory effect on the activities of partially purified corn lipaseand commercially prepared pancreatic lipase. By themselves,they exhibited no lipase activity in the lipase assay at pH 7.5.When increasing amounts of the supernatants were added to theassay medium, the lipase activity was reduced (Fig. 1). In thepresence of roughly equal activities of the two lipases in theassays, the pancreatic lipase was apparently more sensitive to theinhibitors than the corn lipase (Fig. 1; Table I). From the results,the amount of protein in the seed extracts that would give a 50%inhibition of the measured lipase activity was designated as oneinhibitor unit. Of the various oil seeds examined, soybean, sun-flower, cucumber, and peanut possessed inhibitors in a decreas-ing order of contents of inhibitor units per g fresh weight.
Characterization of the Soybean Inhibitors. Since the soybeanextract possessed more inhibitor units per g fresh weight thanother oil seeds examined, it was selected for further studies. Thedialyzed preparation of the supernatant obtained after centrifu-gation of the crude extract at 1 50,000g for 2 h contained essen-tially all of the inhibitors (see later description), and was used asthe inhibitor preparation.The soybean inhibitors inhibited the activities of lipases from
all sources examined, including plants (corn, cotton, mustard,and rape), animal (procine pancreas), and fungus (Rhizopus)(Table II). Furthermore, the degree ofinhibition exhibited by thesoybean inhibitors was independent ofthe amount oflipase usedwithin the range suitable for the enzyme assay (a 6-fold differ-ence). Further characterization of the soybean inhibitors wasperformed using both corn lipase and pancreatic lipase. Similarresults were obtained.Treatments of the soybean inhibitors with RNase and (3-
galactosidase resulted in no loss of their inhibitory effect (TableIII). However, treatments of the inhibitors with proteases (pro-nase and protease V8) reduced their inhibitory effect substan-tially. The time course ofprotease effect on the soybean inhibitorswas followed (Fig. 2). Pronase was incubated with the inhibitorsat 24C and at time intervals, aliquots of themixturewereplaced
obtained with a syringe and layered onto a sucrose gradient (from
930 WANG AND HUANG
in a dialyzing bag and dialyzed against a dilute buffer at 4'C for
Table I. Lipase Activities and Inhibitor Activities in Tissue Extracts ofSeedlings of Various Oil SeedsThe crude extracts and the 150,000g supernatants were used to assay for lipase activities and inhibitor
Soybean cotyledon 6.0 5 200 105 126 241a Inhibitor activity in the first four tissues was not measured due to the presence of high background lipase
activity.'Inhibitor unit: amount of proteins giving 50% inhibition of lipase activity.I pH 4.5, 6.0, 7.5, and 9.0 were used, and the pH in which the highest activity was detected is shown.
01% m~~~~~~~~~~~~~Peanut
Sunfloweo 50Soybean.
a. Soybean0 Cucuimber
Sunflower
0F (A) (B)Jo~~~~~~~~~~~~~~~r
0 200 400 800 0 200 400
Protein (iag)
FIG. 1. Inhibitory effects of proteins from various oil seeds on (A)corn lipase and (B) pancreatic lipase. Activity (100%) of corn lipase was14.8,15.5,25.4, and 17.3 nmol/min in the assay with soybean, sunflower,cucumber, and peanut, respectively. Activity (100%) of pancreatic lipasewas 19.8, 18.6, 18.6, and 18.6 nmol/min in the assay with soybean,sunflower, cucumber, and peanut, respectively. The 150,000g superna-tants from seedlings of various species were used as the sources ofinhibitor proteins.
8 h. The proteins left in the dialyzing bag were examined fortheir lipase inhibitor activity, on a fixed volume basis. After thepronase addition, there was a gradual decrease in the amount ofproteins left in the dialyzing bag and a concomitant loss of theirinhibitory effect (Fig. 2). Apparently, the large polypeptidesremaining in the dialysis bag still possessed some inhibitoryactivities. The effect ofprotease V8 was similar to that of pronase(data not shown). Heating of the inhibitors at 100°C for 15 minalso reduced the inhibitory effect (Table III).The inhibitory effect of the inhibitors was expressed irrespec-
tive of the sequence of addition of the three major components,lipase, substrate, and inhibitors, into the reaction mixture. Theinhibitory effect was immediately and equally expressed whenthe inhibitors were added either before or after the lipase reactionwas in progress (Fig. 3).
Table II. Effects ofSoybean Inhibitors on the Activities ofLipasesfromVarious Sources
The dialyzed preparation of the 150,000g supernatant of cotyledonextract from 6-d-old soybean seedlings was used.
Relative Lipase Activityin the Presence of
Source of Lipase Activity of Various Amounts ofLipase Used Inhibitor Protein (ug)
Although the inhibitory effect of the inhibitors was independ-ent of the amount of lipase present in the assay (Table II), it wasdependent on the amount of substrate added. In our routinelipase assay, 5 mM of sonicated substrate was used which wasalways sufficient to give maximal enzyme activities (Fig. 4). At
Table III. Effects of Various Treatments ofthe Soybean Inhibitors onTheir Inhibitory Activity on Pancreatic Lipase
The dialyzed preparation of the 150,000g supernatant of cotyledonextract from 6-d-old soybean seedlings was used. The treatments werecarried out in a dialyzing bag. After treatment, the materials left in thedialyzing bag were assayed for lipase inhibitor activities on an equalvolume basis. Relative lipase activity (100%) was 15.5 nmol/min.
Time (h)FIG. 2. A time course of pronase digestion of the soybean inhibitors.
The soybean inhibitors (dialyzed supernatant from 6-d-old seedlings)were incubated with pronase at 24°C. Controls contained either theinhibitors or pronase only. At time intervals, aliquots were put in a
dialysis bag and dialysis was performed at 4°C. After standardization ofthe volume ofthe dialyzed samples, they were assayed for protein contentand their inhibitory effect on the activity of pancreatic lipase.
this concentration of substrate, the inhibitors drastically reducedthe lipase activity, and the reduction was roughly proportionalto the amount of inhibitors added. However, the reduction wasgradually relieved as the concentration of substrate increased. Athigher substrate concentrations, the inhibitors exerted no inhi-bition; they actually enhanced the lipase activity. The phenom-enon was observed using corn, pancreatic, and Rhizopus lipases.The enhancement was not due to the presence of lipase activityin the inhibitor preparation which by itself gave no detectablelipase activity. There was no apparent alteration of the substratein the presence of the inhibitors, as judged from the turbidity ofthe assay suspension or the number of micelles per volumeobserved under a light microscope.
Occurrence of Inhibitors in Soybean Cotyledons. In the 7 d of
1.5
0Em
00Co
cc
CU
C.)
.4:
.0-co
1.0
0.5
0 20 40 60
Time(min)FIG. 3. A time course of lipolysis by pancreatic lipase without or with
the addition (arrows) of 600 usg soybean proteins at 0 or 20 min. Thesoybean proteins were from the dialyzed 1 50,000g supematant from 6-d-old seedlings.
postgerminative growth of the soybean seedlings, there was agradual decrease in the content ofthe inhibitors in the cotyledons,concomitant with the decrease in the total proteins (Table IV).In differential centrifugations of the cotyledon extract of 4-d-oldseedlings, essentially all of the inhibitors were recovered in thesupernatant after centrifugation at 1 50,000g for 2 h (Table V).In a (NH4)2SO4 fractionation of the supernatant, most of theinhibitors were present in the precipitates of the 40%, 60%, and80% (NH4)2S04 saturation (Table V).From the dry seeds, the total seed protein was extracted with
1 M NaCl, and separated into three fractions of 1 S, 7S, and 2-4S proteins according to their sedimentation coefficients bysucrose gradient centrifugation (18). The lipase inhibitors werepresent only in the 2-4S protein fraction (Table V). The 1 IS and7S proteins are well-known storage proteins, whereas the 2-4Sproteins are a mixture of storage proteins and other proteinssuch as protease inhibitors (10, 18).Assays of Oil Seed Lipase Activities Using Higher Substrate
Concentrations. Using the extracts of those seedlings known tohave low in vitro lipase activities (Table I), attempts were madeto see if using higher substrate concentrations could eliminatethe inhibitors and thus reveal higher lipase activities. The assayswere carried out at pH values 4 to 1O, with increments of 0.5 pHunit in between, in order to reveal any increase of lipase activityat a particular pH. In the extract of peanut, the activity of lipasein 10 mm substrate was about twice that in 5 mm, and wasfurther enhanced in 15 mm (Fig. 5); the optimal activity was stillat pH 4.5 irrespective of the substrate concentration. This en-hancement increases the detectable lipase activity in the peanutextract to about 400 nmol/min .g fresh weight (Table I), whichis high enough to account for the observed in vivo lipolysis. Inthe extract of soybean, sunflower, and cucumber, there was noenhancement of lipase activity when the substrate concentrationin the assay was increased from 5 to 10 and 15 mm. This lack ofenhancement is most likely due to the presence of excess inhib-itors in the seed extracts. With soybean, the amount of extractused in our assay for lipase activities contained 4000 ;g proteins(final concentration in the assay mixture), which was muchhigher than the 200 ,ug protein for a 50% inhibition of lipaseactivity (Fig. 1). Drastic reduction ofthe extract used in the assaywas not possible due to the limit in the sensitivity of the assay.
FIG. 4. Effect of increasing substrate concentrationson the activities of lipases in the absence or presence ofsoybean proteins of the indicated amounts. A suspen-sion of 25 mM trilinolein in 5% Arabic gum was pre-pared by sonication, and added to the assay medium tomake to the desired concentration in a final volume of1 ml. The soybean proteins were from the dialyzed150,000g supernatant from 6-d-old seedlings.
Table IV. Activities ofLipase Inhibitors in the Cotyledon Extracts ofSoybean Seedlings
Corn lipase was used in the inhibitor assay. The dialyzed preparationsof the 1 50,000g supernatant of cotyledon extracts were used. Relativelipase activity (100%) was 10.0 nmol/min.
Relative LipaseActivity in the
Total Protein in Presence of Various1 50,00Og Amounts of Inhibitor
Table V. Activities ofLipase Inhibitors in Subfractions ofSoybeanCotyledon Extracts
Corn lipase was used in the inhibitor assay. Six-d-old seedlings wereused in experiment I and II and ungerminated seeds were used inexperiment III. Relative lipase activity (100%) was 20.6 (experiment I),15.5 (experiment II), and 17.7 (experiment III) nmol/min.
Relative Lipase Activityin the Presence of
Fraction Total Various Amounts ofProtein Inhibitor Protein (Mg)
FIG. 5. Effect of substrate concentration on lipase activities in peanutcotyledon extract at various pH values. Buffers were succinate-NaOH(4-6), imidazole-HCI (6-7), Tris-HCI (7-9), and glycine-NaOH (9-10).
Also, the use of 15 mm substrate was close to the maximalamount of substrate that could be used in an assay, and thus afurther increase in the substrate concentration was prevented. Insummary, in lipase assays the inhibitors in the extracts of seedscontaining low amounts of inhibitors can be overcome by anincrease in the substrate concentration, whereas for seed extractscontaining high amounts of inhibitors, other means will have tobe devised to eliminate the inhibitor effect.
DISCUSSION
The low amount of in vitro activity of lipase detected in theextracts of several oil seeds examined is due to the presence ofinhibitors. In soybean, the inhibitors are proteins that can berecovered in the soluble fraction in subcellular fractionation.They are not the globulin storage proteins but are present in thefraction of proteins having sedimentation coefficients of 2-4S.They probably represent a group of proteins rather than oneunique protein species, as judged from their widespread distri-bution in various fractions in a (NH4)2SO4 fractionation. It isspeculated that the proteins possess some hydrophobic propertiessuch that they can bind to the surface of the substrate micelles.The binding is immediate, and it prevents the normal functioningof lipase which acts on the interfacial area between the aqueousmedium and the micelle surface. Addition of substrate increasesthe micelle surface and therefore releases the inhibitory effects.
It is unlikely that the inhibitors exert their effect by binding tothe lipase molecules. Such a direct binding would not be releasedby the increase in substrate, and would be released by the increasein lipase molecules; these are contrary to the current experimen-tal results. Thus, the inhibitor proteins are not the classicalenzyme inhibitors or effectors which are supposed to bind to theenzymes directly and affect the function of the enzymes. Someproteins of animal origins, such as serum albumin, are alsoknown to bind to the substrate micelles and thus affect lipaseactivities (2, 3). Whether the inhibitors exert some physiologicalfunctions in the oil seeds or they merely represent proteins ofamphipathic nature that fortuitously can bind to the substratemicelles in lipase assay remains to be seen.
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