Purification and characterization of lipoprotein lipase from pig adipose tissue.

Abstract Lipoprotein lipase was purified from acetone powders of pig adipose tissue. Extraction of acetone powders with 1.2 m NaCl in 0.005 m sodium-barbital buffer, pH 7.4, or heparin (200 units per ml) in distilled water, was 6 times as effective as extraction with 0.025 m NH4OH-NH4Cl buffer, pH 8.6, the commonly used extractant for lipoprotein lipase. At pH values below 7.5, over 85% of the activity extracted into 1.2 m NaCl could be recovered after 4 hours. The partially purified enzyme at later stages was stabilized by the inclusion of 20% glycerol in the buffers. Most of the purification was accomplished by affinity chromatography on Sepharose 4B columns containing covalently bound heparin. At this step, the preparation was purifed 600-fold. This purified enzyme binds reversibly to columns containing concanavalin A covalently bound to Sepharose. Lipolytic activity was eluted from concanavalin A-Sepharose column with 0.2 m α-methyl-d-mannoside, 1.0 m NaCl and 0.005 m sodium-barbital, pH 7.0. At this stage, the enzyme was purified 2100-fold. Isoelectric focusing yielded a single major peak of activity with an isoelectric point of 4.0. Minimum molecular weight determination by gel filtration in buffers containing 1.0 m NaCl and by disc gel electrophoresis in sodium dodecyl sulfate yielded values of 62,000 and 60,000, respectively. The crude enzyme, and that eluted from heparin-Sepharose columns, did not show stimulation by heparin, whereas that obtained after isoelectric focusing exhibited a 60 to 100% stimulation at 22 µg of heparin per ml. Activation by dialyzed serum was dependent on the stage of purification. The crude enzyme showed a 20-fold stimulation by serum but showed some activity in its absence; that purified by isoelectric focusing exhibited a complete dependence on the presence of serum for hydrolysis of triolein emulsions stabilized with gum arabic. Of the three very low density lipoprotein apoproteins studied, only apoLp-Glu could substitute for serum as an activator. In the presence of serum in the assay system, apoLp-Ser was as potent an inhibitor of lipoprotein lipase as apoLp-Ala.

NaCl could be recovered after 4 hours. The partially purified enzyme at later stages was stabilized by the inclusion of 20% glycerol in the buffers.
Most of the purification was accomplished by affinity chromatography on Sepharose 4B columns containing covalently bound heparin. At this step, the preparation was purifed 600-fold. This purified enzyme binds reversibly to columns containing concanavalin A covalently bound to Sepharose.
Lipolytic activity was eluted from concanavalin A-Sepharose column with 0.2 M cr-methyl-D-mannoside, 1.0 M NaCl and 0.005 M sodium-barbital, pH 7.0. At this stage, the enzyme was purified ZlOO-fold.
Isoelectric focusing yielded a single major peak of activity with an isoelectric point of 4.0. Minimum molecular weight determination by gel liltration in buffers containing 1.0 M NaCl and by disc gel electrophoresis in sodium dodecyl sulfate yielded values of 62,000 and 60,000, respectively. The crude enzyme, and that eluted from heparin-Sepharose columns, did not show stimulation by heparin, whereas that obtained after isoelectric focusing exhibited a 60 to 100% stimulation at 22 pg of heparin per ml. Activation by dialyzed serum was dependent on the stage of purification.
The crude enzyme showed a 20-fold stimulation by serum but showed some activity in its absence; that purified by isoelectric focusing exhibited a complete dependence on the presence of serum for hydrolysis of triolein emulsions stabilized with gum arabic.
Of the three very low density lipoprotein apoproteins studied, only apoLp-Glu could substitute for serum as an activator.
In the presence of serum in the assay system, apoLp-Ser was as potent an inhibitor of lipoprotein lipase as apoLp-Ala.
* This work was supported by United States Public Health Service Grants HL-14990, HL-54494, and HL-14197. $ Present address, Rice Hall, Cornell University, Ithaca, New York 14850.
Adipose tissue lipoprotein lipase (glycerol ester hydrolase, EC 3.1.1.3) is considered a major regulating factor in the uptake of chglomicron and very low density lipoprot,ein triglyceride by atlipocgtcs (1). However, there has been little success in purifying adipose tissue lipoprotein lipase, probably because of its instability.
It, was our purpose to isolate a high specific activity adipose tissue lipoprotein lipasc which could be utilized for in v&o drgradation studies of well characterized lipoproteins. Since the lil)oprotein lipasc activities reported for mammalian adipose tissues have been very low (2) or even undetectable according to some ilivcstigators (3, 4), we first explored alternative methods forcstractioii of lil~oproteiii lipase from acetonepowders. The major modality of purification utilized in the present studies was affinity chromatography on Sepharose columns containing covalcntly bound heparin by a method similar to that developed recently for the purification of milk lipase (5) and of a postheparin plasma lipolytic enzyme (6). Affinity chromatography was also conducted on agarose columns containing covalently bound concanavalitl A. The known inhibitors and activators of the crude adipose tissue lipoprotein lipase were re-evaluated with the highly purified enzyme. MATERIALS AND METHODS Lipoprotein lipase activity was assayed in duplicate with a synthetic [14C]triolein substrate emulsified in the presence of gum arabic. Triolein containing  in all three positions was purchased from DHOM Products, North Hollywood, California. The assay system contained the following components in a t,ot,al volume of 0.5 ml: 0.76 wmole of 114C]t,riolein (0.133 PCi per pmole of triolein) ; 2.5 mg of gum arabici 5 mg of albumin; 0.05-ml of normal dialvzed Die. serum: 0.1 mmole of Tris buffer: 0.05 mmole I 1 Y of NaCl ; 5 pmoles of CaClz; and 0 to 0.05 ml of enzyme preparation. In kinetic studies of the effect of substrate concentration on enzyme activity and in activity measurements of enzyme purified by isoelectric focusing, the ['4C]triolein specific activity was increased to 0.53 &i per @mole of triolein. Assays were conducted at pH 8.6 at 30". Enzyme activity was linear with time up to 60 min and proportional to amount of enzyme added. Labeled free fatty acids released were separated from the substrate by the liquidliquid partition system of Belfrage and Vaughan (7). Variation in NaCl molarity (0 to 1.0 M) and in pH (6 to 10) did not affect the partition coefficient of [l-l%]oleic acid.
Sources of Enzyme Aitivity-Swine, fed ad lib&m and weighing between 15 and 80 kg, were anesthetized with pentobarbital and exsanguinated via a catheter placed in the carotid artery. Subcutaneous adipose tissue was dissected from the cervical, dorsal, and lumbar regions, and stored at -20".
Acetone-ether powders were prepared as previously described (8). Lipase activity in acetone powders stored at -20" was stable for up to 2 weeks. Acetone powders were extracted with the following solutions: The results of this experiment ( Table I) demonstrate clearly that considerable residual lipoprotein lipase activity is still present in acetone powders extracted twice with the commonly used NH*OH-NH&l extractant.
The buffered NaCl solutions and the heparin solution extracted, respectively, 2-and 5-fold more lipoprotein lipase activity than the first NHGOH-NH&l extraction.
Furthermore, the specific activity of the enzyme extracted by NaCl was considerably higher than that of the enzyme extracted by NH40H-NH&I.
Heparin solutions appeared to be even more selective as extractants than NaCl solutions, yielding enzyme with more than three times the specific activity.
As shown in Table I, the lipase activity extracted with either NaCl or heparin was serum-dependent and showed marked inhibition when assayed in the presence of 1 M NaCI, properties similar to those of protamine sulfate, however, led to inhibition of all preparations. Graded levels of NaCl and of heparin were tested for their effectiveness in extracting lipoprotein lipase activity relative to that of NHIOH-NH&I.
As shotin in Fig. 1 At their optimal concentrations, NaCl and heparin extract six and seven times more enzyme activity, respectively, than a single NHIOH-NH&l extraction.
All enzymatic assays were conducted in the presence of the same final The complete system is described under "Materials and Meth-0.005 M sodium-Verona1 buffer, pH 7.4 (Extraction 3a), and the reods." Protamine sulfate was added to provide a final concentra-maining 3 samples with heparin, 200 units per ml. All enzymatic tion of 0.3 mg per ml of assay mixture.
NaCl when used as an in-assays were conducted in the presence of the same heparin (17 hibitor was added to supply a 1 M concentration in the final assay units per ml) and NaCl (0.1 M) concentrations. Enzyme activimixture.
Six aliquots of the same acetone powder preparation ties are expressed as micromole of fatty acid released per ml of enwere extracted twice with 0.025 M NHdOH-NH&l, pH 8.6 (Ex-zyme extract per hour at 30". tractions 1 and 2). Three were then extracted wit,h 1.5 M NaCI, -   concentrations of heparin (17 units per ml) and NaCl (0.1 M). Thus, the results reflect esclusively the effectiveness of heparin and NaCl on estraction of enzyme activity.
Moreover, heparin has essentially no effect on the activity of the crude swine enzyme at concentrations up to 20 units per ml. Glucose (1 M) and methyl-a-D-glucopyranoside (1 M) extracted only traces of lipoprotein lipase activity from swine acetone powders.
Purification of Swine Adipose Tissue Lipoprotein Lipase-The results of a representative purification carried out on 14.5 g of acetone powder are presented in Table II. Dialysis of the crude acetone powder extract was accompanied by precipitation of some protein but this contained no enzyme activity.
Recovery of enzyme activity after dialysis varied from 80 to 137%, (107% in the esample shown in Table II).
Raising the NaCl concentration of the dialyzed solution before applying it to the heparin-Sepharose column decreased the mass of protein adsorbed onto the column and allowed loading of up to 285 ml of extract without significant loss of activity. Fig. 2 presents the elution pattern of lipoprotein lipase from a heparin-Sepharose column. The enzyme activity was eluted as a single peak at a salt concentration of 1.1 M. The specific activity of the pooled material in this peak was more than 600 times that of the original estract.
Lipoprotein lipase purified through heparin-Sepharose chromatography was then applied to a concanavalin A column.
The protein not bound to the column contained only traces of activity.
Again, only negligible amounts of activity were eluted during washing of the column with 1 M NaCl.
Fifty-seven per cent of the applied lipase activity was recovered in a single sharp peak by elution with 0.2 M a-methyl-I)-mannoside, 1 M NaCl, 0.005 M sodium-barbital, pH 7.0. Over 807, of the recovered activity was eluted in 14 ml. The specific activity of the pooled One hundred-milligram aliquots of the same acetone powder preparation were extracted for 1 hour at 0" with graded levels of NaCl in 0.005 M sodium-barbital, pH 7.4, or increasing concentrations of heparin in water. Except for the samples extracted with 1 unit and 1000 units of heparin, each point represents the mean plus or minus S.E. of four determinations analyzed in duplicate.
Lipoprotein lipase activity is expressed as a percentage of the activity extracted with 0.025 M NHdOH-NH&l, pH 8.6.
by guest on March 21, 2020 http://www.jbc.org/ Downloaded from material representing the &ire peak (36 ml) was 3.4 times that of t,he enzyme applied and more t,han 2000 times that of the original estract'. Much broader activit'y peaks were obtained when elution was conducted with linear gradientIs of NaCl (0 to 1 M) and cy-methyl-I)-mannoside (0 to 1 M) with a total volume of 60 ml. Met,hyla-I>-glucoPYra~losicie, when tested either in a step elution (0.2 M or 1 .O M) or as a gradient,, produced a broader activity peak, althou& the total act,ivit)y recovered was similar to that observed with cr-methyl-r)-mannoside.
Isoth the presence of QImethyl-I)-mannoside and high NaCl molarity (1 M) are necessary for elution of the enzyme. No significant elutioxl of activitv " occurred in the presence of 0.2 M cr-methyl-I)-mallnoaide with NaCl molarities of 0.15 or 0.5 M, The lipoprotein lipase enzyme preparation obtained at' this stage was weakly stimulat,ed (12 to 25%) by t'he presence of heparin (4 units per ml) in the medium.
Isoelectric lbmsing-A preliminary study in the pH range 3 to 10 yielded a single peak of activity.
Subsequellt analyses were conducted in the 3 to 6 pH rallge. Enzyme preparat8iolls purified by heparin-Sepharose chromatography or sequent,ially by both heparitGepharose and concanavalin A-Sepharose chromatography produced a single activity peak (Fig. 3) with a p1 of 4,O h 0.1 (mean + S.E. of five determinatiolls).
Disc Gel E2ectrophoresis-Samples of the purified enzyme after concanavalin A-Sepharose chromatography yielded a single major ballcl OH gel electrophoresis in the presence of sodium dodecyl sulfate (Fig. 4) (10) + It was found necessary t,o heat t,he protein at 90" for 1 hour wit(h the detergent.
Under these conditions, esamination of t'he gels after staillillg with Cooma,ssie brilliant blue indicated that very little protein remained on the gel surface. In colltrast, when the protein was heat.ed at 37" for 2 hours, only a very small fraction of the protein entered the gel and multiple fine bands were observed.
The major band observed aft,er treatment of t,he protein samples at 90" (Fig. 4) was shown by disc gel scanning to account for over 95% of the total proteins stained by Coomassie blue. Its mobility indicated a molecular weight of 60,000 (average of two estimations on two enzyme preparations run on two occasions with three replicates each, 58,000 and 63,000). Mobility of the reference proteins (transferrin, albumin, H chain of y-globulin, ovalbumin, and L chain of y-globulin) was not changed by treatment at the higher temperature.
Gel Pi&r&on-Gel filtration chromatography on Sephadex G-150 was conducted in the presence of 1 M NaCl, 20% glycerol, 0.005 M sodium-barbital buffer, pH 7.0. In solutions of low ionic strength both the crude enzyme and the purified enzyme (after heparin-Sepharose or concanavalin A-Sepharose steps) bind tightly to Sephades G-150. Sodium chloride at concentratJions of 0.15 or 0.5 M failed to elute the enzyme.
However, in the FIG. 4. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate-containing buffer of porcine adipose tissue lipoprotein lipase purified by heparin-Sepharose and concanavalin A-Sepharose chromatography.
The three faint bands below the major band have mobilities identical to those of the three bands obtained with "blank" eluates and with crystalline concanavalin A (see "Ma-presence of 1 M NaCl in the buffer, the crude enzyme produced a single activity peak (KBY = 0.209) corresponding to a molecular weight of 62,000 (reference proteins: transferrin, serum albumin, and ovalbumin) . Enzyme Stability-Stability of the crude enzyme extract (in absence of glycerol) was studied by extracting aliquots of the same acetone powder pool for I hour with 1.2 M NaCl in 0.005 M sodium-barbital buffers at pH 6.5, 7.0, 7.5, 8.0, and 8.6. The enzyme activities of these extracts, held at O", were then assayed immediately and at various time intervals for 4 hours. Over the range studied, pH did not affect the total amount or specific activity of the enzyme extracted.
The enzyme activity was remarkably stable at pH 6.5 and 7.0 (less than 10% loss in 4 hours).
However, at pH values above 7.5 there was a progressive loss of activity, especially marked at pH 8.6 (30% loss in 4 hours).
Storage for 24 hours at 0" resulted in loss of almost 907; of the activity at pH 8.6, whereas almost 50% was retained at pH 6.5.
Stability of the partially purified enzyme (heparin-Sepharose step) in 1.0 M NaCl at O-3" at pH 7.0 (0.005 M sodium-barbital) was also studied.
Over 40% of the activity was lost during the 1st hour, after which activity was lost more slowly but exponentially for the next 4 hours, with a half-life of approximately 3.5 hours (Fig. 5). However, the purified enzyme in the presence of 1.0 M NaCl could be stabilized by the addition of glycerol to a final concentration of 20%. As shown in Fig. 5, 80% of the original activity was retained after 3 hours in the presence of 20 y0 glycerol.
The purified enzyme could be frozen at -70" with no loss of activity on storage for several days in the presence of bovine serum albumin (1 mg per ml) or glycerol (507,) ( Table III).  a The values given for the NaCl extraction (1.5 M NaCl-0.005 M sodium-Verona1 buffer, pH 7.4) represent the average f S.E. of three determinations on the same pooled sample. Other values represent enzyme activities on separate enzyme preparations. The enzyme preparations eluted from heparin-Sepharose were examined after storage for several days at -70" in the presence of 1 mg of albumin per ml. All other preparations were examined directly without previous storage. Enzyme activity is expressed as micromole of fatty acid released per ml of enzyme solution per hour.
* The complete assay system contained the following components in a total volume of 0.5 ml: ['4C]triolein substrate, gum arabit, CaC12, albumin, Tris buffer, 0.05 ml of dialyzed serum and 0.05 ml of enzyme. c N.D., not detectable (less than 0.01 pmole per ml per hour). For these preparations after isoelectric focusing, the standard assay system was modified to increase sensitivity by increasing incubation time to 1 hour, substrate specific activity to 0.53 pCi per pmole. a.nd enzvme volume to 0.2 ml. phy were stimulated 1% and 25.fold by serum, respectively, but showed definite activity in the absence of serum. This was not true of some preparat.ions obtained after isoelectric focusing (Table IV).
Three of these preparations showed activities of 0.41 f 0.07 pmole per ml per hour in the presence of serum but showed no detectable activity in the absence of serum (less than 0.01 /Imole per ml per hour) ( Table IV). Characterization of Purijied Adipose Tissue Lipoprotein Lipase -Properties of the enzyme obtained after heparin-Sepharose chromatography are summarized in Fig. 6 (unless otherwise specified, the purified enzyme refers to the enzyme obtained after heparin-Sepharose chromatography). This highly purified lipoprotein lipase, at 30", yielded a linear rate of release of fatty acids from gum arabic-stabilized triolein emulsions for at least The effects of protamine sulfate are illustrated in Fig. 7. The enzyme purified by heparin-Sepharose chromatography showed no inhibition at 125 pg per ml of protamine sulfate and was only inhibited by 22a/, at the highest concentration tested (1000 pg per ml). In contrast, the crude preparation obtained by extraction of an acetone powder with 1.2 M NaCl was inhibited 50% at 333 pg per ml of protamine sulfate and 74% at 1000 I.rg per ml. Data for a crude extract prepared from an acetone powder of rat adipose tissue are presented for comparison.
Even 100 pg per ml of protamine sulfate inhibited the rat enzyme by 70%. When the crude pig enzyme was preincubated with protamine sulfate for 1 hour at 30", the inhibition was considerably greater, as shown in Fig. 7. Inhibition was greater than 80% with 300 pg per ml of protamine sulfate.
Effects of Polypeptides Isolated from Human Very Low Density Lipoprotein,sThe activation of the purified lipoprotein lipase by an apolipoprotein prepared from human very low density FIG. 6. Properties of porcine adipose tissue lipoprotein lipase purified by heparin-Sepharose chromatography with respect to pH of assay, substrate concentration, CaC12 concentration, NaCl molarity, incubation time, and heparin concentration in assay. Enzyme activity is expressed as micromole of fatty acid liberated per ml of enzyme solution per hour. Different enzyme preparations were employed with specific activities varying between 900 and 1200 pmoles per mg of protein per hour. 90 min. The pH activity curve showed a single, fairly sharp peak at about pH 8.8. With triolein substrate stabilized with gum arabic, maximal velocity was attained at 1 mM triolein. When the ionic strength in the assay medium was modified by addition of NaCl an optimum at 0.08 M was observed. Higher salt concentrations caused progressive inhibition, maximal at 0.6 M when the enzyme was 80% inhibited.
All enzyme determinations were carried out without prior preincubation at the NaCl molarity being tested.
The heparin-Sepharose purified enzyme, in contrast to the enzyme further purified by isoelectric focusing, was not significantly stimulated by heparin up to 50 pg per ml (9 units per ml). At high levels of heparin (100 pg per ml) there was a small but consistent inhibition. lipoproteins (I 2) (apoLp-Glu2) was investigated.
No serum was added in these experiments (Fig. 8). The activity in the absence of activator was subtracted from that determined with various levels of activator, as suggested by Bier and IIavel (13). The data obtained were found to fit ~lichaelis-12e~ltc~l kinetics with an apparent K, of 3.3 pg per ml (average of two values: 3.6 and 3.1). High concentrations of apoLl)-Glu,? up to 50 ~.rg per ml, were not inhibitory.
The lipoprotein lipase activity measured in the presence of 30 pg of human apoLp-Glu was not significantly different from that measured with porcine serum. A partially purified lipoprotein lipase preparation (hepariii-Sepharose step) gave an activity of 11.56 f 0.36 pmole per ml of enzyme per hour (mean of three observations in the presence of 0.1 ml of porcine serum per ml of assay and 10.33 & 0.11 when the serum was replaced by human apoLl)-Glu. A second polypeptide isolated from human very low density lipoprotein, apoLp-Ala, was inhibitory in the presence of whole serum in the assay system. Fifty per cent inhibition of lipolytic activity was observed at 35 pg per ml (Fig. 8). ApoLp-Ser was an equally potent inhibitor of swine lipoprotein lipase and gave 50y0 inhibition at 36 pg per ml. Human hemoglobin and heart cytochrome c at a level of 35 pg per ml did not affect lipoprotein lipase activity when added to the serum-containing assay system.

DISCUSSIOX
The present results show that extraction of acetone powders of adipose tissue with either high concentrations of NaCl or heparin considerably enhances the yield of lipoprotein lipase. Yields were six times higher than those obtained with the classical method of NH40H-NH&'1 extraction (8 lipase. However, as shown in the present study, the enzyme is not irreversibly inhibited by 1.0 M NaCl provided it is handled appropriately.
13~ keeping the pH near 7, storing at low temperature and incorporating 20% glycerol in the buffer solutions, the enzyme retains activity for many hours. Of course. the NaCl concentration in the assay must be reduced by dilution.
The failure of previous investigators to identify significant lipoprotein lipase activity in acetone powders of adipose tissue of some mammalian species (3, 4) may very well be traced to the inefficiency of NHdOH-NH&l extraction.
Previous physiological studies of lipoprotein lipase levels, using NH4f estraction, may require re-evaluation.
If the small fraction of lipoprotein lipase extracted by NH.++ is a constant fraction of the total the results would be valid, but if the physiologic variables studied influence extractability, the results may not reflect changes in the true tissue content of enzyme.
Heparin-sepharose column chromatography has been utilized previously in the purification of bovine milk lipase (5) and postheparin serum lipase (6). Egelrud (15) has more recently demonstrated the reversible binding of chicken adipose tissue lipoprotein lipase to heparin-Sepharose; however, no quantitative data on yields and no further characterization of purified adipose tissue lipoprotein lipase has been previously reported. In the present study, the heparin-Sepharose chromatography gave a purification of approximately 600-fold. Enzyme activity was eluted along with the tail of a protein peak and use of a less steep gradient might further improve purification in this step.
Concanavalin A covalently linked to agarose was utilized to achieve further purification.
Under the conditions used, all of the enzyme activity was retained during loading of the column. Elution of the enzyme occurred when the eluting buffer contained both 1 M methyl-oc-n-mannoside and 1 M NaCl. The presence of 1 M NaCl alone in the medium was not sufficient to elute enzyme activity.
Concanavalin A is a phytohemagglutinin which binds to specific carbohydrate residues of glycoproteins (16). The tight but reversible binding of the enzyme to concanavalin A suggests that the porcine adipose tissue lipoprotein lipase is itself a glycoprotein or that it is associated with a tightly bound carbohydrate moiety.
Molecular weight estimation by gel filtration of the lipoprotein lipase species present in 1 M NaCl gave a molecular weight (62,000) similar to that obtained by disc gel electrophoresis in the presence of 0.1% sodium dodecyl sulfate (58,000 to 63,000). Thus, the minimum molecular size determined for this adipose tissue lipoprotein lipase is similar to that obtained by Egelrud and Olivecrona (64,000) for the bovine milk lipase (5). The similarity in polypeptide molecular size measured by gel filtration in 1 M NaCl and by disc gel electrophoresis in the presence of sodium dodecyl sulfate strongly suggests that 1 M NaCl dissociates the enzyme to its monomeric form.
In the present study, the isolated lipoprotein lipase appeared to be homogeneous in its affinity for heparin-Sepharose, and yielded a single major peak on gel filtration on Sephadex G-150. Isoelectric focusing also yielded a single peak of activity, either when it followed heparin-Sepharose chromatography or when it was used with preparations obtained after concanavalin A-Sepharose chromatography.
Two species of lipoprotein lipase in extracts of acetone powders of rat adipose tissue have been reported by Garfinkel et al. (17). These were separated by agarose gel chromatography.
These results cannot be directly compared to the present findings since the former were obtained with a different species and employed a different method of acetone powder extraction, using low ionic strength buffer. It should be noted that the present results, showing homogeneity in the presence of 1.0 M NaCl, do not rule out the possibility of multiple aggregate forms in solutions of lower ionic strength.
The present studies afforded the opportunity to compare the properties of a highly purified adipose tissue lipoprotein lipase with those of cruder preparations.
The porcine adipose tissue lipoprotein lipase does not show stimulation by low levels of heparin, either when added to assays of the crude extract or of the heparin-Sepharose purified enzyme. Lipoprotein lipase purified by isoelectric focusing, however, does show a marked increase in activity in the presence of added heparin.
The purified enzyme was markedly inhibited by heparin when present at levels above 100 pg per ml in the assay media as has been reported for purified milk lipoprotein lipase (5). Protamine sulfate added at zero time (without preincubation) inhibited the purified enzyme much less than it did the enzyme in crude 2227 extracts.
Low concentrations (100 to 125 pg per ml) actually increased activity slightly and inhibition was less than 15% at 300 pclg per ml. The crude enzyme was more sensitive to inhibition but less so than crude enzyme prepared from acetone powder of rat adipose tissue. These differences (between purified and crude enzyme and between species) tended to be eliminated by preincubation of the enzyme with the protamine sulfate.
The reasons for the differences are not clear but may reflect in part differences in affinity of t,he enzyme preparations for substrate i.e. in the presence of substrate the enzyme may be partially protected from interaction with protamine sulfate.
The porcine adipose tissue lipoprotein lipase after heparill-Sepharose chromatography exhibited lipolytic activity in the absence of serum. The same behavior was reported for the bovine milk lipoprotein lipase after the same purification step. In the present study, isoelectric focusing yielded an enzyme preparation which is completely dependent 011 serum activator. This increase in the magnitude of stimulation by serum after further purification suggests that traces of act,ivat,or may be tightly associated with lipoprotein lipase, co-chromatographing through the two affinity steps but dissociating in the course of isoelectric focusing.
Of the three very low tlcnsity lipoprotein apoprotcins which were studied, only apoLl)-Glu could substitute for serum as an activator, and this activation was as effective as that produced by whole serum. ApoLp-Ala, when added to a system containing serum, was inhibitory as has been previousl> reported for milk lipoprotein lipase (18). A new and unespectcd finding was the potent inhibitory effect, of apoLp-Ser in the presence of serum in the assay system. At the same levels of substrate and serum in the medium, apoLp-Ser was as potent an inhibitor as apoLp-Ala. The physiological meaning of this finding remains uncertain at this time but might be evaluated by in vitro studies of the degradation of very low density lipoproteins and chylomicrons by purified adipose tissue lipoprotein lipase.