Recovery and activation of hydroxymethylglutaryl coenzyme A reductase from rat small intestine

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1 Reovery and ativation of hydroxymethylglutaryl oenzyme A redutase from rat small intestine Nany L. Young,' Christopher D. Saudek, Sarah A. Crawford, and Stewart L. Zukerbrod Department of Mediine, Cornell University Medial College, 13 York Avenue, New York, NY 121 Abstrat We desribe a method for estimating the total ativity of 3hydroxy3methylglutarylCoA (HMGCoA) redutase (EC ) in the small intestine of rats. An homogenate of the whole small intestine is prepared rapidly and assayed diretly to maximize the yield of enzyme and to minimize opportunity for unontrolled hange in ativity. Fresh homogenate inhibits the expression of redutase in hepati mirosomes, has high HMGCoA leavage ativity, and forms NADPHindependent metabolites whih ontaminate mevalonolatone isolated by thinlayer hromatography. When homogenate is preinubated, these interfering fators are dereased and redutase ativity is inreased. Part of this inrease an be inhibited by F. After freezing and preinubation, total redutase ativity reovered from homogenates of small intestine from 3g male rats at the middle of their dark period is 4 nmol mevalonate per min ompared to 7 from hepati mirosomes. If F is added at the time of homogenization, an ativity of 11 nmol per min is reovered from eah organ. Redutase in intestinal homogenate has an apparent K, for SHMGCoA of 4 pm.young, N. L., C. D. Saudek, S. A. Crawford, and S. L. Zukerbrod. Reovery and ativation of hydroxymethylglutaryl oenzyme A redutase from rat small intestine. J. Lipid Res Supplementary key words holesterol synthesis phosphatase mevalonate hydroxymethylglutarylcoa lyase hydroxymethylglutaryl CoA hydrolase hydroxymethylglutary ICoA deaylase hydroxymethylglutarylcoa leavage enzymes. To assess possible physiologial onsequenes of hange in both HMGCoA redutase speifi ativity and organ weight, a measure of total redutase ativity in the whole organ would be useful. For example, both intestinal weight and the speifi ativity of redutase in muosal mirosomes are inreased in rats with long term insulin defiieny, and these inreases may ontribute to their hyperholesterolemia (1). However, a measure of total redutase ativity is not urrently available, and thus a quantitative evaluation of the net effet of these hanges has not been attempted. To estimate total ativity, maximal yield of ativity is required. Greater yields are obtained from homogenates of small intestine than from mirosomes (2, 3). However, analyses have been restrited to muosal ells isolated only from portions of ileum or jejunum (2, 3). Isolating muosal ells has two inherent disadvantages: redutase ativity hanges during the proedure for isolating ells (4), and the yield of ativity is redued. Finally, previous reports on redutase in muosal ell homogenates (2, 3) show kinetis with time and protein in the assay but do not demonstrate purity of the assay produt, requirement for NADPH, substrate dose response, or absene of interfering fators. The present report desribes a method for the rapid preparation of an homogenate of whole small intestine, allowing total enzyme ativity to be measured. We doument the validity of the assay, and desribe more fully the effets of F inhibitable ativation and of HMGCoA leavage enzymes on the expression of redutase in vitro noted previously in an abstrat (5). Animals MATERIALS AND METHODS Wistar rats, 225 g (Charles River Laboratories, Wilmington, MA) were housed in a room illuminated from 6 PM to 6 AM, and fed Purina Formulab how #58 ad libitum. They were killed after 2 weeks at noon when males weighed a. 3 g and females weighed a. 25 g. Tissue homogenates All solutions were used at 5OC. Rats were anesthetized with ether. The small intestine from the pylorus to the ileoeal juntion and the liver were exised, rinsed with saline, and weighed. Homogenates, 2% (w/v), were prepared in homogenizing medium (HM) with a Tissumizerm (motor SDT, shaft 18K; Tekmar Co., Cin Abbreviations: HMGCoA, 3hydroxy3methylglutaryl oenzyme A; HMG, 3hydroxy3methylglutari aid; MVA, mevaloni aid; MVL, mevalonolatone; TLC, thinlayer hromatography; R,, ratio with the front; R,, ratio with the peak; SEM, standard error of the mean; HM, homogenizing medium (omposition given in text). ' To whom reprint requests should be addressed. Journal of Lipid Researh Volume 23,

2 innati, OH) at speed 25 for 15 se. H M ontained 1 mm KZHPO4, 3 mm Na2EDTA, NaOH to give ph 7.5, NaCl to give 22 mm Na', 1 mm dithiothreitol, and.2% (w/v) soybean trypsin inhibitor (Type 1S, Sigma Chemial Co.). The onentrated homogenate was immediately diluted to 5% (w/v) with HM. To inhibit ativation of HMGCoA redutase in vitro, onentrated homogenate was diluted also into HM ontaining 66.7 mm NaF in plae of an equal onentration of NaCl, to give a final NaF onentration of 5 mm. Total time from start of anesthesia to dilution of homogenate was less than 1 min. Portions of homogenate were frationated (see below) and others were stored in liquid Nz. Subellular frationation All steps were performed at 5 C. Twenty ml of 5% homogenate was entrifuged 2 min at 9, g. The supernatant liquid was deanted and 1 ml was entrifuged 1 hr at 16, g. The Tissumizers (shaft 1N) at speed 25 for 15 se was used to resuspend the pellets. The low speed pellets were resuspended in HM to give a total volume of 2 ml; the high speed pellets of intestine and liver were resuspended in 1 ml and 2.5 ml HM, respetively. Frations were assayed immediately or stored in liquid NZ. Assay of HMGCoA redutase and HMGCoA leavage ativities The method of Shapiro et al. (6) was modified as follows. Substrateofator solution for one assay ontained the following: a) 2 nmol of (3R,S)[3I4C]HMG GOA prepared as desribed by Young and Berger (7) and 23 nmol of HMGCoA (PL Biohemials, In., Milwaukee, WI) to give 5 Ci/mol; 6) an NADPHgenerating system as in (7) exept that all quantities were halved; ) an internal reovery standard of 1 pmol (lo4 dpm) (3R,S)[ 53H]MVA (New England Nulear, Boston, MA); d) NaF to give total amount after adding enzyme sample of 3.75 pmol; e) 5 pl HM at 5 times usual onentration;f) NaOH to give ph 7.5; all in a total volume of 25 pl. Fifty pl of enzyme sample was preinubated at 37 C for 3 min in a.5 ml polyethylene entrifuge tube. The substrateofator solution was added with Vortex mixing, and inubation was ontinued for 15 min (liver mirosomes), 3 or 6 min (intestinal homogenate prepared without or with F, respetively). Control samples were the omplete system minus NADP+, and the omplete system minus enzyme. The reation was stopped by addition of 5 p1 of 6 N HC1. To minimize leavage of HMGCoA by the aid, tubes were kept at 2 C or on ie unless otherwise indiated. After entrifugation (1, g, 1 min, 25"C), 1 pl of the supernatant liquid was hromatographed with bu tanolaeti aidwater 7:2:3 on ellulose thinlayer sheets sored in 16 hannels (System 1, referene 7) to determine the fration of [I4C]HMGCoA that was leaved (7). The assay tube was then inubated at 37 C for 15 min to onvert MVA to MVL. I4CLabeled and 3Hlabeled MVL in 5 pl of the supernatant liquid were isolated by hromatography with aetonetoluene 1: 1 on silia gel thinlayer sheets sored in eight hannels (System 11, referene 7). Radiolabeled produts were measured by sintillation ounting (7). HMGCoA redutase ativity was alulated from the I4Clabeled produts isolated with 'Hlabeled MVL after orreting for 3Hreovery, as usual, and for NADPHindependent 14Clabeled produts. HMGCoA leavage ativity was alulated, for example in fresh muosal homogenate without F (Table 2), as follows. The fration of [I4C]HMGCoA at the end of a 3min inubation of a buffer ontrol,.914, less the fration in homogenate,.67, times the initial amount of substrate orreted for radiohemial purity, 23.8 nmol, gives the amount leaved by homogenate, 7.31 nmol. This amount divided by the weight of tissue in the assay,.25 g, and the assay time gives leavage ativity, 97 nmol/(min X g). Cleavage, as defined here, inludes all metaboli onversions of HMGCoA inluding its redution to MVA (if NADPH was present). However, in intestinal homogenates, redutase ativity was always less than 1% of leavage ativity. Sine eah 5p1 sample was derived from a fixed amount of tissue (homogenate and low speed pellet from 2.5 mg of tissue, high speed pellet of small intestine and of liver from 25 and 1 mg of tissue, respetively), speifi ativity was expressed per g tissue. For the present appliation, where reovery of enzyme ativity is stressed, expressing ativity per g tissue is more useful than per mg protein. However, the latter an be alulated from our data for the former by dividing by protein reovery whih was 143 f 12 mg protein/g tissue in small intestine homogenate, and 23 k 1 mg/g in liver mirosomes. Total ativity per organ was alulated as ativity per g tissue times organ weight. Assay of HMGCoA redutase inhibition Intestinal homogenate, 4 pl, and 1 p1 of liver mirosomes prepared without F, eah from 2 mg of tissue, were assayed separately and together by inubation with substrateofator solution in a total volume of 75 pl at 37 C for 3 min. Inhibition was alulated as the differene between the moles of MVA formed by the mixture and by intestine alone, divided by the moles of MVA formed by liver alone and then subtrated from 1. It was assumed that liver mirosomes did not inhibit redutase. 258 Journal of Lipid Researh Volume 23, 1982

3 RESULTS Chromatography of ["CIHMGCoA metabolites TLC System I separated ['4C]HMGCoA from all I4C1abeled metabolites and therefore was suitable for assay of leavage ativity (Fig. 1). In this ase, both isomers of (R,S)HMGCoA were quantitatively metabolized by intestinal homogenate prepared in Tris buffer to less polar produts. However, in all subsequent experiments, where homogenate was prepared in phosphate buffer, and less tissue was inubated for a shorter time, less than 4% of [14C]HMGCoA was metabolized. In TLC System 11, most leavage produts migrated with Rf less than.5 (not shown). Nevertheless, some leavage produts formed by fresh intestinal homogenate migrated with Rf greater than.5 and were poorly resolved from MVL (Rf.7, Fig. 2). 14CLabeled produts formed in the absene of added NADP+ overlapped but did not omigrate with [3H]MVL(Fig. 2B). This showed that little or no [14C]MVA had been formed, and therefore that endogenous NADP+ did not support [I4C]MVA formation. More ontaminants were formed in the absene than in the presene of NADP+. When leavage ativity was dereased and redutase ativity inreased, as desribed below, the purity of [14C]MVL isolated by TLC was muh improved. The profile of the 14Clabeled produts on TLC orresponded losely with that of the 3Hlabeled standard as the latone, the aid, and the amide (Fig. 3). The ratio of 14C to 3H in the peak did not derease in onseutive TLC separations. NADPHindependent metabolites were present in small amounts (4%) and still hromatographed as in Fig. 2B. 1 I +NADPH A NADPHo r:, :? p I :b p Q U v m w k J 1 Fig. 2. Cohromatography of [I4C]MVL with other metaboiites of [I4C]HMGCoA. Intestinal homogenate (F) was assayed as desribed in Materials and Methods exept that freezing and preinubation were omitted. Radioativity in 1m setions of TLC System I1 is shown. Effet of pretreatment of intestinal homogenate on metabolism of ["CIHMGCoA HMGCoA leavage ativity was highest in fresh homogenate, delined slightly with storage on ie, and delined substantially after freezethawing and inubation at 37 C (Table 1). In ontrast, redutase ativity inreased slightly with storage of homogenate on ie, but dereased over 9% with freezethawing. This loss was more than reovered with subsequent preinubation yielding a 4fold inrease from ativity in fresh homogenate. The fration of I4Clabeled produts isolated with MVL that were NADPHindependent varied from 93% after freezethawing to 11% after preinubation at 37 C. Intestinal homogenate that was not preinubated in 1 MEVALONOLACTONE I MEVALONATE IMEVALONATE a I W z j z Fig. 1. Isolation of [14C]HMGCoA from its metabolites by TLC. ["CIHMGCoA (33 um) was inubated for 1 hr at 37'C with an homogenate of 5 mg of small intestine in 5 mm Tris buffer (ph 7.5), 3 mm mannitol, in a total volume of 11 pl. After adding 5 p1 of 6 N HCI and entrifuging, the supernatant liquid was hromatographed in System I. '%(dpm) in.5m setions of the TLC sheet was divided by "C(dpm) at the peak to give R, for metabolites formed by homogenate () and for stok [I4C]HMGCoA(). Rr Fig. 3. Chromatography of putative [I4C]MVL. Intestinal homogenate (F) was assayed as desribed in Materials and Methods. Panel A shows radioativity in.5m setions of TLC System 11. Material in the region R, was eluted with aetone and methanol, divided in half, dried with a stream of NZ, and inubated at 37OC for 15 min with either 1 pl of.1 N NaOH to form MVA (B) or 1 pl of onentrated ammonia to form MVA and mevalonamide (C). B and C show radioativity in 1m setions of TLC System IV (ellulose sheet; butanolammoniawater 2:l:l in a sandwih hamber). The ratio of IC to 'H was.366 in MVL,.374 in MVA in B, and.379 in C, and.391 in mevalonamide. Young et al. Assay of HMGCoA redutase in rat small intestine 259

4 TABLE 1. Effet of pretreatment of intestinal homogenate on its metabolism of ['4C]HMGCoA HMG HMG CoA ["C]"MVL" CoA Cleavage Redutase Pretreatment Ativity +NAPD+ NADP+ Ativity nmol min X g dpm nmol min X g 2 Min on ie Min on ie Freezethawed Freezethawed C Absent A 1% (w/v) homogenate and a 15min assay were used. Data for ["C]"MVL" are for produts reovered at R,.61 to.84 of TLC System I1 after orreting for ['HIMVL reovery. hibited the expression of redutase in hepati mirosomes by 89% (Table 2). Inhibition delined to about 2% with a 3min preinubation at 37 C. Assoiated with dereased inhibition was a derease in HMGCoA leavage ativity. The inrease in redutase ativity with preinubation was partially bloked by F. F had little or no effet on inhibitory ativity, leavage ativity, or their derease. The data in Tables 1 and 2 thus demonstrate that the inrease in redutase ativity with preinubation was due to the ombined effets of reovery from freezing, ativation of redutase by a Finhibitable proess, and derease in redutase inhibition ativity. The optimal time for preinubation of freezethawed intestinal homogenate was 3 to 6 min, when redutase ativity was maximal and leavage ativity minimal. TABLE 2. Effet of preinubation of small intestine homogenate with and without F on HMGCoA redutase, redutase inhibition, and HMGCoA leavage ativities HMGCoA HMGCoA HMGCoA Redutase Redutase Cleavage Preinubation Speifi Inhibition Speifi Conditions Ativity Ativity Ativity nmol % nmol mtn X g min X g min F.4 f ~ f 41 +F.1 f.2 85 f k 28 3 min F 3.42 k.4 19 iz 6 3 f 8 +F.72 f.9 17 f 7 26 f 4 Data are means f SEM for small intestines from five male rats. Homogenates were freezethawed before preinubation at 37'C. Inhibition ativity was alulated as desribed in Materials and Methods, assuming that liver mirosomes did not inhibit redutase. Alternatively, if inhibition is alulated as moles MVA formed by the mixture of liver mirosomes and small intestine homogenate divided by the sum of moles MVA formed by liver and intestine separately and then subtrated from one, inhibition is 81 and 85% without preinubation, and 17 and 13% after preinubation. Frationation into muosa and musle The possibilities that fators in intestinal homogenate whih interfered in the redutase assay orginated in musle or resulted from freezethawing were tested by assaying HMGCoA leavage and redutase ativities in muosa and musle separated by sraping and pretreated in various ways (Table 3). After freezing and preinubation without F, redutase speifi ativity in muosal homogenate, 5.2 nmol/ (min X g), was higher than in homogenate of intat small intestine, 3.7 nmol/(min X g) (Table 3). On the other hand, total ativity was higher in the latter (36 nmol/ min) than in the former (25 nmol/min). Thus, although inlusion of musle inreased total leavage ativity from 98 to 26 nmol/min, it did not derease total redutase ativity. Freezethawing before preinubation also inreased leavage ativity and did not derease redutase ativity. On the ontrary, freezethawing appeared to inrease Finhibitable ativation of redutase in homogenate of the intat small intestine from 2. to 4.6 fold. The ativation of redutase in muosa after homogenization (1.3) was low ompared to that in homogenate of intat small intestine (4.6). Sine redutase speifi ativity in muosal homogenate prepared with F was high before preinubation, 4.1 nmol/(min X g), it appears that ativation had ourred prior to homogenization, probably during sraping. In all ases, after freezing and preinubation, less than 1% of I4Clabeled produts isolated with MVL were NADPHindependent. Variation in HMGCoA redutase ativity along the length of the small intestine In agreement with previous findings for holesterol synthesis (8) and for redutase ativity in mirosomes (9), redutase ativity in homogenates showed a U shaped variation along the length of the small intestine (Fig. 4). Ativity was highest in the short setion of the proximal jejunum between the pylorus and the entrane of the bile dut. Subellular frationation Most measurable sedimentable redutase ativity from small intestine was reovered in the low speed pellet (Fig. 5) in agreement with previous reports (2, 3, 9), in ontrast to that from liver whih was reovered in the high speed pellet. The yield of redutase from small intestine dereased with frationation, while that from liver inreased. Thus, the yield of ativity from small intestine was maximal in homogenate, while the yield from liver was maximal in mirosomes. It is evident that the expression of redutase in liver homogenate, low speed supernatant, and possibly low speed pellet was inhibited. Re 26 Journal of Lipid Researh Volume 23, 1982

5 I E % $ a P L 71 m e, 2 5 e, e 4 B e m 3 x n 2 Y 13 E z * bz E ** w W e, m 3 3 C w ug a3uul 2 q r: & &Ins Y sz C + mr. zz ( E? 22 e 3 m o m u l m m 3 =: " o w Bx u * m L E E g v I E P N u7 2: 9 E z m b z ;z u mi z h ; : b 5 22 PmnNr. ag N & e ) $ 4 ;" BILE g 2 m & f 71 g z :g DUCT =I 22 i m m 7 a K Y ; x gs &+? sa FRACTION OF INTESTINAL LENGTH l w b b 5 az Fig. 4. Distribution of HMGCoA redutase ativity along the length % L b 6 3 testines was divided into IOm setions (, A), and a third into 12 g.2 " E was setioned just before the entrane of the bile dut and at 3 m W C I m o m setions (m) In the seond experiment, eah of five small intestines m o P $2 O m 2 5 from the ileoeal juntion to give three piees (mean k SEM given by the bars) Eah setion was homogenized (F) and assayed as desribed in Materials and Methods $ $ z o o m k, of the small intestine In the first experiment, eah of two small in $5 g 5 H a m :z z sz 2 H 7 m o 9 n $? dutase ativity in all preparations, exept intestinal low L h x + 5 t. E 2 speed pellet, inreased with freezing and preinubation, " E E *r. QI gu O O Bo" along with a derease in leavage ativity. After freezing 3s n N* 22 and preinubation, the fration of NADPHindependent " 5 3 u P4F7 ul 8 C Eo; sp, m u *= z g q g ;: 2 2 H q "" a+?? 2 N 3: v fz h L. ummm C:mh s;j E k L w m z o om+ = a E I $ = IL" d X Y g m C > u M t.r.m 't; 3 r. m w Gxm 2 b m m o s a x 2 z pn 2s $ m s AS$ 3% LIVER SMALL INTESTINE u E E 22 2 % A =2 4 g. %mr a" " E Zbt; w > ; g g 2: 3 3 m s 9 EzE o U T U T U T UT U T U T h, EE a5 >k e: L 4 $ z o w E 4 d g 5 N u 3gZ a $ Z CTL k X Ova 4' a i q L 5 W W ' ; $ p N I m w m 8p 2 CENTRIFUGATION None First Seond None First Seond b m e m N b b $ s Z Ob Fig. 5. Subellular frationation of liver and small intestine Homogp e) enates (fine stipling), low speed pellet (oarse stipling), low speed sue$s pernatant (lear, enter), high speed pellet (rosshathing), and high = N E speed supernatant (lear, right) were prepared without F and assayed m C g.2 g %5 as desribed in Materials and Methods before (bars labeled U for y z g s <zz untreated) and after (bars labeled T for treated) freezing and prein E222 2: ubating for 15 min at 37OC Young et al. Assay of HMGCoA redutase in rat small intestine 261

6 produts isolated with MVL was 26% for the low speed pellet of small intestine, and less than 1% for all other preparations. 2, I I Kinetis MVA prodution by 2.5 mg of homogenized intestine was linear with assay time up to 6 min (Fig. 6A). MVA produed in 6 min was approximately linear with amount of tissue in the assay up to 2.5 mg (Fig. 6B). When HMGCoA onentration was varied under speial assay onditions whih provided adequate [I4C]MVL reovery while avoiding exessive substrate leavage, MVA prodution showed typial saturation kinetis with a K,,, of 4.1 &.2 pm (3S)HMGGOA (Fig. 7). When the measured deline in substrate onentration during the assay (less than 1% (R,S)HMG CoA) was taken into aount as suggested by Lee and Wilson (lo), the revised estimate, 3.9 k.2 pm, was not signifiantly different. Variability The oeffiient of variation between tripliate assays of redutase ativity in intestinal homogenate in the experiment shown in Fig. 7 averaged 2.8% and did not exeed 6.6%. The oeffiient of variation among 63 rats was 25% for intestinal homogenate ompared with 61% for liver mirosomes. DISCUSSION Preparing homogenates of whole small intestine for assay of HMGCoA redutase has several advantages. Most importantly, the yield of enzyme ativity is maximized thereby permitting estimate of total ativity in the organ. We reover a total ativity from the small intestine of male rats weighing a. 3 g of a. 4 nmol/min I5 2.5 mg 3k 3 6 ASSAY TIME (min) INTESTINE WEIGHT (mg) Fig. 6. Variation of MVA prodution with time and amount of tissue. Intestinal homogenate (F) was assayed for HMGCoA redutase ativity as desribed in Materials and Methods, exept that assay time was varied in A, and onentration of homogenate in the preinubation and assay was varied in B. Fig. 7. Substrate dose response of MVA prodution. Intestinal homogenate (F) was assayed as desribed in Materials and Methods, exept that homogenate was diluted to 1% with HM (F) after preinubation. ['4C]HMGCoA onentration was from.4 to 15 pm and speifi ativity was 122 to 24 dpm/pmol, and all other omponents inluding total assay volume were doubled. Data are means of tripliate assays. Linear regression of data plotted aording to EadieHofstee in the bottom panel gave an estimate for K, of 4.1 f.2 PM (S)HMG CoA and V,n.,of 1.92?.9 nmol MVA/(min X g tissue). (Table 4). Inlusion of musle does not derease the yield (Table 3). In fat, the yield in muosa sraped from musle is about onethird less than in whole small intestine. The yield ontinues to derease with further frationation (Fig. 5 and referenes 2, 3). Panini et al. (3) measured a total ativity of 15 nmol/min in homogenate of epithelial ells from the distal 5 m of ileum. They observed suessive losses of 74% and 56% with isolation of rypt ells and of mirosomes, respetively, giving a final ativity in rypt ell mirosomes of only 1.7 nmol/ min, whih is 5% of the ativity we reover from whole small intestine homogenate. On the other hand, whole small intestine homogenate would be unsuitable where high redutase speifi ativity is required. The speifi ativity we see in homogenate, 28 k 1 pmol/(min X mg protein), is muh lower than that in mirosomes from ileal rypt ells, 45 pmol/ (min X mg) (3). Inidentally, we would predit the highest speifi ativity in mirosomes from the most proximal jejunum (Fig. 4). Ativity in homogenate is, however, 262 Journal of Lipid Researh Volume 23, 1982

7 TABLE 4. Comparison of rates of holesterol synthesis predited from total HMGCoA redutase ativity with those of literature values from inorporation of 'H from tritiated water Liver Small Intestine Total HMGGOA Redutase Ativity +F F +F F Males nmol MVA/min 11 f 1 7 f 5 11 f 1 39 f f 5 g n = 63 mg holesterol/hr unorreted" orretedb Females nmol MVA/min 25 f f f 3 26 f f 5 g n=8 mg holesterol/hr unorreted" orretedb Cholesterol synthesis in vivo' Females mg holesterol/hr f 5 g n=4.26 a Calulated from nmol MVA/min by assuming 6 nmol MVA/nmol holesterol, 6 min/hr, mg holesterol/mmol, and lo6 ng/mg. Correted for 35% reovery of endoplasmi retiulum from liver and 2% inhibition of redutase ativity in small intestine. ' Measured in SpragueDawley rats at 1 hr after injetion of tritiated water by Turley et al. (23). well within the range for reliable miroassay, without heroi inreases in the speifi ativity of [I4C]HMG CoA. At least 1 14Cdpm are reovered in [I4C]MVL, and there is less variation than in liver mirosomes. Another advantage of our method is that, by avoiding the timeonsuming step of isolating muosal ells, it redues the opportunity for unontrolled hange from the in vivo level of enzyme ativity, suh as the ativation that ours during ell isolation (Table 3 and referene 4). This ativation an be prevented with F and reversed with biarbonate in the medium (4), but sine inativation is not ontrolled, the original state of ativation may not be preserved. In ontrast, at the time of homogenization, inativation an be bloked with EDTA and ativation an be bloked with F (11). In our method, intestine is homogenized in a medium with EDTA, then immediately diluted into media with and without F. It is highly unlikely that ativation ould our within less than a minute on ie from the start of homogenization to the dilution into F. Thus, both ativated and nonativated redutase ativity an be assayed in the same small intestine and experimental variation is redued. Use of intestinal homogenate, rather than mirosomes, does require speial are, however. Homogenate ontains HMGCoA leavage enzymes and inhibitors of redutase ativity with potential for interfering in the redutase assay in a number of ways. HMGCoA an be exessively depleted (Fig. l), produts an be formed that ontaminate isolated MVL (Fig. 2), and redutase an be inhibited over 8% (Table 2). However, these interfering fators are redued to tolerable levels by homogenizing in phosphate buffer, preinubating, and limiting the amount of tissue and the time of assay. It is evident that another potential problem with homogenate, the further metabolism of MVA, does not our under our assay onditions. We inlude [3H]MVA in the substrateofator solution, so we an monitor and orret for loss of MVA during and after the inubation. In the nonpolar region of the TLC (Figs. 2 and 3), [3H]MVL is the only 3Hlabeled material present. Thus the NADPHindependent I4Clabeled produts in this region are not metabolites of MVA, and do not inlude A23CH3MVL, the dehydration produt formed. in strong aid and migrating in front of MVL (12). It is unlikely that phosphorylation of MVA, the first step in its onversion to holesterol, ould our during the inubation. This reation requires ATP whih was not added, and Mg2+ whih was helated with EDTA. Finally, the reovery of [3H]MVL after inubation with homogenate is not lower than after inubation with buffer ontrols. Under the onditions we employ, the purity of isolated [14C]MVL is greater than 9% (Fig. 3), redutase inhibition is less than 2% (Table 2), [14C]MVA prodution is linear with time and weight of tissue (Fig. 6), and the K,,, for (3S)HMGCoA is 4 pm (Fig. 7). This estimate is slightly lower than for redutase in intestinal Young et al. Assay of HMGCoA redutase in rat small intestine 263

8 mirosomes, 7 pm (9) and 21 pm (13). Low K,,, is a favorable indiation that substrate leavage is not interfering in the assay (7, 14, 15) and that fators in the homogenate do not affet substrate binding any more than those in mirosomes. In this and the following report (1 6), we used ontrols without NADP+ to orret for the I4Clabeled ontaminants isolated with MVL. It is lear from the TLC data (Fig. 2) that [14C]MVA is not formed in suh ontrols, in spite of the possible presene of NADP. However, sine more ontaminants are formed in the absene of NADP+ than in its presene, this orretion underestimates redutase ativity. In properly treated homogenates, these ontaminants aount for less than 1% of the 14Clabeled produts isolated, so orretion is unneessary. Intestinal homogenates may be stored in liquid N2 before assay of redutase ativity. Freezethawing drastially lowers redutase ativity, but the effet is transient (Table 1). With preinubation, the yield of redutase is atually improved by prior freezing as a result of inreased Finhibitable ativation of redutase (Table 3). Cleavage ativity is moderately inreased by freezing, possibly due to broken mitohondria, but this inrease does not impair the reovery of redutase (Table 3). Although preinubation of intestinal preparations to inrease redutase ativity is ommon pratie (14, 9, 13) and HMGCoA lyase is known to be present (17), the roles of HMGCoA leavage enzymes (5) and of F inhibitable ativation (4, 5) in the inrease were noted only reently. The inrease results from the ombined effets of a 4fold ativation and about a 4fold derease in redutase inhibition (Table 2). Inhibition is not due to phosphorylation of either redutase or MVA by kinases sine these proesses require ATP whih is not added and Mg2+ whih is helated with EDTA. The inhibition might be due to leavage enzymes sine leavage ativity delines along with redutase inhibition during preinubation. However, in this ase, exessive substrate depletion by leavage enzymes annot aount for the inhibition. Even when inhibition is 8%, the onentration of (3QHMGCoA is greater than 3 pm, whih with a K,,, of 4 pm, should provide a rate of MVA formation more than 88% of maximum. On the other hand, the produts of leavage enzymes in small intestine homogenate inlude HMG and free CoA, whih are known to inhibit redutase (1 8, 19). Therefore, leavage enzymes might be inhibiting redutase, not by depleting substrate in this ase, but by forming inhibitory produts. Monitoring onversion of HMGCoA to MVA as suggested by Langdon and Counsel1 (1 5) or to aetoaetate will seriously underestimate total substrate utilization in * Unpublished observation by Albreht Shnieder in our laboratory. the redutase assay of intestinal homogenate. We find that intestinal homogenate forms three major I4Clabeled produts in addition to aetoaetate and has maximal HMGCoA leavage ativity of 85 nmol/(min X g tissue) ompared to lyase ativity of 4 nmol/(min X g) (17). Although leavage ativity is redued to 13 nmol/(min X g) under our redutase assay onditions, it is still higher than redutase ativity of 4 nmol/(min X 9). Measuring total substrate utilization by isolating all metabolites with the simple TLC method I (5, 7) is advisable for substratedose response experiments or when intestinal homogenate is prepared in nonstandard ways. We reover a total redutase ativity in homogenate from intestine of male rats killed at middark of 4 nmol/ min ompared to 7 nmol/min in liver mirosomes (Table 4). When F is added at the time of homogenization, total ativity from eah organ is 11 nmol/min. Our redutase speifi ativity in liver mirosomes prepared without F, 25 pmol/(min X mg protein), is in line with previously reported values for Wistar rats (9) when the lower ativity in males ompared to females (9, 2) and the deline with age (9) are taken into aount. Our reovery of protein in liver mirosomes, 23.6 f.7 mg protein/g tissue, is similar to that reported by Eriksson (21) and Brown et al. (22) of 2 mg/g. However, aording to Eriksson (21), only about 35% of the endoplasmi retiulum is reovered in liver mirosomes as they are usually prepared. If orretions are made for 35% reovery of endoplasmi retiulum from liver and the 2% inhibition of redutase in intestinal homogenate, total redutase ativity before ativation is 3 1 nmol/min in liver and 14 nmol/min in small intestine, and after ativation is 2 and 49 nmol/min, respetively. Thus, by these estimates in males, the total ativity in small intestine is 25 to 45% of that in liver. In females, total redutase ativity in small intestine is 1 to 22% of that in liver. Our major goal has been to estimate total HMGCoA redutase ativity in small intestine. Our reovery of ativity is at least 1 times that of previously published methods, but we still do not know what fration of in vivo redutase ativity we measure in vitro. HMGCoA may not be as aessible to redutase in vesiles of endoplasmi retiulum formed during homogenization as it is to redutase in planar membranes in vivo. It is also unlear whether the ativated or the unativated level of redutase ativity is the more nearly physiologial. Brown et al. (22) addressed the latter question by omparing rates of holesterol synthesis measured in vivo with redutase ativity. They onluded that only the higher, ativated level of ativity ould aount for holesterol synthesis in liver. However, these authors assumed 1% reovery of endoplasmi retiulum. Turley et al. (23) 264 Journal of Lipid Researh Volume 23, 1982

9 reently estimated rates of holesterol synthesis in both liver and small intestine in vivo. Their data are shown with ours in Table 4 for the purposes of disussion, but it should be noted that rates of holesterol synthesis in their rats were probably higher than in ours beause they used younger rats from the faster growing SpragueDawley strain. Given this unertainty, we an be onfident only that at least 2% of the redutase ativity is reovered in both small intestine and liver (Table 4). Studies of redutase ativity and holesterol synthesis in identially treated rats are neessary for a definite answer to these questions.il We would like to thank Bradley Berger, Demosthenes Tambakos, and Maria Lopez for their expert tehnial assistane. This work was supported by grants RR47, HL2488, and RR5396. Manusript reeiued 1 August 1979, in reuisedfom 8 Deember 198, and in rereuisedform 17 August REFERENCES 1. Nakayama, H., and S. Nakagawa Influene of streptozotoin diabetes on intestinal 3hydroxy3methylglutaryl oenzyme A redutase ativity in the rat. Diabetes. 26: Merhant, J. L., and R. A. Heller Hydroxy3 methylglutaryl oenzyme A redutase in isolated villous and rypt ells of the rat ileum. J. Lipid Res. 18: Panini, S. R., G. Lehrer, D. H. Rogers, and H. Rudney Distribution of 3hydroxy3methylglutaryl oenzyme A redutase and alkaline phophatase ativities in isolated ileal epithelial ells of fed, fasted, holestyraminefed, and 4aminopyrazolo(3,4d)pyrimidinetreated rats. J. Lipid Res. 2: Panini, S. R., and H. Rudney Short term reversible modulation of 3hydroxy3methylglutaryl oenzyme A redutase ativity in isolated epithelial ells from rat ileum. J. Biol. Chem. 255: Young, N. L Coping with substrate leavage in the HMGCoA redutase assay. J. Lipid Res. 2: 149. Abstrat. 6. Shapiro, D. J., J. L. Nordstrom, J. J. Mitshelen, V. W. Rodwell, and R. T. Shmike Miroassay for 3hydroxy3methylglutaryl CoA redutase in rat liver and L ell fibroblasts. Biohim. Biophys. Ata. 37: Young, N. L., and B. Berger Assay of S3hydroxy 3methylglutarylCoA redutase. Methods Enzymol. C. 71: Dietshy, J. M., and M. D. Siperstein Cholesterol synthesis by the gastrointestinal trat: loalization and mehanisms of ontrol. J. Clin. Invest. 8: Shefer, S., S. Hauser, V. Lapar, and E. H. Mosbah HMGCoA redutase of intestinal muosa and liver of the rat. J. Lipid Res. 13: Lee, H. J., and I. B. Wilson Enzymati parameters: measurement of V and K,. Biohim. Biophys. Ata. 242: Nordstrom, J. L., V. W. Rodwell, and J. J. Mitshelen Interonversion of ative and inative forms of rat liver hydroxymethylglutarylcoa redutase. J. Biol. Chem Sanghvi, A., and B. Parikh Isolation and identifiation of a mevalonolatone byprodut formed during the assay for 3hydroxy3methylglutaryl oenzyme A redutase ativity. Biohim. Biophys. Ata. 444: Sugano, M., H. Okamatsu, and T. Ide Properties of 3hydroxy3methylglutaryloenzyme A redutase in villous and rypt ells of the rat small intestine. Agri. Biol. Chem. 42: Ness, G. C., and M. H. Moffler Influene of a naturally ourring ompeting enzymi ativity on studies of 3hydroxy3methylglutaryl oenzyme A redutase ativity. Biohim. Biophys. Ata. 572: Langdon, R. E., and R. E. Counsel Determination of the MihaelisMenten onstant for Phydroxymethylglutaryl oenzyme A redutase. J. Biol. Chem. 251: Young, N. L., C. D. Saudek, and S. A. Crawford Total hydroxymethylglutaryl CoA redutase ativity in the small intestine and liver of insulin defiient rats. J. Lipid Res. 23: MGarry, J. D., and D. W. Foster Ketogenesis and holesterol synthesis in normal and neoplasti tissue of the rat. J. Biol. Chem. 244: Moorjani, S., and P. J. Lupien Effet in vitro of 3hydroxy3methylglutari aid on the synthesis of mevalonate and its preursors. Arh. Int. Physiol. Biohim. 85: 11, 19. Edwards, P. A., D. Lemongello, J. Kane, I. Shehter, and A. M. Fogelman Properties of purified rat hepati 3hydroxy3methylglutaryl oenzyme A redutase and regulation of enzyme ativity. J. Biol. Chem. 255: Carlson, S. E., A. D. Mithell, M. L. Carter, and S. Goldfarb Evidene that physiologi levels of irulating estrogens and neonatal seximprinting modify postpubertal hepati mirosomal 3hydroxy3methylglutaryl oenzyme A redutase ativity. Biohim. Biophys. Ata Eriksson, L. C Preparation of liver mirosomes with high reovery of endoplasmi retiulum and a low grade ontamination. Biohim. Biophys. Ata. 58: Brown, M. S., J. L. Goldstein, and J. M. Dietshy Ative and inative forms of 3hydroxy3methylglutaryl oenzyme A redutase in the rat. Comparison with the rate of holesterol synthesis in different physiologial states. J. Biol. Chem. 254: Turley, S. D., J. M. Anderson, and J. M. Dietshy Rates of sterol synthesis and uptake in the major organs of the rat in vivo. J. Lipid Res. 22: Young et al. Assay of HMGCoA redutase in rat small intestine 265