ANALYTICALBIOCHEMISTRY206, 6-11 (1992)

An Aluminum Silicate Binding Assay for Quantitation of Degradation of Cholecystokinin Octapeptide and Other Short Peptides R o m a n M. J a n a s , 1 D a v i d L. M a r k s , a n d N i c h o l a s F. L a R u s s o Center for Basic Research in Digestive Diseases, Department of Internal Medicine, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905

Received February 11, 1992

M o s t a v a i l a b l e t e c h n i q u e s for t h e q u a n t i t a t i o n o f e n zymatic degradation of peptide hormones are time-consuming and require expensive equipment and/or novel r e a g e n t s . O u r a i m h e r e w a s to d e v e l o p a r a p i d a n d s e n sitive assay for the measurement of degradation of chol e c y s t o k i n i n o c t a p e p t i d e ( C C K - 8 ) as w e l l as o t h e r s h o r t , h y d r o p h o b i c p e p t i d e s . T h e p r o p o s e d t e c h n i q u e is b a s e d o n o u r n o v e l o b s e r v a t i o n t h a t i n t a c t C C K - 8 , b u t n o t its d e g r a d a t i o n p r o d u c t ( s ) , b i n d s to L l o y d r e a g e n t , a f o r m of aluminum silicate. When radiolabeled CCK-8 was exp o s e d to r a t l i v e r c y t o s o l c o n t a i n i n g e n d o g e n o u s CCKd e g r a d i n g a c t i v i t y , t h e r e w a s a t i m e - d e p e n d e n t dec r e a s e i n t h e b i n d i n g o f r a d i o l a b e l to a l u m i n u m s i l i c a t e [ f r o m 8 6 to 8% o v e r 6 0 m i n at 3 7 ° C ] . T h e d e c r e a s e i n binding closely paralleled the extent of CCK-8 degradation over time as assessed by high-performance liquid chromatography and immunoprecipitation with specific p o l y c l o n a l a n t i b o d i e s to C C K - 8 . W h i l e a l u m i n u m s i l i c a t e d i d n o t e f f i c i e n t l y b i n d to C - t e r m i n a l a n d N - t e r m i n a l C C K t e t r a p e p t i d e s , m a g n e s i u m s i l i c a t e b o u n d to b o t h t e t r a p e p t i d e s ( > 8 2 % ) , b u t n o t to t h e i r r a d i o l a b e l e d degradation products. Both aluminum and magnesium s i l i c a t e a l s o e x t e n s i v e l y b o u n d ( > 8 2 % ) to o t h e r p e p t i d e hormones including Met-enkephalin, somatostatin, and secretin, but did not bind their degradation products. These binding assays will be useful in studies of peptid a s e s w h i c h d e g r a d e c h o l e c y s t o k i n i n or o t h e r s m a l l , h y d r o p h o b i c p e p t i d e s . © 1992 AcademicPress, Inc.

A variety of analytical techniques has been used for the quantitation of enzymatic degradation of peptide hormones. Included among these methods are: (i) high1Present address: Department of Nuclear Medicine, Child Health Center Hospital, 04-736 Warsaw, Poland.

performance liquid chromatography (HPLC)2; (ii) immunoprecipitation with specific polyclonal or monoclonal antibodies; (iii) receptor binding assays, (iv) thin-layer chromatography, and (v) molecular sieve chromatography (1-6). While these approaches have varying degrees of resolution and sensitivity for detecting and quantitating peptide degradation, they all are time-consuming; moreover, some require expensive equipment or novel reagents. These latter characteristics may be limiting, especially during the initial phases of the purification of tissue peptidases where large numbers of samples are generated which must be screened for specific peptide-degrading activity. Several relatively simple and rapid methods have been developed which distinguish intact from degraded hormones, including trichloroacetic acid precipitation and differential chemical adsorption (7-9). However, the former approach is restricted to relatively high-molecular-weight peptides and the latter has limited sensitivity (5,10-13). Our laboratory has been interested in the hepatic processing of circulating forms of cholecystokinin and other small, hydrophobic peptides. Recently, we demonstrated efficient uptake and extensive, nonlysosomal degradation of cholecystokinin octapeptide (CCK-8) by the isolated perfused rat liver (14,15) and by isolated hepatocytes (16). In order to purify the hepatic, nonlysosomal peptidase responsible for the near-complete degradation of CCK-8 by the liver (17), we developed a rapid, simple, and sensitive method to confidently quantify CCK-8-degrading activity based on the bind-

2Abbreviations used: CCK-8, cholecystokinin octapeptide (ArgAsp-Tyr(SO2)-Gly-Trp-Met-Asp-Phe(NH2);CCK2e_29,C-terminal tetrapeptide of CCK-8; CCK3o_33,desulfated N-terminal tetrapeptide of CCK-8; HPLC, high-performance liquid chromatography; SE, standard error; RIA, radioimmunoassay. 0003-2697/92$5.00 Copyright© 1992by AcademicPress, Inc. All rights of reproductionin any formreserved.

ALUMINUM SILICATE ASSAY FOR QUANTITATION OF PEPTIDES

ing of intact but not degraded CCK-8 to Lloyd reagent, a form of aluminum silicate. MATERIALS AND METHODS

Materials CCK-8 and the C-terminal tetrapeptide of CCK-8 (CCK30_3a) were from Peninsula Laboratories (Belmont, CA). The desulfated N-terminal of CCK-8 (CCK26_29) was purchased from Cambridge Research Biochemicals (Valley Stream, NY). CCK-8, CCKa0_33 and CCK26_29 were labeled with Na~25I monoiodinated Bolton-Hunter reagent (New England Nuclear, Boston, MA), yielding probes of high specific activities (~2000 Ci/mmol) as previously reported (18). ~25I-recombinant human renin was prepared as described previously (19). High specific activity (~2000 Ci/mmol) ~25I-labeled bombesin, Metenkephalin, glucagon, somatostatin, secretin, insulin, and hydroxyphenylpropionic acid were generous gifts from the Radioimmunoassay (RIA) Core Facility (Mayo Clinic, Rochester, MN). CCK-8/gastrin C-terminal antibody (Ab 3438) and CCK-8-specific N-terminal antibody (Ab 216) as well as synthetic peptides (H. T r p A s p - P h e . NH2; H . T r p - N l e - A s p . NH2; H . Gly-GlyP h e - G l y . NH2) were the generous gifts of Dr. S. Powers, Mayo Clinic. Polyphenylalanine (Mr 20005000) was from ICN Biomedicals (Costa Mesa, CA). For binding assays, Lloyd reagent (Pfaltz & Bauer, Inc., Waterbury, CT; hereafter referred to as aluminum silicate) was routinely used. Kaolin (a different form of aluminum silicate; Sigma, St. Louis, MO), hydrous magnesium silicate (talc; Fisher Scientific, Fair Lawn, NJ), and aluminum magnesium silicate (fuller's earth; EM Science, Cherry Hill, NJ) were also tested for peptide binding activity. Bio-Gel P2 was from Bio-Rad (Richmond, CA). Other chemicals used were of reagent grade. Hepatic SubceUular Fractions Adult Sprague-Dawley rats (200-300 g) were killed by decapitation and livers were then perfused in situ with ice-cold saline. Ten grams of liver was minced and homogenized two times in a final volume of 100 ml of 25 mM Tris-HC1, 0.9% NaC1, pH 7.6. Nuclei, unbroken cells, and tissue debris (pellet) were separated from other organelles and cell sap (supernatant or E-fraction) by centrifugation at 1000g for 15 min at 0°C. The E-fraction was then centrifuged at 100,000g for 90 min at 0°C to separate membranes (pellet) from cytosol (supernatant). All fractions were used directly or after freezing at -80°C. Enzymatic Hydrolysis of Radiolabeled Cholecystokinins Radiolabeled cholecystokinins or other peptides (2-7 fmol) were incubated in polypropylene tubes for different periods of time at 37°C in 100 #1 of incubation buffer (25 mM Tris-maleate, 1 mM Ca +2, pH 6.0) with 10-20 #g protein of liver fractions. Controls included radiola-

7

beled peptides with or without heat-inactivated (2-3 min at 100°C) liver fractions or those incubated for 0 min. Enzymatic hydrolysis in the incubation tubes was terminated by 10-fold dilution with aluminum silicate suspension (see below) or by acidification with 10 #1 of 1 M HC1.

Silicate Binding Assay One milliliter of homogenous silicate suspension (100 mg silicate in 1 ml of binding buffer [25 mM Tris-HC1, pH 7.6 buffer]) was added to each tube containing incubate. After vortexing (2-3 min), supernatants and pellets were separated by centrifugation (2000g, 5 min, 4°C). The pellets were resuspended in 2 ml of binding buffer followed by vortexing and recentrifuging as described above. Radioactivity bound (pellet) and unbound (supernatant) to silicate was measured in a Beckman Gamma 5500 gamma counter for 1 min. Results were expressed as (B/T) × 100%, where B is radioactivity in the pellet or supernatant, and T is the total radioactivity used per assay. Other Analytical Methods For analysis by HPLC, samples of intact 125I-CCK-8 and 125I-CCK-8 which had been degraded by liver cytosol were equilibrated with 400 #1 of H P L C buffer (0.1 M acetate, pH 4) and run on a reverse-phase C-18 column (Vydac, The Nest Group, Inc., Southboro, MA) at 0.6 ml/min using H P L C buffer with a 20-50% acetonitrile gradient for 60 min. Similarly, radiolabeled degradation products t h a t did not bind to aluminum silicate were also analyzed by HPLC. After H P L C , radioactivity, CCK-8 immunoreactivity, and aluminum silicate binding of eluates were determined. For immunoprecipitation, samples were equilibrated with RIA buffer and measurements of CCK-8 N-terminal and C-terminal immunoreactivity were performed as described (20,21). The binding of unlabeled proteins, peptides, and amino acids to silicates was measured using the fluorescamine assay as previously reported (22,23). RESULTS A N D DISCUSSION

Aluminum Silicate Binding Assay Aluminum silicate bound intact ~25I-CCK-8 maximally [86(_+3)%, mean _+ SE] at a concentration of 50100 mg m1-1 of silicate (Fig. 1). After incubation (20 min at 37°C) with rat liver cytosol, binding of the label to aluminum silicate decreased by ~80%, suggesting t h a t proteolytic activity generated fragments of CCK-8 which did not bind to this adsorbent. When heat-inactivated cytosol was used or probes were incubated for 0 min, the binding of labeled peptide was not decreased (data not shown). Binding of intact radiolabeled cholecystokinin tetrapeptides (i.e., CCK26_29and CCK30_33) to aluminum silicate was minimal (~90 >90

>90 >90 >90

Proteins and peptides Bovine serum albumin" 125I-reninb Poly-L-PHE° t25I-CCK-8b 12~I-CCK26_2~b 126I-CCK~0_s3b H. GLY-GLY-PHE-GLY • NH2" H* NLE-ASP-PHE • NH2" H. TRP-NLE-ASP-NH2" GLY-GLY"

>90 0 0 0 0 0 0

>90 >90 >90 0 >90 0 0

Other Material Dextran blue c Evan's bluC Coomassie bluC Amino acids"d Dinitrophenol ~ Cr206~ CuS04 ~ Free 125Ib

0 >90 0 0 0 0 0 0

0 >90 0 0 0 0 0 0

Note. Substances were incubated with silicates and centrifuged as described under Materials and Methods. Binding to silicate pellets was assessed by "Fluorescamine, b radioactivity, or c colorimetry. 0, undetectable level of binding. d Amino acids tested were Phe, Met, Asp, Tyr, I-Tyr, His, Cys, Ala, Trp, Gly.

ALUMINUM SILICATE ASSAY FOR QUANTITATION OF PEPTIDES TABLE 3

Binding of the I n t a c t a n d Degraded Labeled Peptides to A l u m i n u m or M a g n e s i u m Silicatea

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peptidases when many samples must be tested for enzym a t i c a c t i v i t y (17). ACKNOWLEDGMENTS

125I-labeled peptide hormone Met-Enkephalin Bombesin Secretin Somatostatin Insulin Glucagon

Aluminum silicate Intact 84 90 92 87 91 93

(---3) (-+2) (-+3) (-+3) (-+2) (-+2)

Degraded

Magnesium silicate Intact

(% Label bound) 14 (--_2) 89 (-+3) 14 (-+5) 90 (-+3) 13 (-+4) 89 (-+6) 18 (-+5) 88 (-+4) 15 (-+4) 89 (-+5) 9 (-+2) 92 (-+3)

Degraded 31 24 28 27 24 20

(-+6) (-+2) (-+7) (-+3) (-+7) (-+3)

a Data are means _+ SE for/>3 experiments.

P o t e n t i a l A p p l i c a t i o n of A l u m i n u m a n d M a g n e s i u m Silicates for S t u d y o f D e g r a d a t i o n o f O t h e r Radiolabeled P e p t i d e s

The potential usefulness of aluminum and magnesium silicates for measuring the degradation of several o t h e r r a d i o l a b e l e d p e p t i d e h o r m o n e s is s h o w n in T a b l e 3. I n t a c t r a d i o l a b e l e d M e t - e n k e p h a l i n , b o m b e s i n , s e c r e tin, somatostatin, insulin, and glucagon were extensively bound by both aluminum and magnesium silicate (84-93%). After incubation of each hormone with rat l i v e r c y t o s o l (30 m i n a t 37°C), a d e c r e a s e d a b s o r p t i o n o f r a d i o a c t i v i t y b y b o t h s i l i c a t e s w a s o b s e r v e d ( T a b l e 3), suggesting that the silicates did not bind to at least some of the radiolabeled degradation products of each horm o n e ; h o w e v e r , a d d i t i o n a l s t u d i e s will b e r e q u i r e d t o f u l l y c h a r a c t e r i z e b o t h b i n d i n g a s s a y s for s t u d i e s u s i n g t h e s e p e p t i d e s . T h e p e p t i d e s l i s t e d in T a b l e 3 w e r e a l l d i r e c t l y o x i d a t i v e l y l a b e l e d w i t h 12~I, in c o n t r a s t t o t h e CCK peptides which were nonoxidatively labeled with 125I-Bolton-Hunter reagent. Thus, the silicate binding assay appears useful for detecting degradation of both oxidatively and Bolton-Hunter-labeled peptides. In summary, a simple, fast, inexpensive, and sensitive technique for the characterization of the enzymatic degradation of low-molecular-weight forms of cholecystokinin and other small peptide hormones has been develo p e d . A l u m i n u m s i l i c a t e (i.e., L l o y d r e a g e n t ) a d s o r b s i n t a c t 125I-CCK-8 b u t n o t i t s r a d i o l a b e l e d d e g r a d a t i o n products. Results with this assay are comparable to those obtained with reverse-phase HPLC and immunoprecipitation using specific polyclonal antibodies. While aluminum silicate does not adsorb intact labeled cholec y s t o k i n i n t e t r a p e p t i d e s (i.e., CCK26_29, CCK30 33), m a g n e s i u m s i l i c a t e (i.e., t a l c ) s u s p e n s i o n a d s o r b s b o t h t e t r a peptides but not their radiolabeled degradation p r o d u c t s . T h e s e s i m p l e b i n d i n g a s s a y s will b e u s e f u l during the initial phases of purification of hormone

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