Extraabdominal serum protein synthesis in the rhesus monkey

EXTRAABDOMINAL IN THE El TZItUGII

,M U L L 1 N S )

M,D,, JUD1TII

SERUM

PROTEIN

RHESUS

MONKEY

Stl ERMAN A,

GENERALLY it is stated that the only proteins present in plasma in amounts detectable by chemical methods which are synthesized outskle the liver are the immunoglobulins [9, 10], In the species investigated, such proteins as albumin and fibrinogen are predominantly or totally derived from the liver [8]. However, recent studies [6, 16] have demonstrated that the hepatect'omized dog incorporates significant amounts of radioactivity into serum proteins with alpha and befa electrophoretic mobility. It was considered desirable to extend these studies to primates for several reasons, "lqm low levels of specific activity of serum proteins obtained in these experiments necessitate the analysis of rather large sampies of serum. Therefore, such studies are more readily performed in larger animals. The serum proteins of primates have been more extensively studied than those of dogs. Finally, there is no method for detection of extrahepatie synthesis of minor serum protein components in man, and studies with monkeys provide the closest available analogy. The use of preparative ~K~,lamide gebelectrophoresis apparatus made it possible to analyze sufficient serum to be able to detect From the Surgery and Metatmlism Branches, National Cancer Institute, National Institutm of Health, Public Health Servi~, Department of Health, Education, and Welfare, Bethesda, Md. Submitted for publication Dec, 21, 1965.

SYNTItESIS

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minor radioactive components. At the same time it provides sufficient resolution so that these components are not obscured by radioactive immunoglobulins. This report presents evidence that a variety of serum proteins present in hepatectomized monk~,s are derived from sources other than the abdominal viscera, METHODS

AND

MATI£RIALS

The animals used in these experiments were 3- to 6-kg. Rhesus inonkeys (Macaca mulatta ) T of either sex obtained from the National Institutes of Health [ rnnate Quarantine I aclhty, Prior to use in this study they were maintained in individual cages and fed standard monkey chow with ,dail') fruit and water ad libitum. ~ e radioactive amino acids, used were 1-C j4 Laleucine (15 cro, tool labek 140 ~Ic,

*

ach ammal ~aas hghtly anesthetized with pentobarbi~ respiration, formed were one-sta~eg he)atectomy[ with portacaval shunt [ 15 ] tacaval shunt and ne with gastroentereet(~ * Obtained from New England Nuclear:C0rlx~ration, B o s t o n , Mass. 3J5

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pancreatectomy; and the latter combination of organ excisions with nephrectomy. The gastrointestinal resection extended from the esophageal hiatus to the rectum. The abdomen was closed with continuous suture, and blood sugar levels were supported posthepatectomy with a constant intravenous infusion of 10% glucose in water at the rate of from 10 to .15 nal. per kilogram per hour. All animals resumed spontaneous respiration and sorne muscular activity. Urine output was maintained in those not nephrectomized. None regained full consciousness and purposeful movement. Sham-operated animals were similarly anesthetized, then subjected to celiotomy with intestinal manipulation and otherwise treated in an identical manner to those undergoing organ removal, After the operative procedure had been completed, each animal was given the radioactive amino acid intravenously. Unless otlierwise hadicated, the experiment was performed with 100 ~,e. of C ~ leucine. At the stated times, the animals were sacrificed by exsanguination. The blood was allowed to clot mad the serum removed by decantation after the specimen had been centrifuged at 20,000 G. for 15 minutes. Serum was dialyzed for 4S to 79. hours at 4°C. against nmltiple changes of the appropriate buffer and stored frozen until analyzed. Ammollium sulfate fractionation of sen~m proteins was performed by the m e t h o d of Peterson et al. [12]. The resulting serum pr orein fractions were again~ dialyzed for 48 hours prior to further, analysis. Preparative ac~Tlamide gel-electrophoresis was performed by the method of Jovin e t al. [5] with a discontinuous tris [tris (hydroxymethyl) aminomethmm] glycine buffer System and an initial pH-of 8.9 in t h e l o w e r gel. Solid sucrose 5 ~ was dissolved in the set,am SP' and they were thel~ the spacer at 60C. Vive-milli-

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2/adioactivity was measured on a low background (2.7 cpm) N~s flow planehet counter w h i c h accommodated planchets with a diameter of 2 inches. Samples were plated at infiliite thinness. The e~ciency of detection of C TM was approximately 28%. Samples ",,,,ere counted sufficiently long to give a statistical counting error of less than ~ 6 % , Sermn from a total of 20 ~mimals submitted to various operative procedures was examined by preparative gel-electrophoresis. Ammonium sulfate supemates and precipitates from two animals, one hepatectomized and one hepatectomJzed and entereetomized, were similaHy analyzed. Serum was examined from animals hepatectomized 3, 4 , and 5 hem's prior to sacrifice; from animals hepatectomized and enterectomized 4 and 5 hours before sacrifice; h'om an animal hepatectomized and nephrectomized 3 ~ hours before sacrifice; and from maimals hepatectomized, enterectomized, and nephrc~ctomized 4 and 5 hours before sacrifice. Serum from 2 sham-operated animals was examined.

RES U LTS The protein incorporation of radioactivity was Considerably lower in the hepatectomized animals (26 to 58 c[)m per absorbancy unit at 280 rap,)than in the shmn-operated animals ( 2 ~ to 240 cpm per absorbancy unit at 280

m~). A representative pattern of protein separation by tlm preparative gel-electrophoresis apparatus:is shown in Figure 1. The initial sharp band Of optically dense, material (tubes 30 to 32) is unidentified and may be an artifact. Material in tubes 40 to 48 is predominantly serum albumin: Tim Rhesus monkey appear s to have variable polym0rphism i n its transferriiL This is shown in Fi~are 2, which illustrates an experiment wt~ere transferrin was found in tubes ~ t o 6 7 ~md 71 to 74. A similar variability in , of transferrins in. a noted ill a : n u m oer o r speems t~l, rlemogl0bin (or h e m 0 , gl0bin-hapt6g!obin Complexes) was eluted in

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approximately tubes 78 to 82. G a m m a globulin was eluted in a diffuse region begimaing at about the position of hemoglobin. In general, this g r o u p of proteins was veD, slowly eluted from the gel. Therefore. the eleetrophoresis was frequently not c o n t i n u e d through the g a m m a globulin region. In the sham-operated animals n~dioaetivity was ineoq~orated into all protein fractions, and the relative m n o u n t of radioactivity tended to parallel the optical density in most regions (Fig. 1). Significant mnounts of radioactivity were T

present in m a n v regions of the e l e e t m p h o r e t i c pattern when serum from hepatectomized animals was analyzed. Very little radioactivity was presm~t in the prealbumin region. T h e center region of the albumin also h a d velT little l~,dioactivity. In contrast to pattmu}s previously obtained with serum from h c p a t e c t o m i z e d dogs, two radioactive components were present, overlapping t h e range of electrophoretie mobility of albmnin, one of slightly higher and the other of. lower mobility than the main portion of the Mbumin (Fig. 2, tubes 38 to 40 a n d 43 to 46). Eve W F

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Fig, 3. Preparative acrylamide electrophoresis of serum from a hepatectomized monkey. The animal was given I00 lie. of C ~* leucine four and o~e-half hours before sacrifice. Closed circles represent absorbency at 280 nag and c~pen circles radioactivity per milliliter of eftluent. fraction b e y o n d the a l b u m i n region eontah~ed m e a s u r a b l e radioactivity in animals allowed to survive 4 or m o r e hours after the openltion I Fig. 3). T h e r e was a distinct suggestion of concentration or p e a k i n g of 'radioactivity in certain c o m p o n e n t s in the slow alpha a n d fast beta region. In contrast to t h e results obtained w i t h dogs, there was less t e n d e n c y of radioactivity to c o n c e n t r a t e in the interm e d i a t e beta region. O n e h u n d r e d microcuries o f C I"~ Iysine were injected into a h e p a t e c t o m i z e d a n d entereetomized a n i m a l T h e animal was saeririced 5 hours later. T h e p a t t e r n o f distribution of isotope w a s very similar to t h a t seen in the leucine e x p e r i m e n t s ( Fig. 4). O n e animal was given 2 ~ g c o f C 14 leu-

cine. This s e r v e d to give a s o m e w h a t better definition of the radioactive c o m p o n e n t s because of t h e higher c o u n t rates obtained. The results verified those seen with lower doses o f isotope (Fig. 5). Various combinations of n e p h r e e t o m y and e n t e r e e t o m y w i t h p a n c r e a t e c t o m y and splen e e t o m y w e r e tried in e x p e r i m e n t a l animals. In no case was there a c l a i r alteration in the distribution of radioactivity in the proteins as c o m p a r e d with t h e h e p a t e c t o m i z e d animal. T h e 50% a m m o n i u m sulfate precipitate of serum from t h e hel)atectomized animals contained over 80% of the radioactivity. '/llis w o u l d b e expected, since all o f t h e maior imm u n o g l o b u l i n fractions a r e p r e c i p i t a t e d by this concentration of salt. E l e~e t r o n h o r ~ s r

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of this fraction of the proteins showed that c o m p o n e n t s with slower alpha mad beta mohility were also precipitated by m n m o n i u m sulfate (Fig. 6). T h e r e was a marked reduction in the a m o u n t of radioactive material with fast alpha and a l b u m i n mobility in this fraction. Analysis of the a m m o n i u m s u l p h a t e s u p e r n a t e (Fig, 7) s h o w e d several radioactive c o m p o n e n t s which a p p e a r e d in part to he more discretely separated than those in the precipitate. DISCUSSION Tim results indicate that the synthesis of serum proteins other than the inwnunoglolmlins is not restricted to tlm liver in Rhesus monkeys. Considered t o g e t h e r with earlier results in dogs, this suggests that significant a m o u n t s of serum protein, at least in t h e o p e r a t e d animal~ may b e derived h'om extrahepatic sources. Since: albumin firmly binds a n m n h e r of substances [2] the possibility must be considergd that radioactivity in some c o m p o n e n t s in othe albu,~in region may represent nonprotein material b o u n d to s e r m n proteins. Such hypothetical c o m p o n e n t s m u s t be completely resistant to long-term.dialysis and would h a v e to derive similar anaounts of radioactivity, from the earboxyl c a r b o n of leucine a n d from u n i f o r m | y labeled lysine: It is also possible that these radkmctive c o m p o n e n t s m a y b e p o l y p e p t i d e s complexed with albumin. F r o m what is known of primate i m m u nog!obulins [14], it is not likely that most of the material of higher mobility be|ongs to this group o f proteins. Because of their larger molecular size, these proteins would b e ret a r d e d considerably b e h i n d transferrins in gel-eleetrol)horesis even tlmugh their mobility in free eleetrophoresis m i g h t exceed that o f transferrin. Actually the imnmnoglobulins s t a n d o u t rather cl£arly as the most radioactive c o m p o n e n t in eleetrophoretie determinations such a s that p r e s e n t e d in Figures 2 or 3. T h e authors are n o t a w a r e of any poiyid so

be d e t e c t e d b y t h e p r e s e n t methods. 3=0

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\Virile this work was in progress a repor: a p p e a r e d describing a glyeoprotein in the rm wMd~ was synthesized outside the liver and a p p e a r e d in i n c r e a s e d concentration in tlu serum following stress [1]. H y d m x y p r o l i n e has b e e n noted to occur in some fractions ol serum prSteins a n d these may be synthesized elsewhere than by the parcnehymal cells ot th, ,iv, r 7j. erunier e, .l. !Ial ol,se,[ d tha, hnman" lymph node, spleen, pancreas, and skeletal nmscle in¢'orporated radioactivity i~ vivo into protein, resembling sermn alpha globulin and transferrin in their l)ehavior on immunoeleetrophoresis.- It is possible .-that these radioactive c o m p o n e n t s are eithm" present in reduced a m o u n t s or absent from normal serum and a p p e a r ill increased alllounts aft~r the stress of th e operative procedure. Analogies for this exist with such serum proteins as C-reactive protein [4, l l J . It is likely that similar alpha and beta globulins exisl in man. Their identification is a difficult p m M e m b u t would 1)e of emasiderable interest and importance. SUMMARY Serum proteins of the FIhesus m o n k e y were separated by preparative acrylamide gel-dectrophoresis. After hepatectomy, the animals still incorporated significant amotmts of radioactivity into a h e t e r o g e n e o u s group of alpha a n d beta globulins. This was not "altered by enterectomy or n e p h r e c t 0 m y . After fractionation of the s e r u m by precipitation with 50% saturated a m m o n i u m sulfate solution, radioactiw~, alpha a n d beta globulins were f o u n d in both the w e e i p i t a t e a n d the supernate.

REFERENCES

2.

Bogden, A. E,, Rosenkrantz. IL, and Gray, J. H. Studies on the origin of a specific a|pha-2-glycoproteiri. Fed. Prom 24:230. 1965. Foster, J.T. Plasma Albumin ~In F. XV. Putnam (Ed.), ',The Plasma Proleh~s. New York: Academic, 19~. pp. [79-239. • e Plasma Transfetriu. In A. G. G. Beam (Eds.), Progress m VoL IL New York: Grmm t~ Stratton, 1962, pp, 34-63.

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1963, 5, ]ovin, T., Chamhaeh, A., and Noughton, b.l. A, An "~r preparative temperature-reD,late{ mide gcl-electmPhOreSts. Anal Bioc 1964. I 1~al, J. C,, Slmrn, J., Loach, J., and V¢inzler, 6. Kuk IL J. "Synthesis of alpha and beta glolmlins in nonr, al and li~;erless dog~. Amer, 1. Pl~ysioL 204:262, 1963.

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S}oemsma, A. A h,,,)droX,ypmline eontai~ing collagen-like protein in plasma and two procedures for its assay~ ] Biol. Chem. 23g:3350, 1964.

8. McFarlane, A. S. Meialmlism of Plasma Proteins. In 1:1. N. Munro and J. IL Allison (Eds.), Mammalian Protein Metab¢;tism. Vol. 1 New York: Academic, 196"4, pp, 29%341, 9, Miller, L. L., and Bale, ~,V. T. Synthesis of all plasma protein fractions except gamma globulins bv the liver. 1. Exp, Med. 99~125, 1956. 10, ~{iller, L. L., Bly, C. G,, and Bale, \V. F, Plasma 99:133, 1956:

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1 1. PetermamL M . L . Alterations of plasma proteins in disease, hi F. W. Putnam '(Ed.), The Plasma Proteins. New York: Academic, 1960, pp. 324 -326. 12. Peterson. E, A,, Wyek¢~/[, ,xl, .M,, and Sober. it. A. Gradim~t chromatography of human serum pm/eir~s and ils application to the examination of "M1)tmfin" and "°globulin" ohtained I~, ammonium sol[ale [raetional~oo. Arch. Biochem. 93:428, 1961. I& Pnmier, J, H.. Beam, k. (L. and C|eve. H, Site of formation of the grmq~ specific cxmqmimnt a r~d eeriain other sermn proteins. Proc. Soc. Exp. Biol. Med. 115:1005, 1964. 14. Singer, S. ]. Structure and Function of Antigen and Antihndy Protein. In it. Neurath ( E d . ) , The Proteins, Vol. I11. New York: AcademiC. I,q~t, pp. °69-357. 15. StmvJ. T. E.. Bernhard. V. M., Benwmulo, R., and Cortes, N. A new method for one-stage hepatectomy for dogs. Surgery 46:880, 1959. 16. Veetssman, S. M , V¢ochner, R. D,, Mullins, F. X,, Veynngate, A., and 'Waldmann, T . A . Synthesis of plasma proteins by lira hepateetomized dog, Amer. 1. Physiol. 210:128, 1966.

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