Protein carboxymethylation: Effects of 2% sodium chloride administration on protein carboxymethylase and its endogenous substrates in rat posterior pituitary

Sciences, protein readily found chemotaxis concentration in plays free 80% Although medulla the suggested enzyme, and studied prolonged in hydrolyzed major of carboxyl asubstrates UVol rats axis carbox its the (16-18) Gagnon ofbetween analysis and brain, role CARBOXYMETHYLATION the Under (13~ (15) axis methyl protein PCM effects 22, carboxymethylase in peak the there ~-tom total that inSalt ONare the stimulation groups and ,pp is testes causing these disappearance specific secretion inof the PROTEIN Julius 1(1-5) There this was widely acceptor ?~ed~al posterior loading and radioactivity carboxymethylase neutral membrane good of 2155-2164 methylated Institute the posterior aconditions of and sodium in methyl 2IN ismethyl Axelrod the progressive substrates CARBOXYMETHYLASE of days activity proteins The distributed, final RAT pituitary also isMaryland (14,15) Research stimulates of and proteins Clinical secretion proteins EFFECTS and pituitary protein-methyl involved chloride of POSTERIOR after of acceptor the pituitary acceptor proteins alkaline evidence form and Ethis Mental methyl there hypothalamo-neurohypoand The for Michael 20014 with salt gland Anterior increase (PCM (MAP) Council May OF Science, 2 treatment of ofprotein peak neutralized the in of was molecular PCM 2% PITUITARY Health, acceptor proteins accounting conditions that chromaffin (6,8) 4, neurohypophysial the loading bacterial AND control hypothalamo-neuro S-adenosylmethiônine highest (8) are aSODIUM after Jesters 1978) ofpituitary progressive greatest in protein ITS present Brownstein is methylates Canada protein For on weight proteins showed salt CHLORIDE ENDOGENOUS and neurophysin negative specific Electrofor are to granules (9-12) PCM this carboxymethylNaCI fal form loadin upa especially unstable peptides activity lhwe reason and (estermethanol charges activities from leukowe and (5), Prass on

Life Printed

.

. .S .A.

PROTEIN ADMINISTRATION SUBSTRATES * Claude

Pergamon

: .

Laboratory National Bethesda, (Received Summary Protein been treated . hypophysial peptides . decrease occurring phoretic single to (11,000) ing After physial carboxymethylase

. . . .

The protein-carboxyl-O-methyltransferase, ifies) the .~ are (6,7) . were Protein cyte ation neurophysins adrenal PCM high investigated *Cénténn~al~éflow

.C .

: .1 .1 .24)

.

thylation . . .

:

2156

Protein Carboxymethylation

Vol . 22, No . 24, 1978

MAP capacity of rat posterior pituitary . Salt loading is well-known to stimulate the hypothalamo-neurohypophysial axis and to release vasopressin and neurophysin (19) . In this report we demonstrate that MAP were rapidly depleted whereas the activity of PCM was progressively increased in posterior pituitary after salt treatment . Materials and Methods Materials . S-Adenosyl-L-methyl- 3 H]methionine 12 .3 Ci/mmole was obtained from New England Nuclear Boston, Mass .) Acrylamide, bisacrylamide, N,N,N',N'-tetramethylenediamine, ammonium persulfate and ß-merca toethanol were purchased from Bio-Red Laboratories (Rockville Center, N .Y .~ . Sucrose (Ultrapure grade) and urea (Ultrapure grade) were purchased from Schwarz/ Mann (Orangebur , N .Y .), chymotr psinogen and aldolase from Pharmacia Fine Chemicals Inc . ~Piscataway, N .J .~ cytochrome C, and bovine serum albumin from Boehringer Mannheim Biochemicals (Indianapolis, Ind .) and myoglobin, phosphorylase a, N-cetylpyridinium chloride and Coomassie brilliant blue from Sigma Chemical Co . (St . Louis, Mo .) . Tissue re aration . Male, Sprague-Dawley rats weighing 150-175 g were kept in our ac t es at least 5 days under a light-dark cycle of 12 h before being used . Experimental groups received 2% NaCI in their drinking water, whereas controls received tap water . At various time intervals, rats were killed by decapitation and pituitaries were removed and immediately dissected under microscope . Posterior lobes (pars nervosa) and anterior lobes (pars distalis) were homogenized respectively in 50 and 300 ul of 0 .3 M sucrose containing 0 .01 M sodium acetate buffer, pH 6 .5 . Microdissected samples of su raoptic nuclei were obtained by the procedure described by Palkovits (20~ and homogenized in 0 .3 M sucrose . PCM and MAP determinations . To reproduce in vivo conditions where soluble and me rane oun prote ns compete with eachof er for the cytosolic PCM, Protein carboxymethylation was whole homogenates were used in assays . measured as described elsewhere (5,15) . This procedure iwolved the incubation of tissue homogenates with S-adenosyl-L-[methyl- H]methionine (5 uM), 0 .05 M sodium acetate buffer, pH 6 .5, and with an exogenous substrate, such as gelatin (10 mg/ml) for PCM activity determination or with purified PCM for MAP determination . After a 10 min incubation at 37°C the reaction was stopped by the addition of 10% TCA . Precipitated proteins were centrifuged and resuspended in 200 ul of 1 M sodium borate buffer, pH 11 .0 . The radioactive methanol formed from the hydrolysis of the protein-methyl esters at alkaline His extracted with a mixture of isoamyl alcohol-toluene (2 :3, by vol~ and counted by liquid scintillation spectrometry before and after evaporation . Results are expressed in pmol/mg protein . GEL electro horesis .~ Carboxymethylated proteins were analyzed in two e ectrop ore c systems . The first system was based on the acetic acidurea system of Devis et al . (21) . The 10% polyacrylamide tube gels contained 5 .4% acetic acid, ~7~ acrylamide, 0 .26% bisacrylamide, 20% glycerol, 0 .5% N,N,N',N'-tetramethylenediamine, 1 .0% ammonium oersulfate and 5 M urea . Gels were pre-run overnight in 3% acetic acid and run in 3% acetic acid containing 0 .03% N-acetylpyridinium chloride as described elsewhere (C . Gagnon, O .H . Viveros, E .J . Diliberto, Jr . and J . Axelrod, submitted Samples for electrophoresis were prepared as follows : for publication) . after protein carboxyrt~ethylation, 0 .6 M acetic acid, 5 M urea, 3% ß-

Vol . 22, No . 24, 1978

2157

Protein Carboaymethylatioa

mercaptoethanol, 2% N-cetylpyridinium chloride were added and the mixture was brought to 95°C for 5 min . The electrophoresis was run at 2 mA per tube gel during 3 hours . Gels were stained, destained, sliced and the radioactivity counted as reviously described (22) . The second system was a modification (pH 6 .5~ of the SDS system of Weber and Osborn(23), ~PCM purification . PCM was isolated from fresh bovine pituitaries or adrena me u a essentially according to a method previously described (5) . Purified PCM from either source was devoid of MAP . Protein determination . Protein concentrations were determined according to t e met o 0 owry et al . (24) using bovine serum albumin as a standard . Results Effect of sodiun chloride treatment on PCM activit and MAP ca acit of ra to ar es . o s mu ate t e ypot a amo-neuro oop s a ax s, rats were su ec e to salt loading . Sodium chloride (2% wjvol~ was added to drinking water and PCM activity and MAP capacity were determined after 5 days of treatment . There was no change in PCM activity in the posterior lobe whereas the MAP capacity was markedly lowered to 27% of the control value (Table I) . This decrease was specific to the posterior lobe since there was no change in NAP capacity nor in PCM activity in the anterior lobe (Table 1) .

TABLE 1 Effects of Five Day Salt Treatment on Pituitary PCM Activity and MAP Capacity

Posterior lobe

Anterior lobe

Control

Salt treated

Control

Salt treated

PCM (pmol/mg)

34 .6+1 .4

35 .2+2 .6

31 .2+0 .6

31 .8+0 .6

MAP (pmol/mg)

44 .0+3 .4

11 .8+1 .6*

17 .5+1 .6

16 .9+1 .4

Rats were given 2% NaCI in their drinking water . Five days later, they were killed, and the posterior and anterior pituitary dissected . PCM activity and MAP capacity were determined on tissue homogenates . Values represent the mean + S .E .M . of H-methyl groups transferred/mg protein obtained from 6 antérior end 6 posterior lobes .

2158

Vol . 22, No . 24, 1978

Protein Carboxymethylation TABLE 2

Time Course of the Loss of MAP Capacity after Salt Treatment Peri od of salt treatment (days)

0

MAP capacity (% of controls) 100 +3

1

2

4

28

79* +5

40** +3

23** +3

15** +1

The values represent the mean + S .E .M . obtained from3 6 posterior pituitaries . The MAP capacity of côntrols was 41 .2 pmol H-methyl groups/mg . protein . *P < 0 .05 when compared to controls . **P < 0 .001 when compared to controls .

Time course of the loss in MAP ca acit in sterior ituita after salt treatment . is were g ven % sa ne an e , , an ays ater o~~capacity determination of the posterior lobe . There was a progressive decrease in MAP capacity with the greatest fall occurring between 1 and 2 days (Table 2) . A plateau was reached after 4 days of treatment and only a small decrease was observed after up to 4 weeks of treatment . Electrophoretic profile of posterior lobe carboxymethylated roteins after sa t trea nt . a xyme by ate prate ns rom pos er or t~esoff' control an sa t treated rats were analyzed on polyacrylamide gel to determine which MAP was disappearing after salt loading . An electrophoresic system under acidic conditions was used to avoid hyrolysis of protein-methyl esters . There was a single major peak of radioactivity accounting for about 80% of the carboxymethylated proteins in posterior lobe homogenate of control rats (Fig . 1) . After 5 days of saline treatment, this peak disappeared whereas the other methylated proteins were much less affected . The molecular weight of the major methylated protein was less than 25,000 and was below the ran e for a r~olecular weight estimation with this electrorophoresic system ~C . Gagnon, O .H . Viveros, E .J . Diliberto, Jr . and J . Axelrod, submitted for publication) . An SDS gel electrophoresis at pH 6 .5 was then used to estimate molecular weight of the methylated protein . Although the recovery of the protein-methyl esters was about one-third (30%) that of the previous system, a single peak of radioactivity was observed whose molecular weight was estimated at 11,000 (Fig . 2) . To further characterize the MAP, 35 S-cysteine was stereotactically infected adjacent to the supraoptic nuclei (areas where neurophysin cell bodies are located) . The animals were killed 24 h later and the posterior pituitary was removed and homogenized in 0 .1 N HC1 . After centrifugation at 26,000 g for 30 min the supernatant was saved for analysis . Norstr~om et al . (25) and Gainer et al . (26,27), using a similar procedure, have shown l:Tia~

Vol, 22, No, 24, 297$

Protein Carboxymethplation

2159

in the supernatant the major 35 S-cysteine labeled protein which accounted far 8O% of the total radioactivity was neurophysin . When the supernatant was an~~yzed with the acid-urea and SOS electropharetic systems, the main peak of S-cysteine labeled pratetn co-migrated in both systems with the major peak of protein-methyl esters (data not shown},

~ I

O=_ ©® I II I I

O !

B.

2Q

'

40

FIG,

SLICES

ß0

aQ

T,

Gel electrophoresic profile of MAP from the posterior pituitary homogenate of control and salt treated rats . Rats were given 2% NaCI in their drinking water for 5 days . Posterior pituitaries were hol!logenix~ in 0 .3 M sucrose. Homogenates were incubated with 5 uM SradenosyT-[methyl- H]methionine and purified PCM for 10 minutes at 37°C . The reaction was stopped by the addition of 0 .6 M acetic acid, 5 M urea, 3% B-mercaptoethanol, and 2% Ncetylpyridinium chloride and the mixture was heated at 95°C for 5 minutes . The electrophoresis was run at 2 mA per tube gel for 3 hours. Gels were stained, destained and sliced as described elsewhere {22), The molecular weight standards used were : (1) phosphorylase a (monomer 94,000}, (2) bovine serum albumin {6$,OOO}, (3} catalase (6ä,O00}, {4} aidolase (monomer 40,000} . (5} chymotrypsinogen (25,000), (6) m~yoglobin (17,000) and (7) cytochrome C ( 12,500) . Controls { ~---~) and salt treated { "--" ) .

Protein Carboaymethylatioa

2160

Vol . 22, No . 24, 1978

30-

20 "

20

40

60

80

SLICES FIG . 2 SDS gel electrophoretic profile of MAP from the posterior pituitary homogenate of control rats . Carboxymethylated proteins from homogenates of posterior pituitary were analyzed on a 12X polyacrylamide gel in the presence of 0 .1~ SDS at pH 6 .5 . Molecular weight standards are identical to those of Fig . 1 .

Effects of lon -term salt treatment on PCM activit . When salt treatment wee s ere was a progressive increase in PCM was con nue or , an specific activity (Table 3) . This increase was confined to posterior lobes with no change in PCM specific activity in anterior lobes . To determine whether the increase might be due to :a chahae in Km, àhe Km for S-adenosyl-methionine was measured after 4 weeks of salt treatment . There was no significant change in Km after salt treatment (Fig . 3) . The increase in PCM specific activity was due to a change in Vmax which was 52~ higher after salt treatment . Effect of salt treatment on PCM activit and MAP ca cit of su rao tic nuc e . e o es o pos er or p u tary nerve e na s are ma n y located in 2 discrete hypothalami nuclei : the supraoptic and the paraventricular nuclei (2g) . To determine whether PCM activity in the cell bodies was also increased, the enzyme activity was measured in microdissected samples of supraoptic nuclei of control and salt treated animals . The PCM

Vol . 22, No . 24, 1978

Protein Carboxymethylatioa

2161

S(NM) FIG . 3 Determination of Km for S-adenosylmethionine and Vm of the methylation reaction in posterior pituitary homogenates from control and salt treated rats . Rats were given normal water or 2% NaCI in their drinking water . After 3 weeks, the animals were killed, the posterior pituitar dissected and homogenized in 0 .3 M sucrose. Haragenates (40 u9 proteins were incubated with a saturating amount of gelatin and S-adenosyl-methionine at 5 concentrations ranging from 0 .88 to 14 uhl . Km for controls is 2.49 + 0.17 uM and for salt treated 2.62 + 0.29 uM . Vmax for controls is 2.76 + 0 .06 pmol/10 min and for salt treated 4 .20 + 0 .16 pmol/10 min. Contr-1 s (f--~ ) and salt treated ( "-" ) .

specific activity was twice that of the posterior pituitary, a tissue which has one of the highest enzyme specific activity . In the supraoptic nucleus, there was ra significant difference in PCM specific activity between control and salt treated animals . Discussion After salt loading we have observed a progressive decrease in MAP capacity of the posterior pituitary which falls to 20% of its normal value within 4 days . This decrease 1n MAP was specific to the posterior lobe since neither MAP capacity nor PCM activity were altered in the anterior lobe after salt loading. Electrophoretic analysis of MAP from whole homogenates of control posterior pituitary shows a single major peak of protein-methyl esters

2162

Vol . 2 2, No . 24, 1978

Protaia Carbo :ymathylation

TABLE 3 Effect of Prolonged Salt Treatment on PCM Activity

Period of Treatment

0

2

3

4

PCM activity (% of control)

100 + 5

120 + 4

141** + 2

162** + 12

Rats were given 2% NaCI in the drinking water for 0 - 4 weeks . The values shown represent the mean + S .E .M . of H-methyl groups transferred/mg protein obtained from 6 posteriorpituitaries . *P < 0 .05 when compared to controls . **P < 0 .001 when compared to controls .

TABLE 4 Effects of Prolonged Salt Treatment on PCM Activity and MAP Capacity of Supraoptic Nuclei

Control

Salt treated

PCM activity (pmol/mg protein

66 + 3

62 + 6

MAP capacity (pmol/mg protein)

22 _+ 1

19 _+ 2

Rats were given 2% NaCI in their drinking water for 4 weeks . Values represent the mean + S .E .M . of H-methyl groups transferred/mg protein obtained from 6 supraoptic nuclei .

amounting to about 80% of the total radioactivit in the gel profile, This indicates that one protein (or class of proteins is methylated preferentially by PGM though the homogenate contains . numerous proteins . Since methyl esters on various roteins have different stability (Gagnon et al ., submitted for publication a true MAP profile, from a complexed mixtûréof MAP, is obtained only in electrophoretic conditions where the recovery of protein-methyl esters is very high . With the system used, the recovery was higher than 90% . However, the position of the main peak of radioactivity was outside the linearity range for that system so that its molecular

Vol . 22, No . 24, 1978

Protein Carboaymethylation

2163

weight could not be estimated . The fact that there was only one major peak of protein-methyl esters accounting for about 80% of all protein-methyl esters with the first electrophoretic system made possible the use of an SDS system to determine the molecular weight . Though the recovery of protein methyl esters ~s low (30%), the molecular weight of this MAP was estimated at 11,000 . Norstrom et al . (25) and Gainer et al . (26,2~~ have shown by several S-cysteine is injected technics includingimmunoprecipitationteat when ~~jacent to the supraoptic nuclei of rats, the major peak of acid-soluble S-cysteine labeled proteins in . the posterior pituitary 24 h after injection is neurophysin . Using a similar preparation, we have observed that this major peak of radioactivity was co-migrating in both acid-urea and SDS electrophoretic systems with the major peak of protein-methyl esters . This fact and the observations that the major peak of radioactivity was lost after salt treatment and that its position on SDS gels corresponded to a rtwlecular weight of 11,000 suggest that this major MAP is neurophysin which has a molecular weight of 10,000-12,000 (25-27, 29) . The physiological importance of neurophysin methylation remains to be elucidated, however, it has been shown that the binding of oxytocin to neurophysin II increases the rate of methylation of neurophysin (30) . PCM activity was unaltered after a few days of salt treatment but after two weeks, there was a progressive increase in PCM specific activity . The long time course of this increase and the unaltered Km for S-adenosyl methionine suggest a specific enzyme induction . No increase in PCM specific activity was detected in the supraoptic nucleus where the cell bodies of posterior pituitary nerve terminals are located . This may be due to the fact that the supraoptic nucleus contains many cell types (neuronal and nonneuronal) or processes not related to posterior pituitary nerve terminals . In other secretioy systems correlations have been established between exocytotic secretion and protein methylation . It has been shown that after stimulation by insulin, in vivo protein carboxymethylation was increased several fold in the adrenâl~ulla (14) . In rat parotid gland, where massive secretion can be achieved within one hour, there was a transient increase in PCM activity within 5 minutes after injection of isoproterenol (Strittmatter, Gagnon and Axelrod, submitted for publication) . This increase in PCM activity was not blocked by protein synthesis Inhibitors . These results suggest that PCM, in response to a stimulus causing secretion, is affected in two ways . In acute stimulation PCM increases its activity possibily through an allosteric conformational change whereas by chronic stimulation a specific enzyme induction may occur . A related phenomenon has been found with the enzyme tyrosine hydroxylase . After brief stimulation, tyrosine hydroxylase has an increased affinity for its substrate and its cofactor and a decreased affinity for its inhibitor (31) . After prolonged stimulation, the synthesis of tyrosine hydroxylase is induced and tyrosine hydroxylase activity remains elevated for several days (32) . References 1. 2. 3.

M . LISS, A .M . MAXAM and L .J . CUPRAK, J . Biol . Chem . _244 : 1617-1622, 1969 . S . KIM and W .K . PAIK, J . Biol . Chem . 245 : 605-608, 1970 . S . KIM and W .K . PAIK, oc dn s ry 0~141-3145, 1971

2164

4. 5. 6. 7. 8. 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 . 18 . 19 . 20 . 21 . 22 . 23 . 24 . 25 . 26 . 27 . 28 . 29 . 30 . 31 . 32 .

Protein Carboaymethylation

Vol . 22, No . 24, 1978

A.M . MORIN and M . LISS, Biochem. Biophys . Res . Commun . _52 : 373-378, 1973 . E.J . DILIBERTO, JR . and J . AXELROD, Proc . Natl . Acad . Sci : U .S :A . 71 : 1701-1704, 1974 . J . AXELROD and J . DALY, Science 150 : 892-893, 1965 . S. KIM and W.K . PAIK, Ex er~fiâ3'L : 982-984, 1976 . E .J . DILIBERTO, JR . an ;J . Neurochem . 26 : 1159-1165, 1976 . E.N . KORT, M.F . GOY, S .H . LARSEN an . , roc . Natl . Acad . Sci . U.S .A . 72 : 3939-3943, 1975 . A~~P1~iNGER and D .E . KOSHLAND, JR ., Proc . Natl . Acad . Sci . U.S .A . 74 : 533-537, 1977 . ~S . SPRINGER, M. F. GOY and J . ADLER, Proc . Natl . Acad . Sci . U.S .A . 74 : 3313-3316, 1977 . ~ SILVERMAN and M. SIMON, Proc . Natl . Acad . Sci . U .S .A . _74 : 3317-3321, 1977 . R.F . O'DEA, O .H . VIVEROS, S . ASWANIKUMAR, E . SCHIFFMANN, B.A . CORCORAN and J . AXELROD, Nature 272 : 462-464, 1978 . O.H . VIVEROS, E .T~TCIB~O, JR . and J . AXELROD, in ~S na~ ses, G.A . Cottrell and P .N .R . Asherwood, eds., Blackie and Son Ltd, London, pp . 368-369, 1977 . E.J . DILIBERTO, JR ., O.H . VIVEROS and J . AXELROD, Proc . Natl . Acad . Sci . U.S .A . 73 : 4050-4054, 1976 . D.H . E and D.B . HOPE, FEBS Letters 49 " 145-148, 1974 . J. AXELROD and E .J . DILIB ., nn-l~I .Y . Acad . Sci . _248 : 90-91, 1975 . D .H . EDGAR and D .B . HOPE, J . Neurochem. 27 : 949-955, 1976 . A.M . MOSES and M . MILLER, in an o0 of~h siolo ,Section 7, Vol . IV, E. Knobil and W.H . Sawyer, e s ., er can ys o ogical Society, Washington, D .C ., pp . 225-242, 1974 . M. PALKOVITS, Brain Res . 59, 449-450, 1973 . R.H . DAVIS, J .~ARAV~R and M .J . CARVER, J . Neurochem . _19, 473-477, 1972 . C. GAGNON, U . OTTEN and H. THOENEN, J . Neurochem. 27 : 259-265, 1976 . K. WEBER and M. OSBORN, J . Biol . Chem : ~-4~T2, 1969 . O.H . LOWRY, N .J . ROSEBRO , . . a~ R .J . RANDALL, J . Biol . Chem . 193 : 265-275, 1959 . .NORSTROM, J . SJOSTRAND, B .G . LISETT, L .O . UTTENTHAL and D .B . HOPE, A Biochem J . 122 : 671-676, 1971 . H.~13FE1 1~ ~SARNE and M .J . BROWNSTEIN, J . Cell Biol . _73 : 366-381, 1977 . H. GAINER, Y . SARNE and M.J . BROWNSTEIN, Science 195 : 1354-1356, 1977 . K. .LEDERIS, in Handbook of Ph siolo ,Sect on ,~ . IY, E . Knobil and W .H . Sawyer, e s., er can Lys ological Society, Washington, D.C ., pp . 81-102, 1974 . M.J . BROWNSTEIN and H. GAINER, Proc . Natl . Acad . Sci . U .S .A . 74 : 4046-4049, 1977 . E.J . DILIBERTO, JR ., J . AXELROD and I .M . CHAI KEN, Biochem . Biophys . Res . Camnwn . 73 : 1063-1067, 1976 . NhO~FT, III, M . BOADLE-BIBER and R .H . ROTH, Proc . Natl . Acad . Y. Sci . U .S .A . 71 : 4283-4287, 1974 . ~N-TR~ERER, ~ Handbook of Ps cho harniacolo ,Vol . 3, L .L . Iversen, S.D . Iversen an . . ny er, e s ., enum ress, New York-London, pp . 443-475, 1975 .