Purification of bovine neurosecretory granule membranes by density gradient centrifugation

ANALYTICAL

157,316-322

BIOCHEMISTRY

Purification

(1986)

of Bovine Neurosecretory Granule Membranes by Density Gradient Centrifugation

CARLOS B. GoNz,&LEz,*,’ CARLOS E. CAORSI,* WILLIAM AND OLGA T. BERRIOS*

G. NoRTH,t

‘Institute de Histologia y Patologt’a, Universidad Austral de Chile, Valdivia, Chile, and fDepartment of Physiology, Dartmouth Medical School, Hanover, New Hampshire 03755 Received February 24, 1986 A procedure for the subfractionation of neurosecretory granules into membrane and content components is described. The procedure involves the hypotonic lysis of the secretory granule fraction and further purification of the membranes by centrifugation through a discontinuous sucrose gradient. The neurosecretory granule membranes represented 5.2% of the total proteins of the neurosecretoty granule fraction and were highly enriched in cytochrome bs6,. Electron microscopic analysis of the purified membranes showed vesicles devoid of electrodense content. Q 1986 Academic KEY

Ress, Inc.

WORDS: neurohypophysis; neurosecretory granules; granule subfractionation; membrane

Neurosecretory granules contain vasopressin and oxytocin along with their respective neurophysins (1). The possibility of obtaining large quantities of neurosecretory granules as a relatively pure fraction (2-4) makes the hypothalamo-neurohypophysial system an attractive model for studying molecular aspects of secretion such as formation and flow of granule membranes. However, the degree of purity of the membranes is crucial for the success of this kind of study. In the present work we describe a procedure to prepare highly purified neurosecretory granule membranes. The membranes are characterized by enzymatic analysis, electron microscopy, and polyacrylamide electrophoresis. MATERIALS

AND METHODS

Pwijication of neurosecretory granules. Bovine neural lobes were homogenized in 0.25 M sucrose, 10 mM Hepes, pH 7.4, containing

and neurosecretory granules were obtained by differential and density gradient centrifugations as described by Dean and Hope (2) with some modifications. The homogenate was centrifuged at 4000g for 1 min in a refrigerated centrifuge. The pellet (Pi) was kept for further analysis and the supernatant centrifuged at 7000g for 10 min to yield a pellet (PZ) and a supernatant. This was finally centrifuged at 26,000g for 30 min to produce a pellet (P,) and a supematant (S,). The pellet P3 was resuspended in 0.25 M sucrose and layered onto a discontinuous sucrose gradient formed by layering 4 ml of 2.0 M, 6 ml of 1.4 M, and 2 ml of 1.3 M sucrose and centrifuged at 48,OOOg for 90 min in an RC-SB Sorvall refrigerated centrifuge equipped with an SS34 rotor. The gradient was fractioned and aliquots were taken for radioimmunoassay, enzyme activity, and protein determination. A sample of the granule fraction was processed for electron microscopy. 0.5 mM EDTA,

Subfractionation of granules into membrane and content. The fractions containing neu-

’ Present address: Institute de Fisiologia, Universidad Austral de Chile, Valdivia, Chile. 0003-2697186 $3.00 copyrisht Q 1986 by Academic Press, Inc. AU rigJtts of repmduction in any form merval.

rosecretory granules (fractions 3,4, and 5, Fig. 316

NEUROSECRETORY

SF? SF1

GRANULE

1.2 M i3M 2.0M

FIG. 1. The appearance of bands after centrifugation of the crude membrane preparation on the sucrose gradient. The designation of the different subfractions is also shown.

1) were diluted to -0.30 M sucrose and centrifuged at 40,OOOg for 30 min at 4°C. The pellet was resuspended in 2 ml 10 mM Hepes, pH 7.4,0.5 mM EDTA and frozen and thawed twice. The suspension was centrifuged at 40,OOOg for 30 min to yield a supematant and a pellet. The cycles of centrifugation and suspension were performed three times more. Finally the pellet was resuspended in 10 mM Hepes, pH 7.4, and placed onto a discontinuous gradient formed by layering 0.5 ml of 2.0 M, 0.5 ml of 1.3 M, 1 ml of 1.2 M, and 0.5 ml of 1.1 M sucrose. Gradients were centrifuged at 115,OOOg for 1 h at 4°C in a Beckmann SW 65 rotor. The appearance of the tube after centrifugation is shown diagrammatically in Fig. 1. The gradient was cut with a tube slicer into three subfractions. The supematants of the lysis procedure were pooled and kept for further analysis. Samples of subfractions were processed for electron microscopy. Aliquots of subfractions were taken for radioimmunoassay, enzyme activity and protein determinations, and polyacrylamide gel electrophoresis. Samples were kept frozen until use. Sodium dodecylsurfate(SDS)2-polyacrylamide gel electrophoresis. Proteins were separated on 12.5% polyacrylamide in the discontinuous buffer system of Laemmli (5). Analytical procedures. Acid phosphatase was determined using pnitrophenyl phosphate as substrate (6). Glutamate dehydrogenase was

MEMBRANES

317

determined following the oxidation of NADH as described by Schmidt (7). Fumarase was estimated by the method of Racker (8). Phospholipids were determined as inorganic phosphate with malaquite green (9) after ashing (10). Protein was measured by the method of Bradford (I 1) using bovine seroalbumin as standard. Bovine neurophysins were assayed using a bovine neurophysin antiserum which recognized vasopressin- and oxytocin-neurophysin at a final dilution 1:20,000. Cytochrome &, was determined by the 429-409 difference in absorbance under oxidized and reduced conditions as described by Zinder et al. (12). Reduced minus-oxidized difference spectra were recorded in a Shimadzu 240 spectrophotometer using a bandwidth of 1 nm. Electron microscopy. Samples were fixed in 4% paraformaldehyde, 2.5% glutaraldehyde in 0.1 M phosphate buffer pH 7.4 for 4 h, postfixed in osmium tetroxide and embedded in Epon-Araldite mixture. Sections were examined in a Phillips EM 300. RESULTS

The distributions of protein, enzymic activities, and neurophysin immunoreactivities in subcellular fractions separated by differential centrifugation are given in Table 1. This shows that fraction 3 contains 6% of the mitochondria, 11% of the lysosomes, and 20% of the neurosecretory granules. This fraction was applied on a discontinuous sucrose gradient as described by Dean and Hope. The distributions of the protein, enzymic activities, and neurophysin immunoreactivities among the different fractions obtained from the density gradient are shown in Fig. 2. Neurophysins showed the characteristic bimodal distribution in this type of gradient ( 13). One peak of immunoreactive neurophysins was found at a density of 1.22 g/ml and the other at 1.17 g/ml. The denser band formed by fractions 3, 4, and 5, which were taken as the granule preparation for further purification of membranes, contained 45% of the immunoreactive ’ Abbreviations used: SDS, sodium dodecylsulfate; S, neurophysins and only 4 and 8% of the total supematant; SF, subfractions.

318

GONZALEZ

ET AL.

TABLE 1 DISTRIBUTION

OF ACTIVITIES

IN SUECELLULAR FRACTIONS OBTAINED OF HOMOGENATES OF BOVINE NEURAL

BY DIFFERENTIAL LUBES

CENTRIFIJGATION

Percentage of recovered activity Fraction Protein (6) Neurophysins (3) Glutamate dehydrogenase (4) Fumarase (3) Acid phosphatase (4)

PI 12.82 11.41 11.48 10.02 22.19

+ 1.8 + 2.9 If: 4.3 * 3.1 + 3.5

p3

p2

16.11 + 17.35 + 30.78 t 25.62 k 23.89 +

2.1 3.2 8.9 6.5 4.9

9.16 19.75 5.81 4.72 10.78

Recovery

s3

+ 0.6 * 2.1 k 0.7 zk 2.9 +- 2.4

61.26 54.37 51.89 59.36 43.08

+ 3.9 3~ 6.7 + 10.5 -+ 8.1 I? 1.2

118.7 95.1 110.0 88.7 91.29

+ * rt + f

12.2 7.3 11.2 6.3 5.6

Note. Values are given as means + SD. The numbers of experiments are given in parentheses.

acid phosphatase and glutamate dehydrogenase activities recovered in the gradient, respectively. The acid phosphatase and glutamate dehydrogenase activities represented 0.44 and 0.48% of that present in the homogenate. The electron microscope showed in this preparation granules, some mitochondria, and membranes which might represent lysed granules (Fig. 3a). The neurosecretory granules have an average diameter of 169 f 12 nm which is in good agreement with the reported values for granules isolated in hypertonic (2) and in isotonic media (3). These purified granules were lysed by repeated suspension in hypotonic solutions obtaining a crude membrane preparation and a supematant (S) which contained the extractable proteins of the granule preparation. The crude membrane preparation was further purified in a density sucrose gradient yielding subfractions 1, 2, and 3 (SFl, SF2, and SF3). An evaluation of the effectiveness of the lysis and subfractionation procedure is shown in Table 2. The majority of the proteins were recovered in the supernatant whereas membrane subfractions SF 1, SF2, and SF3 collectively represented about 13% of proteins of the starting material. Conversely, most of the phospholipids were found in SF1 and SF2: 41.9 and 49% (Table 2) of the neurosecretory granule preparation giving phospholipids to protein ratios of 0.42 and 1.39, respectively. Subfraction SF3 and the supematant S contained only a small amount

of phospholipids. Most of the neurophysins were found in S and a very small amount in membrane subfractions SF1 and SF2. The 68.7% of cytochrome bS6,, thought to be an integral membrane protein of the neurosecretory granule, was found in SF2 (Table 2). Half

50 40 30 20 10

“:4 M Protein

H .I r -

1

20

10

z 4 t 5a 6 7

Nwrophysins n:2

20

H 3

x

10 m

5 Fraction

10 number

Frmctlon

numb.r

FIG. 2. Distribution of marker enzymes, immunoreao tive neurophysins, and protein on a discontinuous density gradient. Fraction P3 obtained by ditTerential centrif@ation was layered on a discontinuous sucrose gradient and centrifuged at 48,000g for 90 min. Activities are.given as percentages of the total amounts applied on the gradient.

NEUROSECRETORY

GRANULE

319

MEMBRANES

RG. 3. Electron micrographs of neurosecretory granules (a) isolated by sucrose density gradient centrifugation, membrane subfractions SF1 (b) and SF2 (c) obtained after repurification of the crude membrane preparation on a sucrose gradient. Magnifications (a) X8277, (b) X 10044 and (c) X10416.

of the glutamate dehydrogenase activity was recovered in SF1 whereas only a small proportion was found in SF2. The specific activity of cytochrome b561in SF2 was 1.4 times higher than that of the crude neurosecretory granule membrane (Table 3). An important decrease in the specific activity of glutamate dehydrogenase was observed in SF2 over the crude granule membranes (Table 3). At the electron microscope level SF1 consisted of membrane vesicles and some whole

granules, and mainly of double membrane structures still recognizable as mitochondria (Fig. 3b). SF2 was composed almost entirely by membrane vesicles devoid of content (Fig. 3~). Analysis of subfractions by polyacrylamide electrophoresis showed that SF1 has a complex polypeptide composition and is somewhat different from SF2. The latter contained fewer proteins than the former and most ranged between 1OOK and 25K. Some polypeptides of

TABLE 2 ASSESSMENTOFTHELYSISANDSLJBFRACTIONATION

PROCEDURES

Percentage of starting material Subfraction

SF1

SF2

SF3

S

Protein (6) Phosphohpids (4) Neurophysins (2) Cytochrome br6, (2) Glutamate dehydrogenase (3)

7.0 f 1.3 41.9 f 2.6 0.28 20.2 52.4 + 3.6

5.2 + 0.6 49.0 f 2.9 0.27 68.7 8.9 f 1.2

1.2 -t 0.3 2.9 + 0.4 8.0 13.8 -+ 2.7

83.6 + 4.3 6.1 + 1.3 99.6 2.3 24.6 f 4.3

Note. Values are given as means -t SD. The numbers of experiments are given in parentheses.

320

GONZALEZ

ET AL.

TABLE 3 SPECIFICACTIVITIES OF MARKERS AT DIFFERENT Neurophysins Granule fraction Crude granule membranes Purified granule membranes (SF2)

368k58' 23.6 37.3

STAGES OF PURIFICATION

Cytochrome bs6,

Glutamate dehydrogenase

0.027' 0.061 0.083

0.51' 0.22 0.018

’ gg Neurophysins I and II/mg protein * A A 429 (reduced-oxidized)/mg protein. ’ pmol NADH/min/mg protein.

proteins from the membrane proteins; and second, the decontamination of the membrane subfraction from membranes of other cellular fractions ( 14). The repeated suspension in hypotonic solution was used to lyse and remove the content from the granule membrane. This treatment has proved to be effective in removing chromogranin A and catecholamines from chroDISCUSSION maffin granules (15) and vasopressin from boThe main problems in the separation of vine neurosecretory granules (16). We found membrane and content of secretory granules that neurophysins were also almost completely are, first, the complete removal of the secretory removed from the membrane subfraction of neurosecretory granules. The two main mema b c d brane subfractions SF1 and SF2 collectively M&J3 contained 0.55% of the total neurophysins of the starting neurosecretory granule preparation. However, enzymes seem to be more dif115 96 ficult to remove from membranes than secretory proteins. In preliminary experiments, we found that the same treatment was able to remove only 70% of the acid phosphatase activity from lysosomes isolated from bovine neural 43 lobes (unpublished results). Similarly, the dopamine /3-hydroxylase activity was only partially removed (50%) from chromaffin granule membranes whereas chromagranin A was completely removed under the same conditions (15). 14 Another source of contamination of granule membrane preparations is membranes of FIG. 4. SDS-polyacrylamide gel electrophoresis analysis other cellular fractions. The mitochondrial of the supematant (a), subfraction SF2 (b), subfraction contamination of our granule preparation, as SF1 (c). Approximately 30 pg of proteins each lane. Mojudged by the enzymic activity, was 0.48% of lecular weight markers (d) were: &galactosidase, phosphorylase B, bovine seroalbumin, ovoalbumin, and ti- the initial activity in the homogenate. This degree of contamination is as low as or lower bonuclease A.

SF1 were not present or were minor components of SF1 . The main protein of the supernatant, a 12K polypeptide, was neurophysin as determined by immunoblotting (not shown). Most of the other proteins of this subfraction had apparent molecular weights between 70K and 30K (Fig. 4).

NEUROSECRETORY

GRANULE

than that of available data (2-4). However, when isolating membranes of secretory granules, this contamination becomes relevant because on one hand, the membrane proteins represent only a small percentage of the total proteins of the granule (14,19,20), and on the other, the amount of membrane per volume of the mitochondrion is relatively high. The decontamination was facilitated by the difference in density of the granule and the mitochondrial membranes (17). The crude membrane preparation was resolved into two main subfractions (SF1 and SF2) and a third minor one (SF3). Cytochrome bsbl was used as a marker for the neurosecretory granule membrane ( 18). The recovery of most of the cytochrome bs6, from the crude membrane fraction in SF2 suggests that this subfraction contains the neurosecretory granule membranes. This suggestion is supported by the observation at the electron microscope level of empty vesicles in this subfraction. Subfraction SF1 contained lysed or partially lysed mitochondria which shows an important mitochondrial contamination of the neurosecretory granule membranes. This subfraction represented 7.0% of the total proteins of the starting material but its contribution to the membrane proteins was as much as 50%, in spite of the low contamination of the granule fraction by mitochondria; 0.48% of the activity in the homogenate. Similar proportions of contamination of membrane proteins by proteins of other subcellular fractions, especially mitochondrial, were observed in secretory granules of the parotid gland. In fact, secretory granules containing 0.2% of the cytochrome oxidase activity of the homogenate yield membranes whose contamination by mitochondrial proteins was estimated to be about 15% (14,19). Analysis of the subfractions on SDS-polyacrylamide gel showed that SF1 and SF2 contain different polypeptide compositions, and there seems to be no cross contamination of SF2 and SF1 since some of the main proteins of SFl, such as a 15,000 mol wt polypeptide, are not present in SF2 and vice versa; a 67,000

MEMBRANES

321

mol wt protein of SF2 is almost absent from SF1 . These differences might be explained by differential proteolitic digestion of these polypeptides during the isolation procedure. The granule membrane subfraction SF2 represented 5.2% of the total proteins of the granule fraction. This percentage falls within the range found in granules of other cells ( 14,19,20). The membrane proteins of SF2 were resolved by SDS-gel electrophoresis into 25 to 30 bands. Comparison of the SDS-gel patterns given by secretory granule membranes from a variety of cells shows that membranes of different granules possess a somewhat distinct polypeptide composition ( 12,15,19,20). Even different types of granules within the same cell seem to have particular sets of proteins (20). However, some proteins have been identified in the membrane of more than one type of granule, such as cytochrome b561( 18,2 I ,22). In addition, common antigenic determinants have been demonstrated in vesicles of neuronal and endocrine cells (23,24). Antibodies raised against some of the proteins of SF2, such as a 67,000 and a 120,000 mol wt protein, selectively stain the neurons of the hypothalamo-neurohypophysial system by immunocytochemistry (Caorsi and Gonzalez, manuscript in preparation). This result suggests that some of the proteins of the neurosecretory granule membrane might be specific to this system. By using a density sucrose gradient to repurify membranes of hypotonically lysed granules, we have been able to obtain highly purified neurosecretory granule membranes. The polypeptide composition of these membranes has been determined and the main proteins have been identified. These results will allow studies on aspects of formation and flow of granule membranes as well as the involvement of some of their components in the interaction with other subcellular structures. ACKNOWLEDGMENTS We thank Mr. Luis Delannoy for technical assistance. This work was supported by Grant RS 83-44 from the Direccidn de Invest&&n y Desarrollo de la Universidad Austral de Chile.

322

GONZALEZ

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