Isolation and characterization of an enriched Golgi fraction from rat brain

284

Biochimica et Biophysica Acta, 542 (1978) 284--295 © Elsevier/North-Holland Biomedical Press

BBA 28600 ISOLATION AND C H A R A C T E R I Z A T I O N OF AN ENRICHED GOLGI F R A C T I O N FROM R A T BRAIN

DIWAKAR S. DESHMUKH, WILLIAM D. BEAR and DAVID SOIFER Institute for Basic Research in Mental Retardation, 1050 Forest Hill Road, Staten Island, N.Y. 10314 (U.S.A.)

(Received December 14th, 1977) (Revised manuscript received March 17th, 1978)

Summary A fraction rich in membranes of the Golgi apparatus was isolated from rat brain by discontinuous density gradient centrifugation. The fraction sedimented at the characteristic Golgi density of 1.11--1.15 (g/cm ~, 5°C) and had specific activities of Golgi-marker enzymes (N-acetyllactosaminyl synthetase, glycoprotein (Fetuin) galactosyltransferase, thiamine pyrophosphatase), 6--7 times over those of the original homogenates. The recovery of the enzyme activities in this fraction ranged from 17 to 31%. The incorporation [3H]fucose into glycoproteins was 3-fold higher than in homogenate. Recovery and relative specific activities of marker enzymes for other subcellular organelles were low. Electron microscopic analysis of the fraction revealed the presence of Golgi structures, namely, large sacs or plates with attached tubules and "blebbing" of the tubules into the vesicles.

Introduction The isolation of Golgi apparatus from liver and other tissues has yielded valuable information regarding the composition and synthesis of components [1--3]. In particular, the glycosyltransferases necessary for the synthesis of glycoproteins and N-acetyllactosamine, and the sulfotransferase-synthesizing sulfatides are predominantly located in the Golgi apparatus isolated from liver and kidney [4--8]. The gangliosides of liver are also synthesized in this organ-

Abbreviations: NAL synthetase, UDPgalaetose-N-acetylglucosaminyl galactosyltransferase; glycoprotein g a l a c t o s y l t r a n s f e r a s e , U D P - g a l a c t o s e - g l y c o p r o t e i n ( F e t u i n ) g a l a c t o s y l t r a n s f e r a s e ; INT, ( 2 - i o d o p h e n y l ) 3 - p - n i t r o p h e n y l - 5 - p h e n y l t c t r a z o l i u m chloride; EDTA, d i s o d i u m e t h y l e n e d i a m i n e t e t r a c e t a t e ; E G T A , ethyleneglycol-bis-(fl-aminoethylether)-N,N'-tetraacetic acid; MES, 2 - ( N - m o r p h o l i n o ) - e t h a n e sulfonic acid; I1EPES, N - 2 - h y d r o x y e t h y l p i p e r a z i n c - N ' - 2 - e t h a n esulfonic acid.

285 elle [9,10]. For the brain, however, no comparable information concerning the role of Golgi is available, although such information would be especially interesting because the different nature of brain glycolipids and glycoproteins requires that the synthetic apparatus must differ from that of other organs in many respects. With the conventional methods of subcellular fractionation of brain, the glycosyltransferases required for the synthesis of gangliosides [11], cerebrosides [12], and glycoproteins [13] have been reported to be localized in synaptosomal fractions and subfractions. However, studies of others [14,15] showed only low levels of glycosyltransferases in synaptosomal fractions. Reith et al. [ 16] suggested that the discrepancies may be explained by contamination of synaptosomal fractions with membranes of Golgi apparatus. Autoradiographic studies have also indicated the Golgi as the site of glycosylation in the liver and brain [17,18]. Shortly after injection, most of radioactive fucose is localized in the Golgi structures of the neuronal cells [17]. Thiamine pyrophosphatase is another Golgi marker and histochemical evidence has indicated the presence of this enzyme in the Golgi apparatus in brain [19,20]. The enrichment of this enzyme activity has also been reported in a Golgi-enriched fraction obtained b y subfractionating synaptosomal preparations [16,21]. Since the conventional procedure of subcellular fractionation of brain did not yield a fraction rich in the Golgi markers, we have tried modifying the published methods [22,23] for the isolation of Golgi from liver. Here we report on the subcellular distribution of Golgi markers and other characteristics of the Golgi-enriched fraction isolated from rat brain. A preliminary report of this work has been presented [24]. Materials

Brains were obtained from 16- to 30-day-old rats purchased from Charles River Breeding Labs (Wilmington, Mass.). UDP-[U-~4C]galactose and L-[6-3H] fucose were obtained from New England Nuclear (Boston, Mass.); fetuin from GIBCO (Grand Island, N.Y.). Fetuin free of sialic acid and galactose (DSGFetuin) was prepared by mild acid hydrolysis followed by Smith degradation according to Kim et al. [25]. Methods

Subcellular fractionation. The procedure for the isolation of fractions rich in Golgi-markers was a modification of the methods described by Schachter et al. [22] and Fleischer [6] for liver Golgi preparations. Four to five brains were chopped into thick slices and suspended in 4 vols. of cold homogenizing medium containing 0.32 M sucrose buffered with 0.05 M HEPES (pH 6.5), 5 mM MgSO4, 25 mM KC1, 2.5 mM EDTA (in some experiments EDTA was replaced b y 2.5 mM EGTA), 14 mM mercaptoethanol and 1% dextran (molecular weight 170 000, Sigma, St. Louis, Mo.). The tissue suspension was left in the cold for 5 min to allow the ingredients of the medium to penetrate the cells and react with the cell constituents. The tissue was then homogenized with 10 gentle up and d o w n strokes of a motor-driven Aldridge homogenizer (800 rev./min) of a

286 clearance o f 0.01 inch [26]. A sample of the h o m o g e n a t e was taken for e n z y m e assays. The molarity o f the sucrose in the hom ogenat e used for fract i o n atio n was adjusted to 0.5 M with the aid of a r e f r a c t o m e t e r (Fisher Scientific Co.). The h o m o g e n a t e was centrifuged in a Sorvall centrifuge with an HB4 swinging b u ck et r o t o r for 10 rain at 9000 ×gmax to obtain the supernatant (Golgi-rich post-mitochondrial supernatant) and the pellet fractions. The pellets (9000 × g • 10 rain) were rehomogenized in 0.5 M sucrose solution (2 volumes o f brain weight) with a glass homogenizer with teflon pestle of 0.026 inch clearance (A.H. Thomas, Philadelphia, Pa.), and then centrifuged as before. The supernatant fractions were combined. A part of the supernatant was removed and its sucrose c o n c e n t r a t i o n was adjusted to 0.32 M from 0.5 M. It was then centrifuged at 17 500 rev./min for 30 min to obtain total membranes of the supernatant fraction (designated as Supernatant in Table II). The rest of the supernatant was used for f ur t he r fractionation as follows. The molarity of the sucrose was adjusted to 0.72 M. 15 ml of the supernatant was layered over a discontinuous gradient consisting of 10 ml of each 1.17, 1.13 and 0.88 M sucrose solutions in 39-ml tubes of a SW-27 (Beckman) rotor. On t op of the supernatant was placed 3--4 ml of 0.32 M sucrose solution. All sucrose solutions contained all the ingredients of the homogenizing medium in the same concentrations. The gradient was centrifuged for 120 min at 25 000 rev./min. This resulted in two distinct bands at the interfaces of 0.72 M/0.88 M and 0.88 M/ 1.13 M, b o th within the range of the characteristic Golgi density of 1.11--1.15 (g/cm 3, 5°C) [4]; these bands and the fraction at the top of 0.72 M and the b o t t o m pellets were collected. No band was obtained at the interface of 1.13 M/1.17 M. Substantial amounts of material were always found at the interface of 0.88 M/1.13 M, while the amounts at the 0.72 M/0.88 M interface were comparatively small and varied greatly from one experi m ent to another. The sucrose solutions of all fractions collected (including the supernatant) were brought to 0.32 M and centrifuged in an angle r o t o r (Sorvall) for 30 rain at 17 500 rev./min. The pelleted fractions were rehomogenized in cold H20 containing 14 mM m er c a pt oe t ha nol , and were used for e n z y m e assays on the same day or distributed over several tubes and stored at --70°C till further use. Subcellular fractionation of brain to prepare the microsome, synaptosomal, mitochondrial, and myelin fractions was done as descibed previously [26,27]. The "large" and "small" myelin fractions isolated by discontinuous sucrose gradient centrifugation of crude nuclear [27] and crude mitochondrial fractions [26] were com bi ne d to yield the total myelin fraction. The nuclear and cell debris fractions [27] were not analyzed. E n z y m e assays. The e n z y m e assays for NAL synthetase and glycoprotein galactosyltransferase were c o n d u c t e d on the day of fractionation. The ot her assays were carried out with frozen fractions thawed on following days. Each assay was p e r f o r m e d at o p t i m u m conditions in regard to time of incubation and protein concentrations. NAL synthetase (N-acetyllactosamine synthetase, EC 2.4.1.-) was assayed as described by Morre [1] and Fleischer et al. [28]. Assay mixtures contained 57 mM MES buffer, pH 7.5, 5 mM MnC12, 25 mM m ercapt oet hanol , 0.11 mM o f UDP-[14C]galactose (1000 d p m / n m ) , 0.46 mM N-acetylglucosamine, 50 pl 6% Triton X-100 solution and 50--200 pg o f protein in a final volume at 0.2

287 ml. Incubations were carried out for 20 min at 37°C. The rest of the procedure was as described in the published methods [1,28]. Glycoprotein (Fetuin) galactosyltransferase was assayed as described by Schachter et al. [22] and Ko and Raghupathy [14]. The assay mixture contained 60 mM MES buffer, pH 7.5, 10 mM MnC12, 15 mM mercaptoethanol, 0.5% Triton X-100, 0.1 mM UDP[14C]galactose (1000 dpm/nM), 1 mg DSG-Fetuin and 50--100 pg of protein in a final volume of 0.2 ml. Incubations were carried out for 20 min at 37°C. Thiamine pyrophosphatase was assayed according to Reith et al. [16]. Inorganic phosphorus released was measured as described by Martin and Doty [29]. Cyclic AMP phosphohydrolase was assayed as previously described [27]. The other enzymes were assayed according to published methods: succinic-INT reductase (EC 1.3.99.1) [1], 5'-nucleotidase (EC 3.1.3.5) [1], NADPH-cytochrome c reductase (EC 1.6.99.1) [30] and (Na++ K~)-dependent ATPase {EC 3.6.1.3) [31]. Electron microscopy. Aliquots of the fraction at the 0.88 M/1.13 M interface were taken before final centrifugation and were appropriately diluted (about 0.5 mg protein/ml) with 0.1 M Tris • HC1 buffer, pH 7.0. A small drop of the sample was placed on a carbon film on a 500 mesh grid. The drop was removed and particles adhering to the carbon were stained with 1% phosphotungstic acid (pH 7 with KOH) [32]. Grids were examined and photographed in a Phillips EM300 electron microscope operating at 80 kV with a 25 pm objective aperture. Analytical techniques. The incorporation of [ 3HI fucose into glycoproteins at 15 min after intracerebral injections was studied according to Sturgess et al. [18]. 5 pCi of [3H]fucose was injected. The protein from the fractions was precipitated with a solution containing 1% phosphotungstic acid and 12% trichloroacetic acid. The precipitate was washed twice, first with 10% trichloroacetic acid solution and then twice with ether/ethanol (3 : 1, v/v). The protein was dried and dissolved in 1 M NaOH. Radioactivity was measured in a Packard scintillation counter. Spectrophotometric assays were performed with a Gilford recording spectrophotometer. Protein was quantitated by the m e t h o d of Lowry et al. [33]. Results

Relative distribution of the activities of enzyme markers was first studied in the fractions prepared by conventional methods of subcellular fractionation. Because the absolute concentrations of the markers varied considerably from one animal to another, and the sum of the concentrations of the markers in the various subcellular fractions was often significantly less than that found in the homogenates, the distribution of the enzyme activities is expressed as percentage and is corrected to 100% recovery. The absolute recoveries are also given. The results of the distribution of the markers in the conventional brain fractions are shown in Table I. Microsomes had the highest relative specific activities, more than 50%, of Golgi-specific enzyme s. This was also true for the in vivo incorporation of [3H] fucose into the glycoproteins. Among the other fractions, synaptosomes displayed significantly high activities of Golgi-marker

288 TABLE I D I S T R I B U T I O N O F G O L G I M A R K E R S IN T H E S U B C E L L U L A R BY CONVENTIONAL METHODS OF FRACTIONATION The

fractions were prepared

and assayed

FRACTIONS

OF BRAIN PREPARED

f o r t h e e n z y m e s as d e s c r i b e d in M e t h o d s . T h e s y n a p t o s o m a l ,

m i t o c h o n d r i a l and t h e m y e l i n f r a c t i o n s were w a s h e d t w i c e by s u s p e n s i o n in 0 . 3 2 M sucrose s o l u t i o n a n d e e n t r i f u g a t i o n . V a l u e s i n t h i s t a b l e are a v e r a g e s o f t w o s e p a r a t e e x p e r i m e n t s . H o m o g e n a t e s p e c i f i c a c t i v i ties were: NAL synthetase, 2.99 nm per mg protein per h; glycoprotein galactosyltransferase, 0.94 nm p e r nag p r o t e i n p e r h ; t h i a m i n e p y r o p h o s p h a t a s e , 0 . 5 p m o l p e r m g p r o t e i n p e r h ; i n c o r p o r a t i o n o f [ 3 H ] fucose, 5 1 0 d p m per m g p r o t e i n per 15 rain; cyclic A M P p h o s p h o h y d r a s e , 2.44 t t m o l per m g p r o t e i n p e r r a i n ; 5 ' - n u e l e o t i d a s e , 0 . 0 3 5 i t m o l p e r m g p r o t e i n p e r r a i n ; (Na + + K * ) - A T P a s e , 2 . 6 5 p m o l p e r m g p r o tein per h; N A D P H - e y t o c h r o m e c reduetase, 0.55 pmol per mg protein per min;succinic INTreduetase, 0.724 AA per mg protein per min. RSA; relative specific activity = specific activity of the fraction/specific activity of homogenate. Markers

NAL synthetase G l y c o p r o t e i n galactosyl-transferase Thiamine pyrophosphatase Incorporation of [3HJfucose Cyclic AMP phosphohydrolase (Na ÷ + K+)-ATPase (ouabain sensitive) NADPH-cycochrome c reductase 5'-Nucleotidase Succinic INT reductase

Fractions Microsomal

SynaptosomaI

Mitochondrial

Myelin

RSA

% *

RSA

2.1 2.3

60 56

2.4

Recovery (%) * *

% ~

RSA

% *

RSA

% *

1.7 1.56

26 27.5

0.25 0.2

12 13.0

0.13 0.18

2.3 3.5

47 46

55.4

1.92

28

0.56

13.0

0.22

3.8

53

1.2

54

0.46

23

0.2

15

0.28

8

30

1.0

21

n.d.

n.d.

--

6.8

79

63

1.5

37

2.2

41

0.5

14

0.32

8.0

35

3.6

64

0.56

14.6

0.43

21.4

Nil

Nil

75

1.7 0.2

37 2.7

0.92 1.3

11.7 23.8

0.77 6.6

17 72

1.6 0.16

27 1.5

60 66

* P e r c e n t o f t o t a l a c t i v i t y i n all t h e f r a c t i o n s a n a l y s e d . ** P e r c e n t a c t i v i t y r e c o v e r e d i n all t h e f r a c t i o n s f r o m t o t a l h o m o g e n a t e s . n.d., not determined.

enzymes though not as high as reported by others [16]. The mitochondrial and the myelin fractions had low activities. In general, the relative distribution of Golgi markers in the fractions agrees with the published reports [14,16,20]. The enrichment of other enzyme activities in the respective subcellular fraction was as expected, i.e. cyclic AMP phosphohydrolase in myelin; (Na*+ K*)ATPase in synaptosomes and microsomes; NADPH-cytochrome c reductase in microsomes; succinic-INT reductase in mitochondria; and 5'-nucleotidase in microsomes and myelin. Attempts to prepare a fraction rich in Golgi markers by subfractionating the microsomal fraction with the use of discontinuous density gradient centrifugation were unsuccessful. Modifications in the method of homogenization of brain were, therefore, used to prepare a Golgi-rich post-mitochondrial supernatant. The concentrations of the various markers in the supernatant are shown in Table II. Only about 10% of the total homogenate protein was recovered in the membranes (sedimented at 17 500 rev./min in Sorvall from 0.32 M sucrose)

289 TABLE

II

CONCENTRATIONS OF 'MARKERS' IN THE ARED BY DIFFERENTIAL CENTRIFUGATION

SUPERNATANT

AND

PELLET

FRACTIONS

PREP-

T h e e x p e r i m e n t a l c o n d i t i o n s for t h e p r e p a r a t i o n o f G o l g i m a r k e r - e n r i c h e d s u p e r n a t a n t w e r e as d e s c r i b e d in M e t h o d s . A s s a y p r o c e d u r e s , s p e c i f i c a c t i v i t i e s o f t o t a l h o m o g e n a t e s a n d d e f i n i t i o n o f R S A w e r e s a m e as T a b l e I. E a c h v a l u e is average o f 3 - - 8 e x p e r i m e n t s -+S.E. Markers

Fractions Supernatant ***

Pellets T

Recovery (%) **

RSA

% *

RSA

% *

NALsynthetase

3.2

+ 0.19

37 + 2.1

0.71 ± 0.02

63 ± 2.1

69

Thiaminepyrophosphatase Glycoproteingalactosyl-transferase

3.4

± 0.38

47 + 3.8

0.59 ± 0.44

5 3 -+ 3 . 8

64

2.8

± 0.32

35 ± 6.4

0.71 ± 0.05

65 ± 6.4

62

1.8

+ 0.18

17 ± 3.7

0.67 ± 0.23

53 ± 3.8

61

0.7

± 0.07

13 ± 4.9

1.31 ± 0.18

8 7 -+ 4 . 9

94

0 . 7 8 -+ 0 . 0 5

15 ± 2.9

1.14 ± 0.18

85 ± 2.9

75

0.7

12 ± 2.1

1.77 ± 0.69

88 ± 2.1

75

2.73 ± 0.22

22 ± 7.5

0.94 ± 0.05

68 ± 4.7

49

0 . 9 5 -+ 0 . 2 8

9 ± 2.4

1.77 ± 0.48

91 ± 2.4

84

[3H]Fucose

incorporation Cyclic AMPphosphohydrolase 5'-Nucleotidase (Na + + K÷)-dePendent ATPase (ouabain

± 0.1

sensitive) NADPH

cytochrome

reductase Succinic INT reductase

c

* P e r c e n t o f a c t i v i t i e s in all t h e f r a c t i o n s . ** P e r c e n t o f a c t i v i t i e s r e c o v e r e d in f r a c t i o n s f r o m t o t a l h o m o g e n a t e s . *** Golgi-rich post-mitochondrial supernatant. T 9000

× g, 1 0 min pellet fraction.

of the supernatant fraction. The relative specific activities of the Golgi specific enzymes in this fraction were consistently higher than those of the microsomes (Table I), while the relative specific activity of the other enzyme markers were considerably lower, with the exception of succinic-INT reductase. However, under these conditions of homogenization only up to about 40% of the Golgi marker activities of the total homogenate could be recovered in the supernatant. Attempts to increase the yield of Golgi markers by repeated homogenization of the pellets resulted in high activities of the non-Golgi markers and a reduction in the relative specific activity of the Golgi markers of the supernatant. The distribution of the Golgi markers in the fractions prepared by discontinuous sucrose density gradient centrifugation of the supernatant are shown in Table III. A clean separation of the fractions on the gradient was only achieved by directly loading the supernatant on the gradient after adjusting its sucrose concentration to 0.72 M, not by sedimenting the membranes from the supernatant prior to the gradient centrifugation. The presence of the ingredients of the medium in the sucrose solutions was also essential. A m o n g the four gradient fractions the membranes sedimenting at the 0.88 M/1.13 M interface had the highest specific activities of Golgi specific enzymes, about 6--7-fold

290 TABLE

III

CONCENTRATIONS

OF GOLGI

The experimental conditions s e p a r a t e e x p e r i m e n t s -+S.E. Markers

MARKERS

IN THE SUCROSE

were as described

in Methods

DENSITY

GRADIENT

FRACTIONS

a n d T a b l e I. E a c h v a l u e is a n a v e r a g e o f 3 ~ 7

Fractions

Thiamine pyrophosphatase [3H]Fucose incorporation NAL synthetase Glycoprotein galactosyl transferase

0.32 M/0.72 M

0.72 M/0.88 M

0.88 M/1.13 M

Bottom

pellets

Recovery ( % ) **

RSA

% *

RSA

% *

RSA

%*

RSA

%*

0.59 ±0.068 0.69 +0.10 0.9 +0.16 1.4 +0.46

7.9 ÷2.25 12.8 _+1.62 11.4 _+1.41 16.9 +2.33

3.37 -+0.55 2.8 -+0.77 5.3 -+1.4 4.61 +0.85

13.5 -+2.3 16.3 -+0.83 23.5 -+2.41 18.2 -+1.86

7.15 -+0.8 3.11 -+0.43 5.93 _+0.07 5.91 +0.93

66.4 +9.01 57.9 -+7.2 45.6 +4.5 53 -+3.58

0.61 -+0.06 0.39 _+0.11 0.77 -+0.088 0.49 z0.0S

12.2 -+3.25 13.1 +1.8 19.5 -+1.92 11.9 _+2.7

80.1 71.2 74.5 84.6

* P e r c e n t o f a c t i v i t y i n all t h e f r a c t i o n s . ** P e r c e n t o f a c t i v i t i e s i n r e c o v e r e d f r a c t i o n s f r o m t h e s u p e r n a t a n t .

enrichment over the homogenate. In vivo [3H]fucose incorporation into the protein of this fraction was 3-fold over the homogenate and about twice of that of the supernatant. Up to 60% of the total activities of the markers in the supernatant was localized in this fraction. The estimated total homogenate activities recovered in this fraction were 17% for NAL synthetase, 19% for glycoprotein galactosyltransferase, and 31% for thiamine pyrophosphatase. The only other fraction with substantially high relative specific activity of Golgi markers was found at the 0.72 M/0.88 M interface. The recovery of the mem-

TABLE

IV

CONCENTRATIONS OF GRADIENT FRACTIONS Experimental

conditions

Markers

THE

OTHER

NADPH-cytochrome c reductase Succinic INT reductase (Na + + K+)-dependent ATPase

MARKERS

were as described in Table III. For definition

IN THE

SUCROSE

DENSITY

o f R S A s e e T a b l e I.

Fractions 0.32 M/0.72

Cyclic AMP phosphohydrolase 5'-Nucleotidase

SUBCELLULAR

M

0.72 M/0.88

M

0.88 M/1.13

M

Bottom

pellets

RSA

% *

RSA

% *

RSA

% *

RSA

% *

2.1 +0.35 0.69 _+0.18 1.18 -+0.073 0.08 _+0.022 0.32 _+0.015

58 -+1.8 33.7 _+4.5 34.2 _+3.92 3.5 _+1.43 17.4 _+0.82

1.18 ±0.33 1.38 -+0.14 0.29 ±0.072 0.19 ±0.10 0.59 -+0.19

22 -+2.1 13.8 -+2.22 0.83 _+0.2 3.6 -+1.47 14.6 -+1.6

0.4 -+0.08 1.03 -+0.18 1.07 ±0.09 0.47 -+0.078 0.6 -+0.16

12 -+3.05 28.9 -+2.85 35.8 ±2.91 9.1 +1.81 18.7 ±4.63

0.42 -+0.2 0.34 -+0.038 0.89 -+0.048 1.62 -+0.10 0.95 -+0.12

8.3 ±1.8 23.5 -+3.5 29.2 -+1.25 83.9 ±3.9 49.3 -+4.8

* P e r c e n t o f a c t i v i t y i n all t h e f r a c t i o n s . ** Percent of activities in recovered fractions from the supernatant.

Recov. ery ( % ) ** 71 59 60 63 61

291

F i g . 1. E l e c t r o n m i c r o g r a p h o f m a t e r i a l f r o m i n t e r f a c e b e t w e e n 0 . 8 8 a n d 1 . 1 3 M s u c r o s e . T h i s c l u m p o f c i s t e r n a e a n d t u b u l a e is t y p i c a l o f t h e s t r u c t u r e s s e e n o n t h e g r i d . B a r i n d i c a t e s 5 0 0 rim.

brane proteins in this fraction was comparatively low and extremely variable from one experiment to another. This fraction also showed considerably high specific activities of cyclic AMP phosphohydrolase and 5'-nucleotidase, indicat-

292

F i g . 2. H i g h e r p o w e r m i e r o g r a p h f r o m a n o t h e r a g g r e g a t e o f t u b u l a e a n d c i s t e r n a e . T h e t u b u l a e o f t e n a p p e a r t o b e c o n t i n u o u s w i t h c i s t e r n a e o r sacs. S o m e t u b u l a e s h o w c o n s t r i c t i o n s s u g g e s t i n g t h e f o r m a t i o n (by b l e b b i n g ) o f vesicles. Bar i n d i c a t e s 500 n m .

F i g . 3. A t y p i c a l " p l a t e " f r o m a 0 . 8 8 M / 1 . 1 3 M s u c r o s e i n t e r f a c e . B a r i n d i c a t e s 5 0 0 n m .

293

ing heavy contamination with myelin and plasma membranes (Table IV). The distribution of the other subcellular markers in the sucrose gradient fractions (Table IV) showed that the myelin-specific marker was predominantly located in the two t o p fractions, and that of the mitochondrial membrane was present predominantly in the pellets. The percent of the total supernatant activities recovered, as well as the relative specific activity of marker enzymes for the other subcellular organelles, were very low in the fraction at the 0.88 M/1.13 M interface, except for 5'-nucleotidase and NADPH-cytochrome c reductase. The estimated total homogenate activities of the last two enzymes recovered in this fraction were 4.35% for 5'-nucleotidase and 7.88% for NADPH-cytochrome c reductase.

Electron microscopy Negative staining of fractions of rat-liver Golgi produces images that appear to be diagnostic of the Golgi fractions [2]. Examination of negatively stained preparations of the rat-brain fraction collected at the 0.88 M/1.13 M interface showed the various types of structures characteristic of Golgi preparations (Fig. 1). Predominant among these were tubular cisternae, large sacs or plates with atttached tubules (Fig. 1 and 2), and flattened discoid sacs (Fig. 3). The tubular structures often showed constrictions suggestive of a blebbing of the tubules into vesicles (Figs. 1 and 2). Discussion

Morphological studies have well d o c u m e n t e d that the Golgi apparatus of mammalian cells is closely associated with the endoplasmic reticulum [34]. In preparing subcellular fractions by the classical methods the membranes of the Golgi apparatus are sedimented in the microsomal fractions and this fraction has been a suitable starting material for the isolation of purified Golgi membrane for liver, kidney and other tissues [4--6,35]. However, in the case of brain this approach has not been successful. The presence of myelin, synaptosomes and their subfractions complicates the fractionation procedures. We have, therefore, modified conditions of homogenization of brain and centrifugation to obtain a suitable starting material (the supernatant, Table II) with significant enrichment of all four Golgi markers and relatively low concentrations of the other subcellular markers. The most important aspects are the nature of the suspension medium, tissue concentration, the initial homogenization of the tissue, and centrifugation. Optimal concentrations of MgSO4, KC1, dextran, mercaptoethanol and sucrose were introduced in the homogenizing medium. As pointed out by others [23], 1% dextran, 20% (w/v) tissue concentration and mild homogenization are useful to preserve the structure of Golgi apparatus, even though a sizable portion of homogenate proteins is lost (unbroken cells, etc.). Mercaptoethanol is helpful to protect Golgi-specific enzymes [6]. The presence of 5 mM MgSO4 and 25 mM KC1 are useful in causing clumping of non-Golgi membranes [36]. Under these conditions myelin vesicles were probably not formed and myelin and synaptosomes appeared to have sedimented at relatively low speed of centrifugation (9000 X g, 10 min), as indicated b y the low relative specific activity of the enzymes cyclic AMP phos-

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phohydrolase, (Na++ K* )-dependent ATPase, 5'-nucleotidase and succinic-INT redutase in the supernatant (Table II). Discontinuous sucrose density gradient centrifugation of the supernatant resulted in a fraction, at the interface between 0.88 and 1.13 M sucrose, which by both morphologic and enzymatic criteria appeared to be enriched in elements of Golgi apparatus. The specific activities of Golgi-marker enzymes in this fraction were 6--7 times that of the original homogenates. The recovery of the enzymic activities ranged from 17 to 31%. The incorporation of [~H]fucose after 15 min of injection (indicator of the site of glycoprotein biosynthesis [32]) was 3-fold over the homogenate, and about 10% of the radioactivity incorporated into total glycoproteins of homogenates were recovered in this fraction. On the basis of these results, supported by the morphological characteristics, the fraction appears to be rich in the Golgi membranes. As for purity, the fraction seems to be contaminated with plasma membrane to an extent of only 4% on the basis of 5'-nucleotidase. On the other hand, contamination with smooth endoplasmic reticulum appears to be more significant as judged by NADPH-cytochrome c reductase. It is possible, however, that these two enzymes may be true components of the Golgi apparatus of brain, as demonstrated for the Golgi of liver [2,37]. The total homogenate activities recovered, as well as the relative specific activity of the marker enzymes for the other subcellular organelles, were very low, suggesting that the fraction is reasonably free of mitochondria, endoplasmic reticulum, myelin and cellular and synaptic plasma membranes. The procedure described here represents a relatively simple means whereby elements of Golgi apparatus of brain can be isolated with fairly good yield and purity. Acknowledgements We thank Dr. H. Brockerhoff for his advice and suggestions in relation to this work. We are also thankful to Mr. L. Black for his help in bibliographic search of the literature. References 1 Morre, D.J. ( 1 9 7 1 ) in M e t h o d s in E n z y m o l o g y ( J a k o b y , W.B., ed.), Vol. X X I I , pp. 1 3 0 - - 1 4 8 , Acad e m i c Press, N e w Y o r k 2 Morre, D.J., K e e n a n , T.W. and M o l l e n h a u e r , H.H. ( 1 9 7 1 ) in A d v a n c e s in C y t o p h a r m a c o l o g y (Clem e n t i , F. a n d Ceccarelli, B., eds.), Vol. I, pp. 1 5 9 - - 1 8 2 , R a v e n Press, N e w Y o r k 3 Morre, D.J., F r a n k e , W.W., D e u m l i n g , B., N y q u i s t , S.E. a n d O v t r a c h t , L. ( 1 9 7 1 ) in B i o m e m b r a n e s ( M a n s o n , L . A . , ed.), Vol. 2, pp. 9 5 - - 1 0 4 , P l e n u m Press, N e w Y o r k 4 E h r c n r e i c h , J . H . , B e r g e r o n , J . J . M . , Siekevitz, P. a n d Palade, G.E. ( 1 9 7 3 ) J. Cell Biol. 59, 4 5 - 7 2 5 B e r g e r o n , J.J.M., E h r e n r e i c h , J . H . , Siekevitz, P. a n d Palade, G.E. ( 1 9 7 3 ) J. Cell Biol. 59, 7 3 - - 8 8 6 Fleischer, B. ( 1 9 7 4 ) in M e t h o d s in E n z y m o l o g y , B i o m e m b r a n e s Part A (Fleischcr, S. and Packer, L., eds.), Vol. X X X I , pp. 1 8 0 - - 1 9 1 , A c a d e m i c Press, N e w Y o r k 7 Fleischer, B. a n d Z a m b r a n o , F. ( 1 9 7 4 ) J. Biol. C h e m . 249, 5 9 9 5 - - 6 0 0 3 8 Wagner, R.R. a n d C y n k i n , M.A. ( 1 9 7 1 ) J . Biol. C h e m . 246, 1 4 3 - - 1 5 1 9 K e e n a n , T.W., Morre, D.J. a n d Basu, S. ( 1 9 7 4 ) J. Biol. C h e m . 249, 3 1 0 - - 3 1 5 10 Wilkinson, F.E., Morre, D.J. a n d K e e n a n , T.W. ( 1 9 7 6 ) J. Lipid Res. 17, 1 4 6 - - 1 5 1 11 Den, M., K a u f m a n , B. a n d R o s e m a n , S. ( 1 9 7 0 ) J . Biol. C h e m . 245, 6 6 0 7 - - 6 6 1 5 12 Basu, S., S c h u t z , A.M., Basu, M. a n d R o s e m a n , S. ( 1 9 7 1 ) J. Biol. C h e m . 246, 4 2 7 2 - - 4 2 7 9 13 B o s m a n n , H.B. ( 1 9 7 2 ) J. N e u r o c h e m . 19, 7 6 3 - - 7 7 8 14 K o , G.K.W. a n d R a g h u p a t h y , E. ( 1 9 7 2 ) B i o c h i m . Biophys. A c t a 286, 3 3 9 - - 3 4 6

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