Effect of acute ethanolic intoxication on the neuraminidase activity of rat liver golgi apparatus

101

Biochimica Biophysica Acta, 497 (1977) 101--111 © Elsevier/North-HollandBiomedicalPress

BBA 28199 EFFECT OF ACUTE ETHANOLIC INTOXICATION ON THE NEURAMINIDASE ACTIVITY OF RAT LIVER GOLGI APPARATUS

GOLLAMUDIS. KISHORE and RAOUL CARUBELLI Biomembrane Research Laboratory, Oklahoma Medical Research Foundation Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City, Okla. 73104 (U.S.A.)

(Received August 9th, 1976)

Summary Neuraminidase and galactosyltransferase were investigated in total Golgi apparatus and in the three fractions of increasing densities (GF,, GF2, and GF3) isolated from the microsomal fraction of rat liver homogenates by flotation in a discontinuous sucrose density gradient (Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45--72). About 50% decreases in neuraminidase content (units/g liver) and specific activity (units/ mg protein) were observed in total Golgi as well as in the three fractions isolated at 45 min, 90 min, 180 min and 16 h after administration of a single oral dose of 50% aqueous ethanol (0.6 g/100 g body weight). Colchicine administration (intraperitoneal injection, 0.5 mg/100 g body weight) caused a similar loss of neuraminidase activity; however, the effect of ethanol plus colchicine was not additive. Golgi galactosyltransferase, on the other hand, experienced marked increases of activity following ethanol administration but, unlike the results reported by others (Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 243, 123--125), significant increases in total activity and specific activity were already quite evident at 90 min after ethanol ingestion. In contrast with the decreased values observed i n Golgi, the total particle-bound neuraminidase was significantly elevated following ethanol administration. Ultrastructural studies revealed increased lysosomal content and detachment of polysomes from the rough endoplasmic reticulum. A model, which takes into account these enzymological and ultrastructural findings and their biological significance, is proposed.

Introduction Rat liver contains soluble neuraminidase in the cytosol [1], while particlebound neuraminidases have been detected in lysosomes [ 1--4], in plasma mem-

102

brane [5,6] and more recently in Golgi apparatus [7]. Acute ethanolic intoxication of rats leads to marked biochemical changes in hepatic Golgi which are manifested by an increase in galactosyltransferase activity [8] and a decrease in the b u o y a n t density of the Golgi apparatus caused by accumulation of very low density lipoproteins [9]. The latter property has been utilized for the isolation and fractionation of Golgi apparatus in three fractions of increasing density b y flotation in a discontinuous sucrose gradient

[lO]. In this paper we describe the effect of acute ethanolic intoxication on the a m o u n t and distribution of neuraminidase and galactosyltransferase in total hepatic Golgi and in its fractions. Materials and Methods Neuramin-lactose, N-acetylneuraminic acid (type IV) and colchicine were products of Sigma. UDP-['4C]galactose (275 Ci/mol) was obtained from New England Nuclear. Male rats of the Holtzman Strain (180--250 g), fed ad libitum on a commercial pellet diet (Teklad Mills), were used for these studies. The animals were fasted overnight and ethanol, 0.6 g per 100 g b o d y weight, was administered as a 50% (w/v) aqueous solution by gastric intubation. The rats were stunned and killed b y decapitation at 45 min, 90 min, 3 h and 16 h after ethanol administration. The livers were quickly excised and chilled in ice-cold 0.25 M sucrose. For experiments involving the study of the effect of colchicine, the rats received an intraperitoneal injection of a 0.1% aqueous solution of colchicine (0.5 mg/100 g b o d y weight) and the animals were killed after 90 min. For experiments involving the combined effect of colchicine and ethanol, the rats received ethanol 45 rain after colchicine and were killed 45 min after ethanol administration. Pooled liver tissues from four rats were used for each experiment. The subcellular fractionations were conducted at 4°C with a Spinco Model L and with a Beckman Model L5-75 ultracentrifuge equipped with rotors 30, 40, SW 25.1 and SW 27 (Beckman Instruments, Inc.) using a modified version of the techniques described by Ehrenreich et al. [10], and by Bergeron et al. [11]. The microsomal pellets obtained from homogenates in 0.25 M sucrose [10] were resuspended in a b o u t 40 ml of 0.25 M sucrose. The sucrose concentration of one third o f this suspension was then brought up to 1.15 M while the remaining two-thirds were adjusted to 1.3 M by adding 2.0 M sucrose (sucrose concentrations were estimated with a refractometer). For the isolation of total Golgi (Gt) 10-ml aliquots of the microsomal suspension in 1.3 M sucrose were overlaid with 10 ml layers of 1.15 M and 0.25 M sucrose, respectively [11], and centrifuged in a SW 25.1 rotor at 2 5 0 0 0 rev./min for 250 rain. The Gt band formed at the 0.25 M/1.15 M sucrose interface was harvested with a Pasteur pipet, sucrose concentration was adjusted to 0.25 M by dilution with distilled water and the Gt fraction was pelleted by centrifuging at 1 0 5 0 0 0 X g for 30 min. 9-ml aliquots of the microsomal suspension in 1.15 M sucrose were placed in the b o t t o m of four centrifuge tubes and overlaid with successive layers of sucrose solutions 0.8, 0.6 and 0.25 M and centrifuged in a SW 27 rotor at 2 4 0 0 0 rev./min for 180 min. Golgi fractions of increasing densities were har-

103 vested with a Pasteur pipet: GF~ at the 0.25 M/0.6 M interface: GF2 at the 0.6 M/0.8 M interface; and GF3 at the 0.8 M/1.15 M interface. Collection of GF3 presented some difficulties because of variable contamination with material suspended in the infranatant solution of sucrose 1.15 M and this led to probblems in reproducibility of recoveries and of specific activities of the enzymes present in GF3. In order to overcome this problem, t w o additional tubes containing 9 ml of microsomal suspension in 1.3 M sucrose overlaid with sucrose solutions of the same molarities mentioned above, i.e. 1.15, 0.8, 0.6 and 0.25 M, respectively, were included in the centrifugal run conducted with the SW 27 rotor. Under these experimental conditions, the microsomal contaminants remain in the 1.3 M layer and a clean, compact, well-delineated GF3 can be harvested from the 1.15 M/0.8 M sucrose interface. The individual fractions were then processed as described above for Gt. Since studies conducted with GF3 obtained b y the modified gradient described above were more reproducible than those obtained with fractions prepared by the original technique [10], all experimental data for GF3 presented here were obtained with specimens isolated by our modified procedure. The supernatant fraction ( 1 0 5 0 0 0 X g, 1 h) of liver homogenates (1 : 4, w/v) in isotonic KC1 was utilized for the assay of the soluble neuraminidase [1]. The total membrane-bound neuraminidase was assayed in an aqueous suspension of the nuclei-free particulate fraction. This fraction was isolated from liver homogenates (1 : 5, w/v) in 0.25 M sucrose/1 mM Na2EDTA. Following low speed centrifugation (800 X g, 10 min), the supernatant fraction was decanted and the pellet was homogenized and centrifuged as above. The combined supernatant fractions were then centrifuged at 1 0 5 0 0 0 X g for 1 h in order to sediment the nuclei-free particulate fraction [12]. The incubation mixtures for the neuraminidase assay, consisted of 135 pl of liver fraction, 15 pl of 1 M acetate buffer of the required pH and 50 pl of substrate solution containing 200 nmol of neuramin-lactose. Enzyme and subst-rate controls were run concurrently. The pH and the length of the incubation at 37°C varied according to the t y p e of enzyme being assayed. The particulate enzyme was incubated for 1 h at pH 4.4, the Golgi enzyme for 2 h at pH 4.2 and the soluble enzyme for 3 h at pH 5.8 [1,7,12]. The free sialic acid released b y the action of the enzyme was assayed by the thiobarbituric acid m e t h o d of Warren [13] as modified by Yeh et ai. [14]. UDPgalactose: N-acetylglucosamine galactosyltransferase was assayed according to Morre et al. [15]. One unit of enzyme activity is defined as the a m o u n t of enzyme that causes the release of 1 nmol of N-acetylneuraminic acid (neuraminidase) or the transfer of 1 nmol of galactose (galactosyltransferase) per h under the conditions of the assays. Protein was determined according to L o w r y et al. [16] using crystalline bovine serum albumin as the standard. For 'ultrastructural studies, the tissue was fixed in 2% glutaraldehyde and postfixed with OsO4. After washing with buffered normal formalin, the material was dehydrated in graded ethanol and e m b e d d e d in Araldite 6005. Tissue sections were then examined in a Hitachi HU-11B electron microscope.

104 Results The content and distribution of galactosyltransferase in Gt and in the three Golgi fractions are shown in Table I. At 45 min after ethanol administration, there are already indications of increased enzyme content (units per g of tissue) in some fractions; this increase became more clear at 90 min. The enzyme content, except for GF3, remained quite elevated after 16 h. The specific activities for the galactosyltransferase of these same fractions are shown in Table II. The results show an early rise of the specific activity in Gt and in all Golgi fractions of ethanol-treated rats. The maximum values were observed between 90 min and 3 h, except for GF2 which showed its peak of activity at 45 min after ethanol administration. In contrast with the results obtained with galactosyltransferase, the neuraminidase c o n t e n t (Table III) experienced a 50% drop in Gt and in the three Golgi fractions. This decrease in neuraminidase content is already quite clear at 45 min and the values remained significantly low 16 h after ethanol treatment. The specific activities for neuraminidase in Gt and in the three fractions dropped to values between 25 and 50% of the controls at 45 min after ethanol administration and the values remained depressed after 16 h (Table IV). The galactosyltransferase content and specific activity in the Gt fraction of rats killed 90 min after colchicine injection (Table V) showed increases similar to those observed in rats 90 min after ethanol administration (Table I). The increments of galactosyltransferase in the Gt fraction of rats given alcohol 45 min after colchicine injection were smaller than those observed with ethanol or with cochicine b y themselves. In the individual fractions (GF1, GF2, and GF3) of animals treated with colchicine and colchicine plus ethanol the values for total activity in general paralleled those observed in Gt, while the specific activities, except for GF3, showed no change or slight decrease. Colchicine injection caused a neuraminidase drop, both total activity and specific activity, in Gt and in all Golgi fractions (Table VI). Ethanol administraTABLE I EFFECT OF ETHANOL ADMINISTRATION ON THE CONTENT AND DISTRIBUTION GALACTOSYLTRANSFERASE ACTIVITY OF THE RAT LIVER GOLGI APPARATUS

OF THE

The animals were killed at the intervals indicated after a single oral dose of ethanol (0.6 g/100 g body w e i g h t ) . T h e d a t a r e p r e s e n t m e a n -+ S . E . o f t h r e e s e p a r a t e e x p e r i m e n t s ( i n e n z y m e u n i t s p e r g l i v e r ) . T h e P v a l u e s w e r e c a l c u l a t e d b y t h e S t u d e n t ' s t - t e s t , n.s., n o t s i g n i f i c a n t .

Golgi fraction

Control

45 rain

90 rain

3 h

16 h

Gt

6 9 . 4 6 +- 1 0 . 2 2

9 1 . 0 9 -+ 1 4 . 0 6 n.s.

108.19 ± 1.21 P <0.01

1 3 9 . 5 3 -+ 7 . 5 9 P <0.005

1 1 0 . 3 2 -+ 5 . 6 2 P <0.025

GF l

3.48±

1.12

10.50-+ 0.43 P <0.005

16.80-+ 3.58 P <0.025

16.15-+ 1.48 P <0.005

1 4 . 2 1 -+ 3 . 3 6 P <0.025

GF 2

16.93 ±

1.93

24.85 ± n.s.

3.35

30.65 ± 4.03 P <0.025

25.23 ± 2.41 P <0.05

23.33 + 3.40 P <0.05

GF 3

41.15-+

5.33

47.66-+ n.s.

7.71

61.32 ± 5.73 P <0.05

65.20 ± 4.52 P <0.025

67.30 ± 11.52 n.s.

105

T A B L E II EFFECT OF ETHANOL ADMINISTRATION ON THE SPECIFIC ACTIVITY TRANSFERASE OF RAT LIVER GOLGI APPARATUS

OF THE GALACTOSYL-

T h e a n i m a l s were k i l l e d a t t h e i n t e r v a l s i n d i c a t e d a f t e r a s i n g l e o r a l d o s e o f e t h a n o l ( 0 . 6 g / 1 0 0 g b o d y weight). The d a t a r e p r e s e n t m e a n ± S.E. of t h r e e separate e x p e r i m e n t s . (in e n z y m e u n i t s per m g G o l g i prot e i n ) . T h e P v a l u e s w e r e c a l c u l a t e d b y t h e S t u d e n t ' s t - t e s t , n.s., n o t s i g n i f i c a n t . Golgi fraction

Control

45 min

90 rain

3 h

16 h

Gt

68.11 ± 5.98

109.85 + 18.48 P <0.05

128.17 ± 15.48 P <0.025

122.00-+ 11.81 P <0.01

102.47 ± 15.36 n.s.

GFI

9 7 . 9 4 +- 6 . 0 7

154.16 + 4.63 P ~0.01

186.32 i 14.41 P ~0.005

1 7 4 . 5 7 -+ 4 . 3 2 P <~0.0005

174.90 ± 11.29 P ~0.01

GF 2

97.10 + 7.18

154.62 + 23.73 P <0.05

143.90 ± 23.55 n.s.

1 1 4 . 0 0 -+ 1 5 . 9 2 n.s.

111.10 ± n.s.

GF 3

50.77 ± 6.87

92.56 ± 17.96 P <0.05

96.80 ± 9.31 P <0.01

71.33 ± n.s.

9.83

9.44

81.16 ± 27.93 n.s.

TABLE III EFFECT OF ETHANOL ADMINISTRATION ON THE CONTENT NEURAMINIDASE ACTIVITY OF RAT LIVER GOLGI APPARATUS

AND

DISTRIBUTION

OF THE

The animals were killed at the intervals indicated after a single oral dose of ethanol (0.6 g/100 g body weight). The data represent mean + S.E. of three separate experiments (in enzyme units per g liver). The P v a l u e s w e r e c a l c u l a t e d b y t h e S t u d e n t ' s t - t e s t , n.s., n o t s i g n i f i c a n t . Golgi fraction

Control

45 rain

90 rain

3 h

16 h

Gt

19.39 ± 0.52

9.63 ± 0.95 P <0.0005

9.61 ± 1.36 P <0.005

10.16 ± 0.62 P <0.0005

13.21 ± 0.72 P ~0.005

GF I

2.44 ± 0.08

1.30 i 0.18 P <0.005

1.46 ± 0.25 P <0.01

1.47 ± 0.16 P <0.005

1.61 ± 0.13 P <0.005

GF 2

5.06 + 0.01

2.25 2 0.30 P <0.0005

2.74 ± 0.23 P <0.0005

3.36 + 0.18 P <0.0005

3 . 6 8 -+ 0 . 7 0 n.s.

GF 3

13.37 i 0.85

5.24 ± 0.72 P <0.005

6.85 + 0.14 P <0.005

7.89 + 0.31 P <0.005

7 . 3 4 -+ 0 . 7 9 P <0.005

TABLE IV EFFECT OF ETHANOL ADMINISTRATION DASE OF RAT LIVER GOLGI APPARATUS

ON THE

SPECIFIC ACTIVITY

OF THE NEURAMINI-

T h e a n i m a l s were k i l l e d a t t h e i n t e r v a l s i n d i c a t e d a t t e r a s i n g l e o r a l d o s e o f e t h a n o l ( 0 . 6 g / 1 0 0 g b o d y w e i g h t ) . T h e d a t a r e p r e s e n t m e a n ± S.E. o f t h r e e s e p a r a t e e x p e r i m e n t s ( i n e n z y m e u n i t s p e r m g G o l g i p r o t e i n ) . T h e P v a l u e s were c a l c u l a t e d b y t h e S t u d e n t ' s t - t e s t . Golgi fraction

Control

45 min

90 rain

3 h

16 h

Gt

20.15 ±

1.68

11.03 ± 1.30 P <0.01

11.42 ± 1.69 P ~0.025

11.16 ± 0.92 P ~0.005

1 4 . 9 8 -+ 0 . 5 8 P <0.025

GF 1

83.96 + 19.47

1 8 . 9 6 -+ 3 . 6 5 P <~0.025

18.13 ± 6.14 P ~0.025

16.40 ± 0.05 P ~0.025

30.14 i 5.91 P ~0.05

GF 2

31.49 ±

14.87 ± 2.04 P ~0,025

13.64 ± 1.68 P <0.01

1 5 . 8 3 -+ 1 . 1 9 P <0.025

18.86 ± 2.51 P ~0.05

GF 3

17.50 ± 0.38

9.30 ± 0.76 P <0.0005

11.29 ± 0.95 P <0.005

10.80 ± 0.84 P <0.005

11.15 ± 2.13 P <0.025

4.12

TABLE V

OF COLCHICINE TREATMENT BY I T S E L F A N D ACTIVITY OF RAT LIVER GOLGI APPARATUS

IN CONJUNCTION

WITH ETHANOL

68.80-+ 2.70 P <0.01

18.07 + 0.49 n.s.

5.29 ± 0.70 n.s.

108.70 ± 8.34 P <0.025

48.38 + 3.83 n.s.

31.21 + 1.77 P <0.005

11.45 ± 0.50 P <0.005

94.52 ± 7.14 n.s.

5 0 . 7 7 +- 6 . 8 7

97.10 + 7.18

97.94 ± 6.07

68.11 + 5.98

83.42 ± 21.78 n.s.

7 8 . 4 3 +- 5 . 5 1 n.s.

52.76 ± 2.19 P <0.005

102.93 + 5.66 P <0.01

a Rats sacrificed 90 rain after intraperitoneal injection of colchicine (0.5 mg/100 g body weight). b Rats given ethanol (0.6 g/100 g body weight) 45 rain after colchicine injection and killed 45 rain after ethanol administration.

4 1 . 1 5 +- 5 . 3 3

GF 3

1.12

1 6 . 9 3 +- 1 . 9 3

3.48 ±

69.46 + 10.22

Colchicine

Control

Colchicine b + alcohol

Control

Colchicine a

Enzyme units per mg Golgi protein

Enzyme units per g liver

GF 2

GF l

Gt

Golgi fraction

70.10-+ 7.67 n.s.

100.08 + 1.49 n.s.

83.24 + 8.61 n.s.

78.40 + 9.98 n.s.

Colchicine + alcohol

ON THE. GALACTOSYLTRANS-

t-test, n.s., not significant.

ADMINISTRATION

T h e d a t a r e p r e s e n t t h e m e a n ± S.E. o f t h r e e s e p a r a t e e x p e r i m e n t s . T h e P v a l u e s w e r e c a l c u l a t e d b y t h e s t u d e n t ' s

EFFECT FERASE

O~

VI

BY ITSELF

5.06-+ 0.01

1 3 . 3 7 -+ 0 . 8 2

GF2

GF 3

8 . 0 9 -+ 0 . 4 2 P <0.005

3.52-+ 0.45 P <0.025

1 . 7 0 -+ 0 . 4 9 n.s.

13.29 + 0.57 P <0.005

Colchicine a

6.13 + 0.27 P <0.005

3.35-+ 0.57 P <0.025

1 . 5 6 -+ 0 . 2 4 P <0.025

9.84-+ 0.99 P <0.005

Colchicine b + alcohol 1.68

1 7 . 5 0 +- 0 . 3 8

3 1 . 4 9 -+ 4 . 1 2

12.31 + 1.08 P <0.01

16.37 + 1.34 P <0.025

17.67 + 1.39 P <0.025

14.50-+ 0.21 P <0.025

Colchicine

u n i t s p e r m g G'olgi p r o t e i n

8 3 . 9 6 -+ 1 9 . 4 7

20.15-+

Control

Enzyme

ACTIVITY

8.60 + 0.11 P <0.0005

1 3 . 0 8 -+ 0 . 8 8 P <0.01

1 1 . 4 7 -+ 1 . 2 1 P <0.025

9.14-+ 0.65 P <0.005

Colchicine + alcohol

ON THE NEURAMINIDASE

t-test, n,s., not significant.

ADMINISTRATION

a Rats killed 90 min after intraperitoneal injection of colchicine (0.5 mg/100 g body weight). b Rats given ethanol (0.6 g]100 g body weight) 45 rain after colehicine injection and killed 45 rain after ethanol administration.

2.44 + 0.08

GFI

units per g liver

1 9 . 3 9 +- 0 . 5 2

Control

Enzyme

Gt

Golgi fraction

WITH ETHANOL

The P values were calculated by the Student's

AND IN CONJUNCTION

T h e d a t a r e p r e s e n t t h e m e a n -+ S . E . o f t h r e e s e p a r a t e e x p e r i m e n t s .

EFFECT OF COLCHICINE TREATMENT OF RAT LIVER GOLGI APPARATUS

TABLE

108

Fig. 1. E l e c t r o n m i c r o g r a p h s o f r a t liver: ( A ) C o n t r o l . (B) 9 0 rain a f t e r e t h a n o l i n g e s t i o n . (C) 16 h a f t e r e t h a n o l i n g e s t i o n . S o m e o f t h e l y s o s o m e s are i n d i c a t e d b y a r r o w s . T h e b a r r e p r e s e n t s 1 /~m.

109 TABLE VII EFFECT

OF

ETHANOL

NEURAMINIDASES

ADMINISTRATION

ON

THE

CYTOSOLIC

AND

MEMBRANE-BOUND

OF RAT LIVER

T h e a n i m a l s w e r e k i l l e d at t h e intervals i n d i c a t e d a f t e r a single oral d o s e o f e t h a n o l ( 0 . 6 g / 1 0 0 g b o d y w e i g h t ) . Tissue f r a c t i o n s a n d e n z y m e a s s a y s w e r e p e r f o r m e d as d e s c r i b e d u n d e r Materials a n d M e t h o d s . T h e d a t a r e p r e s e n t m e a n -+ S.E. o f t h r e e s e p a r a t e e x p e r i m e n t s . T h e P values w e r e c a l c u l a t e d b y t h e S t u d e n t ' s t-test, n.s. n o t s i g n i f i c a n t . Rat liver f r a c t i o n

Cytosol fraction Control 90 min 16h

Total activity ( u n i t s / g liver)

Specific activity (units/mg protein)

2 0 8 . 1 4 -+ 1 6 . 6 0 197.02-+ 6.27 n.s. 249.42 ± 14.51

2 . 2 0 -+ 0 . 1 5 2.19 +0.14 n.s. 2.55 + 0.21

n.s.

Membrane fraction Control 90 min 16h

187.90 + 13.68 316.29 + 7.03 P <0.005 341.34 ± 28.92 P <0.005

n.s.

2.15 3.25 P 3.57 P

-+ 0 . 2 3 + 0.08 <0.01 ± 0.28 <0.01

tion enhanced the depression of neuraminidase activity caused by colchicine; however, the combined effect of colchicine plus ethanol was less than the sum of the loss of neuraminidase activity caused by these two agents by themselves. Since it is generally accepted that the Golgi apparatus plays a role in the biogenesis of lysosomes and plasma membranes, and in the biosynthesis of some cytoplasmic components, particle-bound and cytosolic neuraminidases were measured [12] in control and ethanol-treated rats in order to establish if the changes observed in Golgi neuraminidase resulted in a subsequent drop of neuraminidase activity in other cellular compartments. However, these experiments showed that the neuraminidase content and specific activity remained virtually unchanged in the cytosolic fraction, whereas the particle-bound enzyme actually showed elevated values after ethanol administration (Table VII). This elevation of particle-bound neuraminidase appears to correlate with the ultrastructural studies which showed a marked increase of lysosomes in the livers of ethanol-treated rats (Fig. 1). Discussion Since Golgi galactosyltransferase plays a role in the biosynthesis of the glycoprotein moiety of plasma lipoproteins, the elevated enzyme activity observed after ethanol administration can be regarded as part of an adaptive response of the Golgi apparatus [8] designed to eliminate the excess lipid which accumulates in the liver cytosol following ethanolic intoxication. Unlike Gang et al. [8], who found marked increases in galactosyltransferase after 16 h but essentially no changes 90 min after ethanol ingestion, our results show increases as early as at 45 min while at 16 h there are indications that the

110 enzymic activity is returning to control levels (Tables I and II). The decreases in Golgi neuraminidase are quite remarkable in magnitude, appear at early stages and the values remain depressed even 16 h after ethanol administration (Tables III and IV). It is tempting to speculate that the decrease in the activity of this catabolic enzyme may reinforce the increased net production of glycoproteins reflected by the increased biosynthetic enzyme galactosyltransferase. However, since in our previous studies we found that Golgi neuraminidase does n o t act upon fetuin and ovine submaxillary glycoprotein [7], this explanation remains as an unlikely possibility which must await evaluation of the possible effect of this enzyme on the sialoglycoprotein moiety of the hepatic lipoproteins. Since colchicine administration, which blocks the release of lipoproteins from liver, also causes accumulation of Golgi vesicles containing lipoprotein granules [17], the effect of colchicine alone and of colchicine plus ethanol on the activity of these two enzymes was also investigated. As shown on Tables V and VI the effect of colchicine alone resembles, in general, the effect of ethanolic intoxication. However, the combined effects of both treatments are n o t additive suggesting that both drugs, at the doses utilized, are eliciting maximal or near maximal response. Since the pharmacological actions of ethanol and colchicine are so different, the enzymic changes observed may represent a liver response to the abnormally elevated levels of hepatic lipids that occur in both cases. The increased levels of total particle-bound neuraminidase (Table VII) are consistent with the marked increase in lysosomes (Fig. 1) which could easily mask the drop in Golgi neuraminidase (the latter accounts for only 3--10% of the total membrane-bound activity). The low levels of Golgi neuraminidase probably reflect an accelerated and preferential segregation of acid hydrolases from Golgi apparatus into the lysosomal compartment. This enhanced production of lysosomes would appear to be required for the increased autophagic processes needed to dispose of altered subcellular organelles in the ethanoldamaged cells. Furthermore, since sialic acids have been implicated in the attachment of polysomes to the membranes of the rough endoplasmic reticulum [18], the release of hepatic ribosomes observed by us and by others [19] during ethanolic intoxication could conceivably be a consequence of the high levels of lysosomal neuraminidase. Acknowledgements The authors wish to express their appreciation to Mr. Steve A. Graham for his excellent technical assistance. The ultrastructural studies were performed by Dr. Robert E. Nordquist. This work was supported in part by United States Public Health Service Grant NS 09176. References 1 2 3 4

T u l s i a n i , D . R . P . a n d C a r u b e l l i , R . ( 1 9 7 0 ) J. Biol. C h e m . 2 4 5 , 1 8 2 1 - - 1 8 2 7 M a h a d e v a n , S., N d u a g u b a , J . C . and Tappel, A . L . ( 1 9 6 7 ) J . Biol. C h e m . 2 4 2 , 4 4 0 9 - - 4 4 1 3 H o r v a t , A . a n d T o u s t e r , O. ( 1 9 6 8 ) J . Biol. C h e m . 4 3 8 0 - - 4 3 9 0 A r o n s o n ~ J r . , N . N . a n d De D u v e , C. ( 1 9 6 8 ) J. Biol. C h e m . 2 4 3 , 4 5 6 4 - - 4 5 7 3

111 5 Schengrund, C.-L., Jensen, D.S. and Rosenberg, A. (1972) J. Biol. Chem. 247, 2742--2746 6 Visser, A. and E m m e i o t , P. (1973) J. Membrane Biol. 14, 74--84 7 Kishore, G.S., Tulsiani, D.R.P., Bhavanandan, V.P. and Carubelli, R. (1975) J. Biol. Chem. 250, 2655--2659 8 Gang, H., Lieber, C.S. and Rubin, E. (1973) Nat. New Biol. 2 4 3 , 1 2 3 - - 1 2 5 9 Stein, O. and Stein, Y. (1965) Isr. J. Med. Sei. 1 , 3 7 8 - - 3 8 8 10 Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 45--72 11 Bergeron, J.J.M., Ehrenreich, J.H., Siekevitz, P. and Palade, G.E. (1973) J. Cell Biol. 59, 73--88 12 Carubelli, R. and Tulsiani, D.R.P. (1971) Biochim. Biophys. Acta 237, 78--87 13 Warren, L. (1959) J. Biol. Chem. 234, 1 9 7 1 - - 1 9 7 5 14 Yeh, A.K., Tulsiani, D.R.P. and CarubeUi, R. (1971) J. Lab. Clin. Med. 78, 771--778 15 Morre, D.J., Merlin, L.M. and Keenan, T.W. (1969) Biochem. Biophys. Res. C ommun. 3 7 , 8 1 3 - - 8 1 9 16 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 1 9 3 , 2 6 5 - - 2 7 5 17 Stein, O. and Stein, Y. (1973) Biochim. Biophys. Acta 3 0 6 , 1 4 2 - - 1 4 7 18 Scott-Burden, T. and Hawtrey, A.O. (1973) Biochem. Biophys. Res. C ommun. 54, 1 2 8 8 - - 1 2 9 5 19 Porta, E.A., Koch, O.R. and Hartroft, W.S. (1970) Exp. Mol. Pathol. 1 2 , 1 0 4 - - 1 3 2