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High density lipoprotein and low density lipoprotein catabolism by human liver and parenchymal and non-parenchymal cells from rat liver
586
Biochimica et Biophysics Q Elsevier/North-Holland
Acta, 486 (1977) 586-589 Biomedical Press
BBA Report BBA 51204
HIGH DENSITY LIPOPROTEIN AND LOW DENSITY LIPOPROTEIN CATABOLISM BY HUMAN LIVER AND PARENCHYMAL AND NON-PARENCHYMAL CELLS FROM RAT LIVER
THEO
J.C. VAN
BERKEL,
JOHAN
F. KOSTER
and WILLEM
Department of Biochemistry P.O. Box 1738, Rotterdam
I, Faculty of Medicine, (The Netherlands)
(Received
1976)
November
12th,
Erasmus
C. HULSMANN University
Rotterdam,
Summary The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprdtein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a lotimes higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.
A correlation between high serum cholesterol levels and atherosclerotic heart disease has been clearly demonstrated [ 11. In the blood, cholesterol is carried mainly by low density lipoprotein (LDL) and high density lipoprotein (HDL). A positive correlation exists between internal lipoprotein cholesterol accumulation and the levels of LDL in the blood [Z] . HDL, however, was found by Berg et al. [3] to be relatively low in the blood of patients with coronary heart disease when compared to normal individuals. Studies concerning the effect of diet and drugs on LDL metabolism in humans indicated that there is a linear correlation between plasma LDL concentration and its fractional catabolic rate [4]. In a literature survey Levy and Eisenberg [4] concluded that the physiological control of the plasma Abbreviations:
Protein.
density
LDL, range
low
density
1.063-1.21.
lipoprotein,
density
range
1.019-1.063;
HDL,
high
density
lipo-
587
LDL level is determined by its rate of catabolism. Studies with cultured human fibroblasts [ 51 and rat 161 and human [7] aorta smooth muscle cells have led to a model for this catabolic process [ 51, with binding of the lipoprotein to membrane receptors, and active endocytosis and degradation of the protein and cholesterol esters in the lysosomes by the action of cathepsins and acid lipase, respectively. However, the relative contributions of the various tissues in which LDL and/or HDL are degraded are still unknown. Hay et al. [S] concluded from the observed distribution of radioisotope-labeled LDL in the organs, that liver is the main organ for LDL uptake and degradation and also participates actively in the catabolism of HDL [9] . However, the liver is not a homogeneous tissue and consists of parenchymal cells, which carry out the typical liver functions (for example maintenance of glucose levels in the blood [ lO])and non-parenchymal cells (mainly Kupffer cells [ 111). Although on a cell-number basis these nonparenchymal cells make up about 35% of the liver cells, due to their smaller size their protein contribution to total liver is only about ,lO% [ 121 . It is well known that these non-parenchymal cells can actively take up particles from the blood [13] and our earlier work [14,15] indicated that these cells are especially enriched with cathepsin D and acid lipase compared with parenchymal cells. These investigations suggested to us that especially the nonparenchymal liver cells might be involved in the liver uptake and catabolism of the LDL and HDL particles. To test this possibility we isolated from rat liver pure and intact parenchymal and non-parenchymal cells and determined in the homogenates the LDL and HDL breakdown capacities. Parenchymal cells were prepared by perfusing the liver in vitro with 0.05% collagenase (Sigma, Type I) in Hank’s solution. After filtering (90 nm) the parenchymal cells were purified by differential centrifugation for 2 min at 50 X g. Non-parenchymal liver cells were purified by incubating the cells left on the filter with 0.25% pronase (Calbiochem). All parenchymal cells were destroyed after 1 h and the suspension was then centrifuged at 600 X g for 5 min and washed four times [15]. More than 90% of the isolated parenchymal cells and over 95% of the non-parenchymal cells excluded trypan blue. The purity of each different cell preparation was tested biochemically as described earlier [ 151 . The method employs the distribution of isoenzymes and maximal activities of the cytosolic enzyme pyruvate kinase [16] . The isolated cell types and whole human and rat liver were frozen at -80°C and after thawing sonicated twice for 30 s at 21 kHz. The homogenized cells and whole human and rat liver homogenates were incubated with the freshly isolated LDL and HDL [ 171. Routinely after 60 min incubation at pH 4.2 the amount of trichloroacetic acidsoluble reactive amino groups liberated from the protein moieties of LDL and HDL was determined [ 181. The reactions were found to be linear with time (up to 4 h measured; data not shown). Table I shows that human HDL can be hydrolyzed by the human liver homogenates in contrast to human LDL. Tabel I further shows that the parenchymal cell homogenate is able to hydrolyze protein from HDL. In contrast, no measurable amount of trichloroacetic acid-soluble reactive amino groups is formed from the protein present in LDL, indicating that the enzymes responsible for protein break-
TABLE
I
DEGRADATION OF HDL AND LDL BY HOMOGENATES OF WHOLE HUMAN LIVER, LIVER AND ISOLATED RAT PARENCHYMAL AND NON-PARENCHYMAL CELLS
RAT
LDL and HDL were isolated in a swinging-bucket rotor from fasted volunteers or rats, by the method of Redgrave f171. The density range of LDL was between 1.019 and 1.063 and of HDL between 1.063 and 1.21. The fractions were concentrated in a hollow fiber microconcentrator (Biomed. Instruments). Both LDL and HDL were free from albumin as tested immunologically. The freshly isolated lipoproteins were incubated with the sonicated homogenates for 60 min in sodium acetate buffer pH 4.2, with 4 mM dithiothreitol, at 37’C [18]. The reaction was stopped by the addition of 5% trichloroacetic acid. With the blank the lipoproteins were added after addition of trichloroacetic acid. After centrifugation for 5 min at 6000 X g the supernatants were used for the ninhydrin reaction 1191 with phenylalanine as standard. The activities are expressed as nmol amino group equivalents released per min per mg homogenate protein. The amount of HDL and LDL protein in the assays varied from 1.3 to 2.8 mg for the HDL and from 1.1 to 2.9 mg for LDL. Within this lipoprotein range the measured activities are V values (unpublished results). The amount of homogenate protein in the assays varied from 0.17 to 0.25 mg. Under these conditions degradation values are obtained which are proportional to the amount of homogenate protein and linear with time up to 4 h (unpublished results). __ __ ~__~~~~_.__ Source
of homogenate
Human HDL (* S.E.M.
Whole human liver Whole rat liver Parenchymal cells Non-parenchymal cells Ratio non-parenchymal cell activity/parenchymal
8.46 6.74 3.46 33.4
(n))
* 0.45 * 0.50 + 0.51 ?- 0.6
cell activity 9.7
_
(4) (4) (4) (4)
LDL (’ S.E.M.
(n))
<0.30 (4) 0.42 i 0.10 co.05 (4) 2.59 ? 0.24
>52
-__
(4) (4)
HDL (’ S.E.M.
1.97 0.77 7.19
(n))
f 0.18 * 0.13 i 0.47
(3) (3) (3)
9.3
down (presumably lysosomal cathepsins) in parenchymal cells are able to discriminate between the two lipoprotein particles, although equal amounts of LDL and HDL protein were present in the assays. The capacity for HDL protein breakdown of non-parenchymal cells is also listed in Table I. This activity is about lo-times higher than that of parenchymal cells, and the non-parenchymal cells are also able to catabolize LDL protein, but to a much lower extent than HDL protein. On the basis of the relative protein contribution to the whole rat liver of parenchymal cells (90%) and non-parenchymal cells (10%) the expected capacity of the whole rat liver homogenate to catabolize HDL and LDL can be calculated (for HDL 0.9 X 3.46 + 0.1 X 33.4 = 6.45). This value, compared with the experimental value for the total homogenate (Table I), indicates that no changes in protein degradation capacities are introduced by the cell-isolation procedures. With rat HDL the same relative distribution of the breakdown capacity is obtained as with human HDL, although the absolute activities are lower (see Table I). No studies with rat LDL were performed because the material obtained from rat serum was not sufficient for these assays. The catabolic activities reported here of human liver and both parenchymal and non-parenchymal rat liver cells indicate that the parenchymal liver cells are not able to catabolize intact LDL to a significant extent, at least under the applied assay conditions. Whether LDL breakdown in vivo, in which neutral (phospho)lipases may prepare the particle prior to lysosomal attack, is also low or absent, remains to be determined. This finding
589
contrasts with the general view, but is in agreement with the recent findings of Sniderman et al. [ZO] , who observed a ‘Paradoxical increase in the rate of catabolism of LDL after hepatectomy’. It seems likely then that LDL is the main transport vehicle of cholesterol to extrahepatic tissues [ 211. The importance of the non-parenchymal liver cells for the regulation of the total blood LDL levels cannot be deduced from our experiments. However, it is of interest to note that administration of estrogens lowers the blood LDL level and stimulates the uptake in the liver [8] . Perhaps this effect can be related to stimulation of the endocytotic process in the non-parenchymal liver cells [ 22,23,24] contributing in this way to the lower incidence of atherosclerosis in females [l] . In contrast to the pathogenic nature of high LDL levels, high levels of HDL seem beneficial [ 3,25,26] . As shown here, parenchymal cells are able to catabolize HDL. Also the non-parenchymal liver cells possess a high capacity to degrade HDL. It can be calculated from Table I that even more than 50% of the liver capacity for HDL protein breakdown (and cholesterol ester hydrolysis [ 151) is located in these active endocytosing cell types. Therefore it can be concluded that the liver is suited for an effective removal of cholesterol from the blood, in which HDL can be the transport vehicle [21] for cholesterol from the periphery to the liver. The authors thank Miss M.H.M. Verduin and Mr. J.K. Kruijt for excellent technical assistance. Miss A.C. Hanson is thanked for the preparation of the manuscript. The Dutch Heart Foundation is acknowledged for partial financial support. References 1
Dawber,
T.R.,
Kannel,
W.B.,
Revotskie,‘N.
and
Kauan,
A.
(1962)
Proc.
R. Sec.
Med.
55.
265-271 2
Smith,
3
Berg,
K.,
E.B.
4
Levy,
R.I.
5
Goldstein,
and
Slater,
Borresen, and
R.G.
A.L.
Eisenberg.
J.L.,
Dana.
(1972)
and
Lancet,
Dahlen,
S. (1974)
S.E.,
Ann.
Faust,
463
G. (1976)
J.R.,
Lancet,
Biol.
Clin.
Beaudet.
499-501
32,
A.L.
l-8 and
Brown,
M.S.
(1975)
J. Biol.
Chem.
250.8487-8495 6
St&O.
and
Stein,
I
Bierman,
E.L.
and
8
Hay,
R.V.,
Biophys. 9
Y.
Pottenger, Res.
(1975)
Albers. L.A.,
Commun.
Rachmilewitz.
D..
Circ.
J.J.
Res.
(1975)
36,
436-443
Biochim.
Reingold,
A.L.,
Biophys.
Getz,
Acta
G.S.
388.
and Wissler,
198-202 R.W.
(1971)
Biochem.
44.1471-1477
Stein.
0..
Roheim,
P.S.
and
Stein,
J.F.
Hiilsmann,
Y.
(1972)
Biochim.
Biophys.
Acta
270,
414-425 10
Van
Berkel,
Th.J.C.,
Koster.
and
W.C.
(1972)
Biochim.
Biophys.
Acta
276,
425-429 11
Wisse,
12
Weibel.
E. (1972)
13
Rahman,
14
Van
Berkel,
Th.J.C.
15
Van
Berkel.
Th.J.C..
Kruijt,
16
Van
Berkel,
Th.J.C.,
Koster,
E.R.,
J. Ultra&u&..
Stiubli,
Y.
W.
Res.
GnZigi,
and Wright,
B.J.
(1974)
38,
H.R.
(1975)
F.A.
Biol.
Biophys.
and
J.F.
Hess,
J. Cell
Biochem. J.K.
528-562
and
Kiister.
and
(1969)
65, Res.
J.F.
Commun.
(1975)
Hiilsmann,
J. Cell
Biol.
42,
68-91
112-122 Eur.
61,
204-209
J. Biochem.
W.C.
(1973)
Biochim.
(1975)
Ann.
Biochem.
58,
145-152
Biophys.
Acta
321.
171-180 17
Redgrave.
18
Nakai,
19
Barrett,
20
Sniderman,
21
Glomset,
T..
T.G.,
Roberts,
Otto,
P.S.
A.J.
(1972) A.D.,
J.A.
Kelly, Conning,
D.M.
24
Widman,
J.J. and
25
Miller,
26
Stein,
and
G.J. Y..
and Whaync,
T.F.
(Dinglr,
(1968)
22
and
T.E..
J. Lipid
Dobson, and
E.L.
Miller,
(1971)
H.D.
N.E. M.C.,
9,
(1975)
J.G.
ed.),
and
Biophys.
Acta
PP. 118-119.
Steinberg,
65,42-49 422.
380-389
North-Holland.
D. (1974)
Science
Amsterdam 183.
526-528
155-167 J. Exp.
(1966)
(1976)
Biochim.
J.T.,
Brit.
A.G.
Fainarue,
C.E.
(1976)
Chandler, Res.
Heppleston,
Fahimi,
Glangeaud,
and West,
in Lysosomes Carew,
23
106-118
L.S.
D.C.K.
Brit.
Lab.
Lancet
Invest.
Pathol.
52.
J. EXP.
Pathol.
34.
88-89 47,
388-400
141-149
i, 16-19
M. and
Stein.
0.
(1975)
Biochim.
Biophys.
Acta
380,
Biochimica et Biophysics Q Elsevier/North-Holland
Acta, 486 (1977) 586-589 Biomedical Press
BBA Report BBA 51204
HIGH DENSITY LIPOPROTEIN AND LOW DENSITY LIPOPROTEIN CATABOLISM BY HUMAN LIVER AND PARENCHYMAL AND NON-PARENCHYMAL CELLS FROM RAT LIVER
THEO
J.C. VAN
BERKEL,
JOHAN
F. KOSTER
and WILLEM
Department of Biochemistry P.O. Box 1738, Rotterdam
I, Faculty of Medicine, (The Netherlands)
(Received
1976)
November
12th,
Erasmus
C. HULSMANN University
Rotterdam,
Summary The capacity of the homogenates from human liver, rat parenchymal cells, rat non-parenchymal cells and total rat liver for the breakdown of human and rat high density lipoprotein (HDL) and human low density lipoprdtein (LDL) was determined. Human HDL was catabolized by human liver, in contrast to human LDL, the protein degradation of which was low or absent. Human and rat HDL were catabolized by both the rat parenchymal and non-parenchymal cell homogenates with, on protein base, a lotimes higher activity in the non-parenchymal liver cells. This implies that more than 50% of the total liver capacity for HDL protein degradation is localized in these cell types. Human LDL degradation in the rat could only be detected in the non-parenchymal cell homogenates. These findings are discussed in view of the function of HDL and LDL as carriers for cholesterol.
A correlation between high serum cholesterol levels and atherosclerotic heart disease has been clearly demonstrated [ 11. In the blood, cholesterol is carried mainly by low density lipoprotein (LDL) and high density lipoprotein (HDL). A positive correlation exists between internal lipoprotein cholesterol accumulation and the levels of LDL in the blood [Z] . HDL, however, was found by Berg et al. [3] to be relatively low in the blood of patients with coronary heart disease when compared to normal individuals. Studies concerning the effect of diet and drugs on LDL metabolism in humans indicated that there is a linear correlation between plasma LDL concentration and its fractional catabolic rate [4]. In a literature survey Levy and Eisenberg [4] concluded that the physiological control of the plasma Abbreviations:
Protein.
density
LDL, range
low
density
1.063-1.21.
lipoprotein,
density
range
1.019-1.063;
HDL,
high
density
lipo-
587
LDL level is determined by its rate of catabolism. Studies with cultured human fibroblasts [ 51 and rat 161 and human [7] aorta smooth muscle cells have led to a model for this catabolic process [ 51, with binding of the lipoprotein to membrane receptors, and active endocytosis and degradation of the protein and cholesterol esters in the lysosomes by the action of cathepsins and acid lipase, respectively. However, the relative contributions of the various tissues in which LDL and/or HDL are degraded are still unknown. Hay et al. [S] concluded from the observed distribution of radioisotope-labeled LDL in the organs, that liver is the main organ for LDL uptake and degradation and also participates actively in the catabolism of HDL [9] . However, the liver is not a homogeneous tissue and consists of parenchymal cells, which carry out the typical liver functions (for example maintenance of glucose levels in the blood [ lO])and non-parenchymal cells (mainly Kupffer cells [ 111). Although on a cell-number basis these nonparenchymal cells make up about 35% of the liver cells, due to their smaller size their protein contribution to total liver is only about ,lO% [ 121 . It is well known that these non-parenchymal cells can actively take up particles from the blood [13] and our earlier work [14,15] indicated that these cells are especially enriched with cathepsin D and acid lipase compared with parenchymal cells. These investigations suggested to us that especially the nonparenchymal liver cells might be involved in the liver uptake and catabolism of the LDL and HDL particles. To test this possibility we isolated from rat liver pure and intact parenchymal and non-parenchymal cells and determined in the homogenates the LDL and HDL breakdown capacities. Parenchymal cells were prepared by perfusing the liver in vitro with 0.05% collagenase (Sigma, Type I) in Hank’s solution. After filtering (90 nm) the parenchymal cells were purified by differential centrifugation for 2 min at 50 X g. Non-parenchymal liver cells were purified by incubating the cells left on the filter with 0.25% pronase (Calbiochem). All parenchymal cells were destroyed after 1 h and the suspension was then centrifuged at 600 X g for 5 min and washed four times [15]. More than 90% of the isolated parenchymal cells and over 95% of the non-parenchymal cells excluded trypan blue. The purity of each different cell preparation was tested biochemically as described earlier [ 151 . The method employs the distribution of isoenzymes and maximal activities of the cytosolic enzyme pyruvate kinase [16] . The isolated cell types and whole human and rat liver were frozen at -80°C and after thawing sonicated twice for 30 s at 21 kHz. The homogenized cells and whole human and rat liver homogenates were incubated with the freshly isolated LDL and HDL [ 171. Routinely after 60 min incubation at pH 4.2 the amount of trichloroacetic acidsoluble reactive amino groups liberated from the protein moieties of LDL and HDL was determined [ 181. The reactions were found to be linear with time (up to 4 h measured; data not shown). Table I shows that human HDL can be hydrolyzed by the human liver homogenates in contrast to human LDL. Tabel I further shows that the parenchymal cell homogenate is able to hydrolyze protein from HDL. In contrast, no measurable amount of trichloroacetic acid-soluble reactive amino groups is formed from the protein present in LDL, indicating that the enzymes responsible for protein break-
TABLE
I
DEGRADATION OF HDL AND LDL BY HOMOGENATES OF WHOLE HUMAN LIVER, LIVER AND ISOLATED RAT PARENCHYMAL AND NON-PARENCHYMAL CELLS
RAT
LDL and HDL were isolated in a swinging-bucket rotor from fasted volunteers or rats, by the method of Redgrave f171. The density range of LDL was between 1.019 and 1.063 and of HDL between 1.063 and 1.21. The fractions were concentrated in a hollow fiber microconcentrator (Biomed. Instruments). Both LDL and HDL were free from albumin as tested immunologically. The freshly isolated lipoproteins were incubated with the sonicated homogenates for 60 min in sodium acetate buffer pH 4.2, with 4 mM dithiothreitol, at 37’C [18]. The reaction was stopped by the addition of 5% trichloroacetic acid. With the blank the lipoproteins were added after addition of trichloroacetic acid. After centrifugation for 5 min at 6000 X g the supernatants were used for the ninhydrin reaction 1191 with phenylalanine as standard. The activities are expressed as nmol amino group equivalents released per min per mg homogenate protein. The amount of HDL and LDL protein in the assays varied from 1.3 to 2.8 mg for the HDL and from 1.1 to 2.9 mg for LDL. Within this lipoprotein range the measured activities are V values (unpublished results). The amount of homogenate protein in the assays varied from 0.17 to 0.25 mg. Under these conditions degradation values are obtained which are proportional to the amount of homogenate protein and linear with time up to 4 h (unpublished results). __ __ ~__~~~~_.__ Source
of homogenate
Human HDL (* S.E.M.
Whole human liver Whole rat liver Parenchymal cells Non-parenchymal cells Ratio non-parenchymal cell activity/parenchymal
8.46 6.74 3.46 33.4
(n))
* 0.45 * 0.50 + 0.51 ?- 0.6
cell activity 9.7
_
(4) (4) (4) (4)
LDL (’ S.E.M.
(n))
<0.30 (4) 0.42 i 0.10 co.05 (4) 2.59 ? 0.24
>52
-__
(4) (4)
HDL (’ S.E.M.
1.97 0.77 7.19
(n))
f 0.18 * 0.13 i 0.47
(3) (3) (3)
9.3
down (presumably lysosomal cathepsins) in parenchymal cells are able to discriminate between the two lipoprotein particles, although equal amounts of LDL and HDL protein were present in the assays. The capacity for HDL protein breakdown of non-parenchymal cells is also listed in Table I. This activity is about lo-times higher than that of parenchymal cells, and the non-parenchymal cells are also able to catabolize LDL protein, but to a much lower extent than HDL protein. On the basis of the relative protein contribution to the whole rat liver of parenchymal cells (90%) and non-parenchymal cells (10%) the expected capacity of the whole rat liver homogenate to catabolize HDL and LDL can be calculated (for HDL 0.9 X 3.46 + 0.1 X 33.4 = 6.45). This value, compared with the experimental value for the total homogenate (Table I), indicates that no changes in protein degradation capacities are introduced by the cell-isolation procedures. With rat HDL the same relative distribution of the breakdown capacity is obtained as with human HDL, although the absolute activities are lower (see Table I). No studies with rat LDL were performed because the material obtained from rat serum was not sufficient for these assays. The catabolic activities reported here of human liver and both parenchymal and non-parenchymal rat liver cells indicate that the parenchymal liver cells are not able to catabolize intact LDL to a significant extent, at least under the applied assay conditions. Whether LDL breakdown in vivo, in which neutral (phospho)lipases may prepare the particle prior to lysosomal attack, is also low or absent, remains to be determined. This finding
589
contrasts with the general view, but is in agreement with the recent findings of Sniderman et al. [ZO] , who observed a ‘Paradoxical increase in the rate of catabolism of LDL after hepatectomy’. It seems likely then that LDL is the main transport vehicle of cholesterol to extrahepatic tissues [ 211. The importance of the non-parenchymal liver cells for the regulation of the total blood LDL levels cannot be deduced from our experiments. However, it is of interest to note that administration of estrogens lowers the blood LDL level and stimulates the uptake in the liver [8] . Perhaps this effect can be related to stimulation of the endocytotic process in the non-parenchymal liver cells [ 22,23,24] contributing in this way to the lower incidence of atherosclerosis in females [l] . In contrast to the pathogenic nature of high LDL levels, high levels of HDL seem beneficial [ 3,25,26] . As shown here, parenchymal cells are able to catabolize HDL. Also the non-parenchymal liver cells possess a high capacity to degrade HDL. It can be calculated from Table I that even more than 50% of the liver capacity for HDL protein breakdown (and cholesterol ester hydrolysis [ 151) is located in these active endocytosing cell types. Therefore it can be concluded that the liver is suited for an effective removal of cholesterol from the blood, in which HDL can be the transport vehicle [21] for cholesterol from the periphery to the liver. The authors thank Miss M.H.M. Verduin and Mr. J.K. Kruijt for excellent technical assistance. Miss A.C. Hanson is thanked for the preparation of the manuscript. The Dutch Heart Foundation is acknowledged for partial financial support. References 1
Dawber,
T.R.,
Kannel,
W.B.,
Revotskie,‘N.
and
Kauan,
A.
(1962)
Proc.
R. Sec.
Med.
55.
265-271 2
Smith,
3
Berg,
K.,
E.B.
4
Levy,
R.I.
5
Goldstein,
and
Slater,
Borresen, and
R.G.
A.L.
Eisenberg.
J.L.,
Dana.
(1972)
and
Lancet,
Dahlen,
S. (1974)
S.E.,
Ann.
Faust,
463
G. (1976)
J.R.,
Lancet,
Biol.
Clin.
Beaudet.
499-501
32,
A.L.
l-8 and
Brown,
M.S.
(1975)
J. Biol.
Chem.
250.8487-8495 6
St&O.
and
Stein,
I
Bierman,
E.L.
and
8
Hay,
R.V.,
Biophys. 9
Y.
Pottenger, Res.
(1975)
Albers. L.A.,
Commun.
Rachmilewitz.
D..
Circ.
J.J.
Res.
(1975)
36,
436-443
Biochim.
Reingold,
A.L.,
Biophys.
Getz,
Acta
G.S.
388.
and Wissler,
198-202 R.W.
(1971)
Biochem.
44.1471-1477
Stein.
0..
Roheim,
P.S.
and
Stein,
J.F.
Hiilsmann,
Y.
(1972)
Biochim.
Biophys.
Acta
270,
414-425 10
Van
Berkel,
Th.J.C.,
Koster.
and
W.C.
(1972)
Biochim.
Biophys.
Acta
276,
425-429 11
Wisse,
12
Weibel.
E. (1972)
13
Rahman,
14
Van
Berkel,
Th.J.C.
15
Van
Berkel.
Th.J.C..
Kruijt,
16
Van
Berkel,
Th.J.C.,
Koster,
E.R.,
J. Ultra&u&..
Stiubli,
Y.
W.
Res.
GnZigi,
and Wright,
B.J.
(1974)
38,
H.R.
(1975)
F.A.
Biol.
Biophys.
and
J.F.
Hess,
J. Cell
Biochem. J.K.
528-562
and
Kiister.
and
(1969)
65, Res.
J.F.
Commun.
(1975)
Hiilsmann,
J. Cell
Biol.
42,
68-91
112-122 Eur.
61,
204-209
J. Biochem.
W.C.
(1973)
Biochim.
(1975)
Ann.
Biochem.
58,
145-152
Biophys.
Acta
321.
171-180 17
Redgrave.
18
Nakai,
19
Barrett,
20
Sniderman,
21
Glomset,
T..
T.G.,
Roberts,
Otto,
P.S.
A.J.
(1972) A.D.,
J.A.
Kelly, Conning,
D.M.
24
Widman,
J.J. and
25
Miller,
26
Stein,
and
G.J. Y..
and Whaync,
T.F.
(Dinglr,
(1968)
22
and
T.E..
J. Lipid
Dobson, and
E.L.
Miller,
(1971)
H.D.
N.E. M.C.,
9,
(1975)
J.G.
ed.),
and
Biophys.
Acta
PP. 118-119.
Steinberg,
65,42-49 422.
380-389
North-Holland.
D. (1974)
Science
Amsterdam 183.
526-528
155-167 J. Exp.
(1966)
(1976)
Biochim.
J.T.,
Brit.
A.G.
Fainarue,
C.E.
(1976)
Chandler, Res.
Heppleston,
Fahimi,
Glangeaud,
and West,
in Lysosomes Carew,
23
106-118
L.S.
D.C.K.
Brit.
Lab.
Lancet
Invest.
Pathol.
52.
J. EXP.
Pathol.
34.
88-89 47,
388-400
141-149
i, 16-19
M. and
Stein.
0.
(1975)
Biochim.
Biophys.
Acta
380,