BIOCHEMICAL SOCIETY TRANSACTIONS
560
becomes covalently bonded to the fibrin lattice by transglutaminase that it might promote the endocytosis of fibrin
and fibrin degradation peptides. In this study, fibrin degradation peptides were generated from native and fibronectindepleted plasma clots and the endocytosis of these peptides
measured in vitro.
Fresh blood was collected from volunteers into EDTA at a
final concentration of 3.5 mM. The blood was centrifuged at
1000 g for 20 min and the plasma aspirated and then dialysed
against phosphate-buffered saline for 24 h at 4°C. Aliquots
of plasma (10 ml) were passed down columns (50 ml) containing gelatin-Ultrodex (LKB, Sesdon, Surrey) to remove
fibronectin. This removes all fibronectin as demonstrated by
immunoblotting with polyclonal anti-(fibr0nectin)serum as
described previously (Knox et al., 1986). Control serum was
passed down similarly sized columns of uncoupled Ultroclex.
After concentration to starting volume, control and depleted
plasma were sterilized with 0.22 p m filters before use.
To promote clotting, calcium chloride was added to give a
final concentration of 2 mM. At the time of clotting 25 pl
containing 5 x loh c.p.m. of radioiodinated fibrinogen
(Sigma, Poole, Dorset) was added to each tnillilitre of plasma.
Aliquots (1 ml) of control or depleted plasma were clotted at
37°C in 35 ml tissue culture grade Petri dishes followed by
the addition of 100 p g of streptokinase (Sigma). Incubation
for 1 h at 37°C brought about complete lysis of the clots.
P388DI cells (a macrophage-like cell line) were routinely
cultured in Ham's F10 medium supplemented with 10% (v/v)
fetal calf serum (Gibco, Uxbridge, Middx.). Replicate 35 ml
Petri dishes containing 5 x l o 4 cells were used for measurement of endocytosis. Radiolabelled fibrin degradation peptides (50 pl) was added to each Petri dish. At time intervals,
Petri dishes were washed five times with phosphate-buffered
saline and to determine the amount of isotope remaining
associated with the cells, 1 M-sodium hydroxide was used to
remove the latter and the resulting suspension was counted
with a LKE3 8000 y-spectrometer.
Fig. 1 shows a typical set of results. The Figure demonstrates that there are no significant differences in the rate of
uptake of fibrin degradation peptides derived from control
and fibronectin-depleted plasma clots. The results indicate
that fibronectin is not necessary nor does it enhance the rate
of endocytosis of fibrin degradation peptides.
Knox, P., Crooks, S. & Rimmer, C . S. (1986) J. Cell Riol. 102,
23 18-2323
Saba,T. M. & Jaffe, E. (1980)Am. J . Med. 68,577-583
Yamada, K. M. & Olden, K. (1 978) Nufure (London) 275, 179- 184
Received 27 November 1987
Low density lipoprotein inhibits the cell adhesion stimulating properties of human serum
HELEN RlGG and PETER KNOX
Department of Biochemistry, St George S Hospital Medical
School, Cranmer Terrace, London S W l 7 ORE, U.K.
Raised levels of circulating cholesterol are a predisposing
factor for the development of atherosclerotic lesions and
resulting cardiovascular disease (Armstrong et al., 1974).
The majority of the cholesterol and cholesterol esters in
plasma are found in the class of lipoproteins known as low
density lipoprotein. At the site of atherosclerotic lesions,
histological examination shows the presence of coagulum
and/or fibrous connective tissue and these findings suggest
that an inadequate wound-healing response is in some way
involved in the pathogenesis of the lesions. As part of the
normal wound response cells from the neighbouring healthy
tissue migrate over the surface or through the coagulum.
These migrating cells will eventually effect repair of the
damaged area of blood vessel wall.
Plasma and serum contain two proteins that stimulate
various aspects of cellular adhesion (Knox & Griffiths,
1980).Thus these proteins promote the adhesion of cells on
to a number of biological surfaces such as collagen and
fibrin, and they cause cells to adopt specific morphologies.
These proteins also promote the migration of cells over a
variety of surfaces. One of the proteins is plasma fibronectin
(Yamada & Olden, 1978), a dimeric structure with a molecular mass of 450 kDa. The other is vitronectin, an unrelated
protein of molecular mass 70 kDa (Knox & Griffiths, 1982;
Hayman etaf.,1985).
The adhesion stimulating properties of plasma are likely
to play a significant role in the wound response and in this
study the effect of low density lipoproteins on these adhesive
events was examined.
Fresh blood taken from volunteers was anticoagulated
with EDTA (final concentration 3.5 mM), centrifuged at 4°C
for 15 rnin at 2000 g and the plasma aspirated. The density
loo
I
01
10
20
Cholesterol concn. (mM)
Fig. 1. Effect of low density lipoprotein on cell adhesion
BCL-D1 cells were seeded out in the presence of different
concentrations of low density lipoprotein. The degree of cell
attachment was measured at 3 h. Results represent the mean
of three Petri dishes and the maximum variation between
dishes was 6%.
of the plasma was adjusted to 1.006 g/ml and centrifuged at
4°C for 16 h at 100000 g. Lipoproteins with densities less
than 1.006 g/ml were removed from the top of the tubes. The
remaining infranatant was adjusted to a density of 1.063 g/ml
using solid sodium bromide and then re-centrifuged at
100000 g overnight. The low density lipoprotein could be
seen as a yellow oily band at the top of each tube and was
removed and then dialysed exhaustively against phosphate1988
625th MEETING, LONDON
56 1
buffered saline. The cholesterol content of the low density
lipoprotein was estimated using the choloxidate kit no. 1
from BDH Chemicals (Poole,Dorset).
The cells used in the study were BCL-D1, a strain of
diploid fibroblasts derived from human embryonic lung.
Cells were routinely cultured in Ham's F10 medium supplemented with 10% (v/v) fetal calf serum (Flow Laboratories,
Irvine, Ayreshire). To monitor cell adhesion, a cell suspension of BCL-Dl cells in Ham's F10 and 3% (v/v) human
serum at a cell density of 5 x 104/mlwas prepared. Different
concentrations of low density lipoproteins were added to aliquots of the suspension and these were plated in 35 mm
tissue culture grade Petri dishes. To determine the proportion of cells that had adhered to the surface of the Petri dish
at anv time Doint. dishes were decanted and washed once
with ml of'phosphate-buffered saline. The decanted fluid
and the wash were combined and the number of cells present
determined with a haemocytometer. The percentage of
attached cells remaining in the Petri dish was then calculated.
i
Fig. 1 shows that low density lipoprotein inhibits the adhesion of BCL-D1 cells, and at concentrations of cholesterol
that are found in hypercholesterolaemic patients there was a
greater than 50% reduction in cellular adhesion. If there is
similar reduced adhesion in vivo, then the presence of
increased low density lipoprotein might have the effect of
delaying the migration of cells into the area of the wound.
Armstrong, M. L., Megan, M. B. & Warner, E. D. (1974)Circ. Rex,
34,447-454
Knox, P. & Griffiths, S.( 1980)J. CeNSci. 46,97-112
Knox, P. & Griffiths,S. (1982)J.CellSci. 55,301-316
Hayman, E. G., Pierschbacher, M. D., Suzuki, S. & Ruoslahti, E.
(1985)Exp. Cell Res. 160,245-258
Yamada, K. M. & Olden, K. ( 1978) Nature (London) 275,179- 184
Received 27 November 1987
A colorimetric microvolume assay for nucleoside diphosphatase activity in Gold membrane
preparations
HELEN M. JAMES,
GORDON A. NICHOLAS, CHRISTOPHER A. SMITH
and ALAN J. SWEETMAN
Department of Biological Sciences, Manchester Polytechnic,
Manchester, MI 5GD, U.K.
Nucleoside diphosphatase (NDPase; EC 3.6.1.6.) of the
endoplasmic reticulum (microsomes) has been purified and
well characterized (Yamazaki & Hayaishi, 1968; Kuriyama,
1971; Ohkubo et al., 1980, 1982). However, the NDPase of
Golgi membranes has not been examined to the same extent.
Most studies on NDPase activity of Golgi membranes
have used subcellular fractions enriched in Golgi apparatus.
Kuhn & White (1977) showed NDPase of rat mammary
gland Golgi to be most effectively stimulated by Ca2+,and to
only a slightly lesser extent by Mg2,Mn2+and Co2+.Enzyme
2
4
6
8
10
activity in the presence of Ca2+ did not appear to follow
Michaelis-Menten kinetics. Brandan & Fleischer ( 1982)
Calcium concn. (mM)
showed that rat liver Golgi NDPase was also maximally
stimulated by Ca2+,but was not stimulated by Mgz+,Mn2+
160 (4
or Co*+.In contrast, stimulation by Ca2 appeared to follow
Michaelis-Menten kinetics.
Since Golgi membranes can only be isolated in relatively
small amounts there is a need for a sensitive, microvolume
assay for NDPase of Golgi membranes. We have developed a
colorimetric microvolume assay for determining NDPase
activity. To assess the effectiveness of this assay we have used
it to re-investigate the cation activation of the enzyme.
All chemicals were purchased from the Sigma Chemical
Company, Poole, U.K., except polyvinyl alcohol which was
brought from the Aldrich Chemical Company, Inc., Milwaukee, U.S.A.
Subcellular fractions enriched in Golgi apparatus were
prepared from lactating mammary glands and liver of mouse,
I
rat and guinea-pig by the method of Morre et al. (1970) as
2
4
6
8
10
modified by Smith & Brew (1977). The NDPase assay was
Calcium concn. (mM)
carried out in 0.7 ml Eppendorf tubes. The standard assay
mixture contained 50 m-sodium cacodylate, pH 6, 10 mMFig. 1. A plot of NDPase activity against Ca2+concentration
CaCl,, 0.1% (w/v) Triton X-100, 10-20 pg of Golgi protein
and 5 mM-UDP (final volume 100 pl). The reaction was ( a ) Rat liver Golgi fraction, ( b ) Rat mammary gland Golgi
fraction. Lines of best fit to data and kinetic parameter (see
Abbreviation used: NDPase, nucleoside diphosphatase (EC the text) were determined using the method of Beynon
3.6.1.6).
(1985).
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