Primary amines inhibit recycling of α2M receptors in fibroblasts

Cell, Vol. 20. 37-43, May 1980. Copyright 0 1980 by MIT

Primary Amines Inhibit Recycling of a,M Receptors in Fibroblasts

Fred Van Leuven, Jean-Jacques Cassiman and Herman Van Den Berghe Division of Human Genetics Department of Human Biology University of Leuven Minderbroedersstraat 12 B-3000 Leuven, Belgium

Summary

Receptor-mediated endocytosis of cY&&protease complexes by normal human skin fibroblasts in culture was inhibited by the primary amines, methylamine and mono-dansylcadaverine. The latter was effective at a concentration two orders of magnitude lower than methylamine. Several observations indicated an intracellular mode of action of these amines, excluding interference with binding of c&l to the receptor. Inhibition of endocytosis was dis- sociated from lysosomotropic effects of these amines by kinetic analysis of endocytosis. As op posed to chloroquine and alkaline incubation conditions (pH 8.0), the amines reduced the maximal rate of endocytosis considerably, indicating a reduction in number of available receptors. This was confirmed by measuring binding at 4°C. Continuous recycling of receptors is indicated by the absence of down-regulation of number of receptors under conditions saturating for endocytosis of anM complexes. We postulate that the primary amines, methylamine and mono-dansylcadaverine, interfere with the recycling of a2M receptors, while the chemical nature of the amines and the conditions under which inhibition becomes apparent point to the involvement of transglutaminase in this recycling.

Introduction

We have previously demonstrated that human skin fibroblasts in culture are capable of internalizing (Ye macroglobulin &M) by a cell surface receptor which recognizes, binds and effectively internalizes only (Ye- M-protease complexes (Van Leuven, Cassiman and Van Den Berghe, 1978a, 1979). The kinetic parameters of the internalization of (YAM complexes had many features in common with the LDL receptor system, such as the number and apparent affinity of the receptors, the extremely high rate of internalization under saturating conditions and the rapid and nearly complete intracellular degradation of the proteins in- gested (Goldstein, Anderson and Brown, 1979).

It was recently shown that a2M and EGF become located in coated pits once bound to cell surface receptors and are internalized via formation of coated vesicles (Willingham, Maxfield and Pastan, 1979) confirming the hypothesis (Goldstein, Anderson and

Brown, 1979) that receptor clustering and coated pit formation might be a general mechanism for receptor- mediated endocytosis.

Certain primary amines have been shown to affect receptor-mediated endocytosis. Examining this prop- erty by fluorescent and immunochemical techniques, Maxfield et al. (1979a) showed the clustering of apM receptors to be inhibited by primary amines, such as methylamine. These authors assumed the action of methylamine to be exerted via intracellular pH or by inhibition of transglutaminase. Their findings were ex- tended to other amines (Maxfield et al., 1979b), and a close correlation between transglutaminase inhibition and inhibition of clustering of cllsM and EGF was demonstrated (Davies et al., 1980).

In the present study we quantitatively examine the effect of primary amines on receptor-mediated endocytosis of ‘251-cu2M-trypsin complexes by normal human skin fibroblasts in culture. Using methylamine and mono-dansylcadaverine (m-DNSC), we show that these amines affect the binding of (YAM to the cell surface by decreasing the number of available receptors during a preincubation period with the amines. These results indicate interference with continuous recycling of (Y*M receptors. Our results rule out the possibility that changes in intracellular pH are responsible for the observed inhibition, and indicate the involvement of transglutaminase in receptor recycling.

Results

Effect of Primary Amines on a& Endocytosis Cell layers pretreated with methylamine for 1 hr showed a reproducible inhibition of cr2M-trypsin ((YAM- T) endocytosis. This inhibition was dependent on the concentration of the amine (Table I), whereas simultaneous addition of methylamine (30 mM) and a2M-T to the cells gave only a 13% inhibition as compared to controls (no drug added). Other primary amines were found inhibitory under similar assay conditions: am- monium acetate (20 mM), 34% inhibition; 6-amino- hexanol(20 mfvl). 48% inhibition; putrescine (10 mM), 21% inhibition; glycyl-glycine (40 mM), 19% inhibition. Chloroquine or alkalinization (pH 8.0) of the medium was without effect on the uptake of (u*M-T.

Since amines affect the intracellular pH and lysosomal function, the rate of intracellular degradation of (YAM was examined in the presence of these amines (Table 1, Figure 1). The intracellular half-life of a2M-T was increased under all circumstances (Table 1).

To further dissociate the effect of methylamine on endocytosis from its intracellular effect, the kinetic parameters of endocytosis were examined after methylamine or alkaline pH pretreatment (Figure 2). Under control conditions, maximal rate of endocytosis (VE) was 2.7 pg a2M-T per hr and per mg cell protein. The apparent affinity of the endocytosis mechanism (KE,

Cell 38

Table 1. Effect of Methylamine. Chloroquine and pH on Endocytosis and Intracellular Degradation of ‘25l-a&f-T Complexes by Fibroblasts in Culture

Degradation Uptake (%) T1/2 0-W

Control 100 2.2

Methylamine 10mM 66

20 mM 46 5.3

30 mM 31

Chloroquine 0.05 mM 121 6.7

0.1 mM 102 10

pH 8.0-HCOa- 5mM 110 6.3

Uptake was measured over 30 min at 100 Pg ‘25l-a&f-T per ml medium and is tabulated relative to control cell layers. Degradation is expressed as intracellular half-life of ‘25l-a2M-T, in hr. derived from curves shown in Figure 1.

Confluent cell layers were pretreated with methylamine and chloroquine at the final concentrations indicated for 1 hr prior to the assay. Drugs or HCOs- remained present during assays for uptake and degradation. All data are the mean of determinations on at least two separate cell layers.

at half maximal rate of endocytosis) was 37 nM or approximately 27 pg (w2M-T per ml medium. After pretreatment of the cells with methylamine (1 hr, 30 mM), VE was decreased to 0.7 pg cunM per hr and per mg cell protein with a KE = 15 nM. Under alkaline conditions VE was 2.8 pg per hr and per mg cell protein and KE = 25 nM. Chloroquine (0.1 mM) had only a small effect on the kinetic parameters of endocytosis, with unchanged VE and a slight increase in affinity (KE = 30 nM). Thus methylamine decreased the maximal rate of uptake and increased the affinity of endocytosis, while alkalinization influenced only affinity, as did chloroquine.

Effect of m-DNSC on (~&l Endocytosis Since methylamine and the other primary amines tested were effective in inhibiting (r2M-T endocytosis only at higher concentrations compared to chloroquine, a search was made for amines with higher affinity for the endocytosis mechanism. m-DNSC, a fluorescent derivative of the diamine cadaverine, was found to be a very potent inhibitor of the endocytosis of (YAM. The extent of the inhibition of QM endocytosis by m-DNSC was both time- and concentration-dependent (Figure 3). Simultaneous addition of labeled (Y~M-T and m-DNSC (0.2 mM) to the medium resulted in a 28% inhibition of endocytosis (Figure 38). Prein- cubation of the cells with m-DNSC in the absence of (YAM complexes for increasing time periods resulted in a gradual decrease of endocytosis (Figure 38).

The effect of m-DNSC was reversible; removal of the drug from the medium restored the internalization

rate to approximately 80% of control after 2 hr. A similar phenomenon was observed with methylamine. In this case recovery was somewhat faster; it took

2.0

1.8

1.6

1.4L 1 1 1 1 1 0 1 2 3 4

TIME (HOURS) Figure 1. Intracellular Degradation of ‘25l-n&f-T Complexes

Separate fibroblast monolayers were pulsed with ‘251-a2M-T for 30 min. After removal of the label (zero time), cell layers were further incubated in medium containing the drugs. At the times indicated cells were assayed for intracellular content of ‘? (see Experimental Procedures). Cell layers were pretreated with drugs for 1 hr (or 30 min for m-DNSC) prior to pulse. Data are expressed relative to intracellular amount of ‘*“l-a&t at zero time, on a logarithmic scale. Half-lives (Table 1) were calculated from the straight lines fitted to the experimental points graphically. Symbols: t-j control: (u) m-DNSC (0.2 mM); (x-x) methylamine (20 mM): (C--.-O) chloroquine 0.05 mM; CO--.*) chloroquine 0.1 mM; H pH 8.0 tHC03- 5 mM).

only 1 hr after removal of the drug to restore the endocytosis rate to more than 90% of control.

The effect of m-DNSC on the kinetic parameters of endocytosis of (Y*M complexes was comparable to that observed with methylamine (Figure 4). At 0.2 mM m-DNSC, the maximal rate of endocytosis (VE) was decreased to 0.7 pg per hr and per mg protein with an apparent affinity (KE) of 13 nM. The complexity of the effect was illustrated by the results obtained at a lower concentration of m-DNSC. At 0.1 mM, VE was 1.8 pg per hr and per mg protein and KE = 37 nM. Appar- ently, maximal rate of uptake and affinity are influenced to a different degree by m-DNSC, resulting in a nonlinear relation between inhibitor concentration and the endocytosis parameters.

Qualitative and Quantitative Analysis of m-DNSC Uptake by Cells When fibroblast monolayers were treated with m- DNSC they became fluorescent nearly immediately after addition of m-DNSC to the medium (Figure 5). The fluorescence increased with time and stabilized after lo-20 min of incubation. The fluorescent cells were visible even with m-DNSC still present in the medium, suggesting that the interaction with the cells led to a change in the emission spectrum of m-DNSC

Amines and Endocytosis of anM Complexes 39

after ingestion by the cells. Free m-DNSC in solution showed a peak emission around 540 nm (excitation at 340 nm).

Fluorescence spectra of SDS-solubilized control fibroblasts, excitation at 340 nm, showed high fluorescence at 480 nm. This autofluorescence decreased sharply towards higher wavelengths (Figure 5A, broken curve). Cells treated with m-DNSC (30 min, 0.2 mM) showed a fluorescence spectrum with a peak at 520 nm, and a shoulder at 540 nm. The cell-associated fluorescence at 520 nm increased with duration of incubation with m-DNSC (Figure 5B), reaching sat- uration after 30 min of incubation.

The addition of SDS (0.2 mM, same final concentration as used to solubilize the cells) to m-DNSC did, however, shift the 540 nm emission peak to 520 nm and increased its relative fluorescence emission. Al-

, 1

I I I 1 1 1 0 10 20 30 40 50

t03x (pg o2M/ml medium)” Figure 2. Effect of Methylamine and pH on Kinetic Parameters of Endocytosis of ‘251-a2M-T by Fibroblasts in Culture

Double reciprocal transformation of initial endocytosis rate at different concentrations of ‘%a2M-T. The cells were pretreated for 1 hr prior to assay. (0) Control; (0) methylamine 30 mM; (Cl) pfi 8.0. The lines were fitted to the experimental points by the least-squares method.

100

00

60

0 0.2 0

Im-DNSCI (mM1

though cells solubilized in NaOH (0.01 N) instead of SDS still showed the blue shift to 520 nm, fluorescence was considered unreliable as a quantitative measure of m-DNSC internalization.

Effect of Amines on a&l Binding Binding of ‘251-labeled (Y~M-T complexes to fibroblast monolayers was measured at 4°C. Pretreatment of cells at 37°C with methylamine and m-DNSC resulted in a greater than 50% decrease of specific binding (Table 2). Cells treated in a similar manner at 4°C showed normal binding. Chloroquine and alkalinization to pH 8.0 were without effect on the extent of specific binding. These findings thus paralleled the results obtained on endocytosis. Pretreatment of the cells with a2M complexes was ineffective in counter- acting the inhibitory potency of m-DNSC on endocytosis or binding (Table 3; see also Figure 3B). Prein- cubation of monolayers for 4 hr with concentrations of (Y~M-T complexes high enough to saturate at least 90% of the endocytosis capacity (Van Leuven et al., 1979) did not affect the binding at 4°C or the endocytosis rate at 37°C (Table 3). A similar treatment in the presence of cycloheximide was also without sig- nificant effect; binding and uptake were somewhat decreased.

Discussion

The results show that preincubation of fibroblasts at 37’C with primary amines, such as methylamine, inhibits the internalization of azM-protease complexes, whereas simultaneous addition of the amine and labeled (u~M-T has only a minimal effect. This would indicate that the amines need to enter the cells for inhibition to become apparent. From the kinetic analysis it is clear that the effect of methylamine amounts to a reduction in maximal rate of uptake, while the affinity is increased. The latter finding and the binding studies at 4’C further indicate that binding of the cllnM complexes to the receptor is not interfered with.

Ol 0 7 4

TIME (HOURS)

Figure 3. Endocytosis of ‘25l-a&l-T by Fibro- blasts in Culture in the Presence of m-DNSC

Fibroblast monolayers were pretreated with either (A) different concentrations of m-DNSC for 30 min prior to assay or (6) 0.2 mM m- DNSC for increasing duration. In the latter case, zero time denotes the addition of m- DNSC and ‘*%a2M-T simultaneously to the cells. During the’assay (30 min). m-DNSC becomes inhibitory, although not as extensively as after 30 min pretreatment of the cells [com- pare to 0.2 mM m-DNSC on graph (A)]. Results are expressed relative to endocytosis of untreated cells and are the mean of two determinations on separate cell layers.

Cell 40

= t P

2 1.0 a” 0 a.

I I

0 10 20 30 40 50

103x 1 cg o2M/ml medium)”

Figure 4. Effect of m-DNSC on Kinetic Parameters of Endocytosis of ‘%a2M-T by Fibroblasts in Culture

Double reciprocal transformation of initial endocytosis rate at different concentrations of ‘%azM-T. The cell layers were pretreated for 30 min prior to assay. (0) Control: (0) m-DNSC 0.1 mM; (0) m-DNSC 0.2 mM. The lines were calculated from experimental data by the least-squares method.

Endocytosis and intracellular pH Primary amines affect intracellular (lysosomal) degradation and turnover of proteins presumably by raising the lysosomal pH after diffusion of the uncharged amines into the cell and the lysosomes. Leakage from the lysosome or any other process might then lead to a generalized cytoplasmic pH increase.

Several observations exclude the possibility that an intracellular pH change is responsible for the decreased endocytosis. Whereas methylamine affects degradation of anM complexes (T,,* = 5.3 hr), the results with chloroquine and pH 8.0 indicate that the effects on uptake and degradation are not functionally linked. Indeed, chloroquine (at 0.1 mM) inhibited degradation of apM intracellularly (TI12 >lO hr) but was ineffective in inhibiting uptake. At lower concentrations (0.05 mM), chloroquine even increased uptake of a*M complexes while remaining effective in block- ing degradation (TIIP = 6.7 hr). By addition of HCOs- without CO2 equilibration during the assays, the medium pH was raised to about 8.0 (5 mfvl HC03- added). Under these conditions intracellular degradation of a2M was inhibited (T1j2 = 8.3 hr) to about the same extent as with 30 mM methylamine or 0.05 mM chloroquine, while internalization of a2M complexes was unaffected. Kinetic analysis showed the effect of alkaline pH to be expressed by an increased affinity, while maximal uptake remained unchanged. M-DNSC was a much more potent inhibitor of uptake of a2M-protease complexes by the cells than methylamine, while intracellular degradation of anM complexes was only slightly affected by m-DNSC. This

again indicates that the effect of the different drugs on uptake and degradation are separate phenomena.

From these findings we conclude that the inhibitory effect of amines is not exerted via increased intracellular pH (Maxfield et al., 1979a) and is independent from the effect on intralysosomal degradation of a&t.

Amines and Number of Receptors Previous findings (Van Leuven et al., 1979) showed a close parallel between binding of a2M-T complexes to the cell surface receptors on the one hand and their subsequential internalization on the other. Thus, the amount of a&l complexes taken up is dictated by the fractional occupancy of the receptors, while maximal uptake is reached when all receptors are occupied by ligand, hence by the number of receptors available. Moreover, we have shown that the apparent affinity of internalization of a2M complexes corresponds to the apparent affinity of binding to the receptor (Van Leu- ven et al., 1979). Data on initial rates of uptake are therefore related to the number of receptors exposed at the cell surface, while affinity of uptake reflects affinity of binding of a2M complexes.

The inhibiting effect of amines is reflected by a decreased maximal rate of uptake, suggesting that the number of receptors exposed at the cell surface after treatment with amines is decreased. This inter- pretation is confirmed by direct measurements of the number of receptors at 4°C after preincubation with amines at 37°C.

Since the affinity of binding is not significantly al- tered, we conclude that the inhibitory action of amines on receptor-mediated endocytosis is due to a decrease in the number of receptors available to bind aeM-protease complexes.

Continuous Recycling of wM Receptors Our results show that the continuous exposure of cells to a2M complexes over 4 hr does not influence the capacity of the cells to internalize the complexes. This would mean that under these experimental conditions, there is no decrease in the number of receptors (down regulation) as was observed with receptor-mediated endocytosis of EGF (Carpenter and Cohen, 1976) and insulin (Gavin et al., 1974).

These and previous observations (reviewed by Goldstein et al. 1979) make the a2M system very similar to LDL. Continuous internalization and recycling of the receptor back to the surface is postulated by these authors. Indeed, a fast recycling could ac- count for the extremely high rate of uptake, with only a limited but nearly constant number of receptors exposed at the cell surface at any given moment. Synthesis of receptors seems only minimally involved, in view of the limited effect of protein synthesis inhib- itors such as cycloheximide, on uptake of a,M-complexes and LDL.

Our results further indicate that m-DNSC was


460 520 560 600

WAVELENGTH (nm)

B

TIME (MINUTES)

Figure 5. Ceil-Associated Fluorescence after Treatment of Fibroblast Monolayers with m-DNSC

Confluent cell layers were incubated with 0.2 mM m-DNSC at 37°C. At the times indicated cell layers were washed 5 times with cold

Table 2. Specific Receptor Binding of ‘25l-~$l-T Complexes to Fibroblasts in Culture

Specific Binding

Control

Methylamine (20 mM)

Absolute

23.8

Relative (%I

100

37T

4oc

Chloroquine (0.1 mM)

pH 8.0 tHCOJ- 5 mM)

m-DNSC (0.2 mM)

10.5 44

22.4 98

25.0 105

23.1 97

37% 8.8 37

4°C 23.6 99

Confluent cell layers were incubated for 1 hr (30 min for m-DNSC) with the different drugs tabulated before being cooled down to 4OC to measure binding or cooled down before addition of drugs. Specific binding, determined as described in Experimental Procedures, is expressed in ng a&f-T bound per mg cell protein. Determinations on two separate cell layers were averaged for total and nonspecific binding (Van Leuven et al., 1979).

equally effective in inhibiting endocytosis in the presence as in the absence of (Y~M-T complexes. If LY~M complexes would trigger the internalization of receptor, one would expect that under these circumstances depletion of receptors would occur faster than in the absence of ligand. Apparently this is not the case. We therefore propose that internalization and recycling of receptor is a continuous process, largely independent of occupation of receptor by apM complexes. One probable explanation for the effect of amines would be their interference with the recycling of receptor, ultimately leading to a depletion of receptors from the cell surface.

Amines, Substrates for Transglutaminase Our results clearly show a great similarity in the action of methylamine and m-DNSC. Both primary amines are only effective after preincubation of the cells, while removing the drugs results in a slow return to normal internalization of cYnM-trypsin complexes. From the kinetic analysis of inhibition it is obvious that the inhibition is due to a decreased maximal uptake rate, while the apparent affinity of internalization is increased. These similarities would indicate that meth-

medium and solubtized by incubation for 1 hr at 37’C in PBS containing 0.2% SDS. The resulting solutions were cleared by centrifugation (30 min. 10,000 x g). Fluorescence was measured in a Perkin Elmer fluorimeter with excitation wavelength at 340 nm. (A) Fluorescence emission spectrum of untreated cells (broken line) and cells treated with m-DNSC (0.2 mM. 30 min). (B) C&l-associated fluorescence at 520 nm with increasing time of pretreatment with m- DNSC (0.2 mM). Zero time indicates cells which did not-receive m- DNSC. No correction for autofluorescence was applied. (C) Fluores- cence microscopy of fibroblast monolayers treated with m-DNSC for 1.5 and 20 min. Magnification 500X.

Cell 42

from the present study, might in itself be sufficient to explain the decreased clustering of receptors into coated pits observed by Maxfield et al. (1979a) and Davies et al. (1980). On the &her hand, we have not excluded the possibility that inhibition of clustering and recycling of receptors are closely linked phenomena. It is even probable that receptors which do not become clustered into coated pits would escape the normal recycling route. How transglutaminase is involved in receptor-mediated endocytosis and at what particular step of receptor-recycling must await further experimentation.

Experimental Procedures

Table 3. Effect of Pretreatment of Cell Layers on Uptake and Binding of ‘251-a2M-T Complexes

Additions in Preincubation Uptake (%) Binding

w

None 100 100

a2M-bypsin 98 104

a2M-trypsin + cycloheximide 89 90

uZM-trypsin + m-DNSC (30’) 41 37

m-DNSC (30’) 35 39

Preincubation of cell layers was for 4 hr at 37°C. The additions were to final concentrations: a*M-T 100 pg/ml, cycloheximide 20 pg/ml, m-DNSC (added 30 min prior to assay) 0.2 mM. Uptake (at 37°C) and binding (at 4°C) of ‘25l-a2M-T were measured as described in Experimental Procedures and are expressed relative to control cultures kept under identical conditions, omitting the drugs and a&I-T. Figures shown are average of at least two determinations on separate cell lavers.

ylamine and m-DNSC act via a common mechanism. Lorand et al. (1979) found m-DNSC to be the most specific and active amine-substrate for transglutaminase. The fact that we found m-DNSC approximately two orders of magnitude more potent as an inhibitor compared to methylamine corresponds with the apparent affinities of these amine8 fTIea8Ured with purified preparations of the enzyme (Lorand et al., 1979). Moreover, as m-DNSC seems to enter the cell freely (Pincus et al., 1975, this work), it is noteworthy that we observed 50% inhibition of uptake at 0.2 mM m- DNSC, which is very close to the reported K,,, value for transglutaminase (Lorand et al., 1979). Finally, serine-borate (both at 40 mM), known to be a transition state inhibitor of transglutaminase (Tate and Meis- ter, 1978) and at these concentrations effective on transglutaminase in living cells (Spielberg et al., 1979), inhibits (r2M endocytosis to approximately 20% (results not shown).

These observations support an involvement of transglutaminase in endocytosis of (TIM complexes. Since EGF internalization was inhibited by m-DNSC to a similar extent (40%) as (YAM (results not shown), our results suggest that transglutaminase might be a common denominator in receptor-mediated endocytosis. Based on immunofluorescence, immuno-peroxidase and enzyme activity measurements, Maxfield et al. (1979a) and Davies et al. (1980) came to similar conclusions.

In these studies, however, primary amines were shown to inhibit clustering of (YAM and EGF receptors without affecting the amount pf binding. This finding contradicts our results, but since fibroblasts do bind anM in a “nonspecific” manner to an equal or even larger extent than the amount of a&complexes bound specifically (Van Leuven et al., 1979), the estimates of binding obtained by fluorescence microscopy might not be reliable.

A decreased number of exposed receptors, obvious

Materials Antibodies were obtained from Dakopatts (Denmark). Standard serum for a&t was from Behring Werke (Germany). m-DNSC. bovine serum albumin and crystalline trypsin (2X. bovine pancreas) were from Sigma. Carrier-free Na ‘? was from the Radiochemical Centre (Amer- sham, England).

Cell Culture Diploid human fibroblast cultures were obtained from skin biopsies of the forearm of healthy adult donors. The cells were grown in Dul- becco’s modified Eagle’s medium (DME; Grand Island Biological Co., New York), containing 10% (v/v) heat-inactivated newborn calf serum (NCS; Sera-Lab, England), 1 g/l NaHC03. -15 mM N tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid (Tes) and 4-(2-hydroxyethyl)-l -piperazineethane-sulfonic acid (HEPES) buffered to pH 7.4 with 1 N NaOH. All cell lines were karyotyped and monitored regularly for mycoplasma contamination. Between the tenth and fifteenth passage, an equal number of cells of each of six donors was pooled. After two subcultures the cells were plated in disposo trays (Linbro, Flow Laboratories, Scotland) at a density of 80,000 cells per well in DME containing 10% (v/v) newborn calf serum and placed in a 37’C incubator in an atmosphere of 5% CO*. 95% air and 100% humidity. After 48 hr the cells were used in the experiments.

Purification of d2M aZM was purified from human plasma. Only plastic labware was used and the entire procedure was done at 4°C. The plasma was treated with BaCln and BaSO, as described by Van Leuven et al. (1979). a&4 was precipitated between 4 and 8% (w/v) polyethylene glycol8000. The precipitate was suspended, dialyzed against PBS and fraction- ated on a column (5 x 94 cm) of Ultrogel ACA 22 (LKB, Sweden). The final purification step consisted of affinity chromatography on a column (2.6 x 90 cm) of Blue Sepharose (Pharmacia, Sweden). Eluent was a sodium phosphate buffer (25 mlvl. pH 7.7).

Ouantitation of apM after each step and in column fractions was carried out by rocket immunoelectrophoresis (Van Leuven. Cassiman and Van Den Berghe. 1978b). Protein was determined by the method of Lowry et al. (1951). with bovine serum albumin as standard. A typical purification procedure starting with 300 ml plasma resulted in a preparation containing 97% uZM. in a yield of 58%.

‘25l-a2M and ‘251-aaM-T Complexes aIM was iodinated using Na’? and chloramine-T as described by Van Leuven et al. (1979). After labeling, u&l was reacted with a 3 fold excess crystalline ‘tr/psin (on a molar basis). Complexes were separated from free trypsin as described by Van Leuven et al. (1979). Labeled a*M was stored at 4°C.

Uptake and Degradation of n,M at 37°C The cell layers grown in DME-newborn calf serum were washed 3 times with 1 ml serum-free medium without HCO.-, followed by a 15 min incubation at 37°C in this medium. The medium was then replaced


by 0.5 ml prewarmed DME containing ‘*‘l-a2M-T at the appropriate concentration. The trays were floated on a 37°C waterbath for the desired time period, the medium was removed and the cell layers were washed 4 times with ice-cold PBS. After an additional wash with EDTA (0.7 mM) in tris-buffered saline [2 mM Tris. 0.16 M NaCl (pfi 7.4)]. 0.5 ml of crystalline trypsin (0.5 mg/ml in EDTA solution) was added for 10 min at 37’C. The trypsinized cells were removed and cooled on ice. Each well was washed with 0.5 ml of cold PBS and this wash-fluid was combined with the cell suspension. The cell suspension was centrifuged (800 x g. 10 min) and the resulting cell pellet was resuspended and washed with 5 ml of cold PBS. After centrifugation the cell pellet was counted in an automatic y scintillation counter (PRIAS. Packard). Intracellular degradation of ‘?-c~~M was followed during an 4 hr period. The cell layers were pulsed for 30 min with ‘251-~2M-T. washed 3 times and further incubated in complete medium containing 10% (v/v) serum. At the time periods indicated, the cell layers were trypsinized as described above and counted.

Binding of ‘*%a2M at 4°C Binding studies were carried out at 4°C with the trays placed on crushed ice and for longer incubation in a 4°C refrigerator. The incubation media used in binding studies contained albumin (2 mg/ ml) to decrease nonspecific binding of apM. The cell layers were washed 3 times with cold DME containing bovine serum albumin (0.2%. w/v) and left in this medium for 15 min. The medium was removed and 0.5 ml DME containing ‘251-02M-T (2 pglml) was added. Specific binding was calculated from data with excess unlabeled a?M- T as described bv Van Leuven et al. (1979).

Acknowledgments

This work was supported by grants from the Belgian Cancer Fund (Algemene Spaar en Lijfrente Kas) and from Fonds voor Geneeskun- dig Wetenschappelijk Onderzoek. and by a grant from the Research Fund to K. U. Leuven. F. V. L. is a Post Doctoral Research Fellow of the American Cystic Fibrosis Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received December 3. 1979; revised January 28. 1980

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Primary amines inhibit recycling of α2M receptors in fibroblasts

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