The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.     

Effects of weak bases on the degradation of endogenous and exogenous proteins by rat yolk sacs.

In rat yolk sacs incubated in vitro, the rates of degradation of endogenous [3H]leucine-labelled proteins and of pinocytically ingested 125I-labelled bovine serum albumin were both decreased in the presence of either ammonium, methylammonium or ethylammonium ions (0-20 mM) or much lower concentrations of chloroquine (0-500 microM). These effects were also accompanied by an inhibition of pinocytosis, as measured by the rate of uptake of 125I-labelled polyvinylpyrrolidone, and by a fall in the [ATP]/[ADP] ratio within the tissue. Re-incubation in inhibitor-free medium of yolk sacs previously exposed to a weak base restored pinocytic and proteolytic capacities, except for tissues exposed to chloroquine at concentrations above 0.1 mM (these appeared to be cytotoxic); an attendent rise in [ATP]/[ADP] ratios to near normal values was also observed. Weak bases, at concentrations that fully arrested the breakdown of 125I-labelled albumin, failed to inhibit by more than 45% the degradation of [3H]leucine-labelled endogenous proteins. Since 125I-labelled bovine serum albumin has been shown to be degraded entirely intralysosomally by yolk sacs, this suggests either that the hydrolysis of endogenous proteins is shared between lysosomes and some other site or that, unlike 125I-labelled albumin, some endogenous proteins can be degraded within lysosomes at abnormally high pH.     

Effects of microbial proteinase inhibitors on the degradation of endogenous and internalized proteins by rat yolk sacs.

1. The effects of leupeptin and other microbial proteinase inhibitors were measured in rat yolk sacs on the uptake and degradation of formaldehyde-denatured 125I-labelled bovine serum albumin as well as on the degradation of 3H-labelled endogenous protein. 2. Leupeptin, at concentrations between 1 and 100 micrograms/ml, inhibits the degradation of added albumin without affecting pinocytic uptake. Accordingly large amounts of undegraded albumin accumulate within the tissue. 3. Removal of leupeptin produces a rapid recovery of the capacity to degrade albumin. 4. Endogenous protein degradation is rapidly inhibited by leupeptin, but to a far lesser extent than the breakdown of albumin. However, the inhibition is only slightly reversed on removal of leupeptin. 5. Degradation of both albumin and endogenous protein in intact yolk sacs is inhibited by the microbial proteinase inhibitors in the order: leupeptin greater than antipain greater than chymostatin; elastatinal, pepstatin and bestatin are ineffective. 6. Similar results are found when albumin is incubated in yolk-sac homogenates at pH 4 with the inhibitors. 7. The marked inhibitory effects of leupeptin, antipain and chymostatin suggest that cathepsin B and possibly cathepsin L participate in the degradation of 125I-labelled albumin in yolk sacs. By comparison, the smaller inhibitory effects of the proteinase inhibitors on endogenous protein breakdown imply a minor role of lysosomal cathepsins in this process.     

Ethanol-induced inhibition of pinocytosis and proteolysis in rat yolk sac in vitro

Pinocytic capture of 125I-labelled polyvinylpyrrolidone and of formaldehyde-denatured 125I-labelled bovine serum albumin by 17.5-day rat visceral yolk sacs incubated in vitro was rapidly and strongly inhibited by low concentrations (0.01 and 0.05%, v/v) of ethanol. The induced inhibition of pinocytosis was readily reversible, but a marked lag was observed before ethanol-exposed tissue regained its full proteolytic capacity towards the exogenous protein. These observations suggest that the acute administration of ethanol to a pregnant rat may give rise to concentrations of ethanol in the maternal blood and/or uterine fluid that induce dysfunction of the yolk sac. In late gestation such inhibition of yolk-sac function may interfere with the transfer of passive immunity across the yolk sac. If similar dysfunction is induced earlier in gestation, in the period before the chorioallantoic placenta is functional, this could cause a transient period of inhibition of histiotrophic nutrition that may be important to the pathogenic mechanism of action of ethanol as a teratogen.     

Metabolic interactions between animal cells through permeable intercellular junctions.

The isolated perfused rat liver was used to study the degradation of ¹²⁵I-labelled protein supplied in the perfusion medium. Formaldehyde-denatured proteins (human serum albumin, bovine serum albumin and especially rat liver phosphoenolpyruvate carboxykinase (GTP)) were taken up by the liver and degraded at high rates. Native human serum albumin was not degraded at significant rates by the perfused liver, while native phosphoenolpyruvate carboxykinase (GTP) was catabolised at about one-fourth the rate of the denatured enzyme. The degradation rate of denatured human serum albumin increased markedly as protein was added up to 0.7 mg, and more gradually with further increases in added protein. The biphasic nature of concentration dependence probably reflects the contribution of different cell types in the liver. Autoradiographic examination of serial biopsies taken during perfusion of the liver with formaldehyde-denatured, ¹²⁵I-labelled bovine serum albumin showed that at the cellular level the radioactivity was located predominantly in Kupffer and other non-parenchymal cells; and at the subcellular level the radioactivity was largely in endocytic vesicles, lysosomes and occasionally in the sinusoidal spaces. No significant radioactivity was found associated with other cytoplasmic organelles or the nucleus. It is concluded that lysosomes of the non-parenchymal cells are primarily responsible for the degradation of denatured extracellular protein that enters the liver.     

Mechanism of stimulation of pinocytosis by trypan blue

Trypan blue at 50 microgram/ml stimulates the pinocytic uptake of 125I-labelled PVP, but not of colloidal 198Au or formaldehyde-denatured 125I-labelled bovine serum albumin, by the 17.5-day rat visceral yolk sac incubated in vitro. Neither Trypan blue nor a combination of the dye with 125I-labelled PVP stimulated the rate of pinocytosis of liquid by the tissue. Trypan blue itself was shown to enter the yolk-sac cells by adsorptive pinocytosis. It is proposed that an interaction between Trypan blue and 125I-labelled PVP enables the latter substrate to enter the cells adsorptively by so-called 'piggy-back' pinocytosis.     

Rate of pinocytic capture of macromolecular substrates by rat yolk sac incubated in serum-free culture medium.

Pinocytic activity was quantified for rat yolk sacs incubated in a medium that was either serum-free or contained 10% (v/v) of calf serum. Absence of serum from the medium caused a small increase in the rate of pinosome formation, as determined by the rates of capture of both 125I-labelled poly(vinylpyrrolidone) and [14C]sucrose. In contrast, the rates of uptake of substrates ingested by adsorptive pinocytosis were greatly enhanced when serum proteins, which compete for the same binding sites on the plasma membrane as used by adsorbing substrates, were absent. Elimination of such competition greatly simplifies the quantitative analysis of the binding process, and permitted a detailed study of the binding to the plasma membrane of formaldehyde-denatured bovine serum albumin, a protein that is rapidly digested within the lysosomal system after its pinocytic capture. Binding obeyed Michaelis-Menten kinetics and showed a dissociation constant of approx. 1 micron, indicating the high affinity of this protein for binding sites on the surface of actively pinocytosing yolk-sac cells.     

A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.

The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis.     

Quantitative studies of pinocytosis. II. Kinetics of protein uptake and digestion by rat yolk sac cultured in vitro

Pinocytic uptake of 125I-labeled bovine serum albumin by 17.5-day rat visceral yolk sac cultured in vitro has been examined. Uptake was followed by intracellular digestion and, after an initial period, the content of radioactivity in the tissue itself remained constant during the incubation. Radiolabel was returned to the culture medium predominantly as (125I)iodotyrosine; exocytosis of undigested protein did not occur. The rate of uptake of labeled protein, which was constant within an experiment and reproducible between experiments, was much higher than that of a nondigestible macromolecule, 125I-labeled polyvinylpyrrolidone. The higher rate of uptake was a consequence of the protein entering the cells chiefly by adsorption to the plasma membrane being internalized; 125I-labeled albumin did not stimualte, nor did 125I-labeled polyvinylpyrrolidone inhibit pinocytosis. Different preparations of 125I-labeled albumin had characteristically different rates of uptake, probably reflecting differences in affinity for plasma membrane receptors. The physiological significance of the findings is discussed.     

Inhibition of pinocytosis in rat yolk sac by trypan blue.

Day 17.5 yolk sacs from rats injected with partially denatured 125I-labeled bovine serum albumin (I-BSA) were cultured in vitro by a raft technique. The rates of release of [125I]iodotyrosine were similar in control yolk sacs and in yolk sacs from rats preinjected with trypan blue. Day 17.5 rat yolk sacs were also cultured in medium containing I-BSA. Following pinocytic uptake the substrate was degraded intracellularly and [135I]iodotyrosine released into the medium. Trypan blue, when present in the medium in concentrations above 100 mug/ml, inhibited pinocytosis of I-BSA and so decreased the rate of [125I]iodotyrosine production. Trypan blue similarly decreased the rate of pinocytic uptake of 125I-labeled polyvinylpyrrolidone. Pinocytic uptake of macromolecules was not decreased in yolk sacs from rats pretreated with trypan blue. The relevance of these results to the mechanism of teratogenic action of trypan blue is discussed. It is proposed that if trypan blue in teratogenic doses similarly inhibits pinocytosis by the yolk sac during the organogenetic period teratogenesis might result from a transient interruption in the flow of metabolites through the yolk sac to the embryo.     

A comparative study of the effects of polyamino acids and dextran derivatives on pinocytosis in the rat yolk sac and the rat peritoneal macrophage

Poly-l-lysine, poly-l-α-ornithine, poly-l-glutamic acid, dextran, DEAE-dextran and dextran sulphate all fail to affect greatly the rate of pinocytic uptake of ¹²⁵I-labelled polyvinylpyrrolidone by 17.5-day rat visceral yolk sac or rat peritoneal macrophages cultured in vitro. It is concluded that these agents do not much affect the rate of pinocytic ingestion of liquid. Polycations accelerate the accumulation of colloidal ¹⁹⁸Au in both systems, and this is ascribed to the formation of substrate · modifier complexes which either adsorb to plasma membrane, and thus gain rapid entry, or initiate another mode of endocytosis. Pinocytic uptake of formaldehyde-denatured ¹²⁵I-labelled bovine serum albumin was accelerated by poly-l-lysine and poly-l-ga-ornithine in the macrophage, buth inhibited in the yolk sac.     

Rates of pinocytic capture of simple proteins by rat yolk sacs incubated in vitro.

The rates of pinocytic uptake of a number of small 125I-labelled simple proteins (insulin, ribonuclease A and lysozyme) by rat yolk sacs incubated in vitro were determined both before and after treating these proteins with reagents that are known to increase the rate of capture of 125I-labelled bovine serum albumin. Uptake of the untreated forms of all three proteins was extremely rapid, indicating that adsorptive pinocytosis is the principal mechanism by which yolk-sac cells capture these simple proteins, but these rates show no simple correlation with molecular charge. In contrast with albumin, the rates of uptake of treated proteins were either unchanged or lower than that of the corresponding untreated protein preparations; polymeric forms of 125I-labelled lysozyme larger than dimers were ingested at rates significantly lower than that of the monomer.     

Pinocytosis and degradation of exogenous proteins by cystinotic fibroblasts

Lysosomes of cystinotic human fibroblasts contain over 100-times the normal concentration of cystine. The high cystine concentration (probably in the millimolar range) might be expected to inhibit intralysosomal protein breakdown. A comparison of pinocytosis and degradation of five ¹²⁵I-labelled proteins (bovine serum albumin, formaldehyde-denatured bovine serum albumin, bovine pancreatic ribonuclease A and porcine lactate dehydrogenase isoenzymes H4 and M4) by human fibroblasts has been made, using one cystinotic and two normal cell-lines. The proteins each entered fibroblasts by adsorptive pinocytosis and were then degraded within the lysosomes by enzymes susceptible to leupeptin, the thiol-proteinase inhibitor. Each protein was captured by the fibroblasts at a characteristic rate, which was not different in cystinotic cells. Normal and cystinotic fibroblasts did not differ in their proteolytic capacity, as measured in extracts of disrupted cells. In intact fibroblasts, four of the five proteins were rapidly and fully digested following pinocytosis, in both cystinotic and normal cells. However, with formaldehyde-denatured albumin, the most resistant to degradation of the proteins tested, or with some other proteins in the presence of leupeptin, when the proteolytic capacity of lysosomes is diminished, intralysosomal degradation of pinocytosed protein was incomplete. Moreover, under these conditions, cystinotic cells demonstrated a lower rate of protein digestion than normal cells. It is concluded that pinocytic capture, rather than intralysosomal proteolysis, is commonly the rate-limiting step in the overall process of uptake and degradation of proteins by fibroblasts, and that intralysosomal cystine inhibits digestion of pinocytosed protein only in circumstances when degradation becomes the rate-limiting step.     

Pinocytosis in the rat visceral yolk sac. Effects of temperature, metabolic inhibitors and some other modifiers.

Low temperature,2,4-dinitrophenol and moniodoacetate could each completely abolish the pinocytic uptake of 125I-labelled polyvinylpyrrolidone, 125I-labelled bovine serum albumin or colloidal 198 Au by 17.5-day rat visceral yolk sac cultured in vitro. Cytochalasin B and colchicine caused a partial and dose-dependent inhibition. It is concluded that the mechanism of pinocytic uptake of these substrates is not micropinocytosis as conventionally defined. Removal of extracellular calcium or the presence of theophylline inhibited liquid-phase pinocytosis by the rat yolk sac, whereas addition of ouabain caused a biphasic response: a slight stimulation of pinosome formation at a low concentration, and an inhibitory effect at a higher concentration.     

Evidence that suramin and aurothiomalate are teratogenic in rat by disturbing yolk sac-mediated embryonic protein nutrition

Suramin (250 mg/kg) and sodium aurothiomalate (100 mg/kg) both induced congenital malformations in the offspring following treatment of pregnant rats at either 8.5 or 9.5 days of gestation. Conceptuses from 9.5-day pregnant rats were cultured for 48 h in homologous serum to which either suramin or sodium aurothiomalate was added for the final 6 h. The presence of suramin up to 5 mg/ml had no effect on the protein content of yolk sacs at harvesting, but at 10 mg/ml caused a significant decrease. In contrast sodium aurothiomalate increased the protein content of yolk sacs at harvesting, in a concentration-dependent manner up to 100 micrograms/ml. Neither suramin nor sodium aurothiomalate significantly affected embryo protein content. When 125I-labelled polyvinylpyrrolidone was added to the culture serum for the final 6 h of culture, radioactivity was found in the yolk sac at harvesting, but not in the embryo. When suramin (2-10 mg/ml) was present for the final 6 h of culture, the quantity of radioactivity measured in the yolk sac at harvesting was significantly decreased in a concentration-dependent manner. No radioactivity was detected in the embryos. Sodium aurothiomalate had no effect on the uptake of 125I-labelled polyvinylpyrrolidone. When rat serum whose proteins were labelled with [3H]leucine was used as culture medium, radioactivity was found in the conceptus (both yolk sac and embryo) at harvesting. Suramin (5 mg/ml), present for the final or penultimate 6 h, significantly decreased the uptake of radioactivity into conceptuses and caused a significant increase in the proportion of the captured radiolabel that was associated with the yolk sac. Sodium aurothiomalate (25 or 500 micrograms/ml) had no effect on the total uptake of radio-label but caused a significant increase in the proportion of total radioactivity captured that was associated with the yolk sac. These data indicate that suramin, by interfering with both the uptake and intralysosomal digestion of protein, and sodium aurothiomalate, by inhibiting digestion of captured protein, disturb the normal pathway of yolk sac-mediated protein utilization with a consequent diminution of the supply of amino acids to the conceptus. The effects of suramin are seen only at high concentration, those of sodium aurothiomalate at much lower concentrations. It is likely that the two drugs exert their teratogenic action by their effects on the yolk sac nutritional pathway with resultant amino acid deprivation of the conceptus at a critical stage of development.     

The effect of chemical treatments of albumin and orosomucoid on rate of clearance from the rat bloodstream and rate of pinocytic capture of rat yolk sac cultured in vitro.

Portions of a 125I-iodinated bovine serum albumin preparation were exposed to freezing, acetic acid (pH 3.5, 3.0 or 2.5), urea or formaldehyde, and the effect of these treatments on the rates of pinocytic uptake by yolk sacs from 17.5-day-pregnant rats cultured in vitro and of clearance from the rat bloodstream were studied. Uptake of albumin by the yolk sac was followed by rapid release of [125I]iodo-L-tyrosine into the culture medium. Similarly clearance of albumin in vivo was accompanied by the appearance of trichloroacetic acid-soluble radioactivity in the bloodstream. In both systems the rates of uptake of modified albumin preparations formed a series: formaldehyde or urea greater than acetic acid greater than freezing. The increased rates of uptake of modified albumin preparations could not be ascribed to the formation of aggregates nor, in the yolk-sac system, to an increase in the rate of pinosome formation. It is concluded that the various treatments to which the albumin was subjected increase to varying degrees the affinity of the albumin molecule for binding sites on that region of the plasma membrane from which pinocytic vesicles are formed. Some comparable experiments with native and desialylated human orosomucoid indicate that the rat yolk-sac epithelial cells do not possess the recognition system for uptake of asialoglycoproteins that exists on the surface of hepatic parenchymal cells.     

Degradation of insulin by human fibroblasts: effects of inhibitors of pinocytosis and lysosomal activity

The role of the pinosome-lysosome pathway in the degradation of 125I-labelled bovine insulin by cultured human fibroblasts was examined by comparing the effects of various known inhibitors of pinocytosis and lysosomal degradation on the uptake and degradation of 125I-labelled polyvinylpyrrolidone, formaldehyde-denatured bovine serum albumin and bovine insulin by these cells. Fibroblasts incubated with polyvinylpyrrolidone steadily accumulate this substrate, whereas incubations with insulin or denatured albumin led to the progressive appearance in the culture medium of [125I]iodotyrosine. Inhibitors of pinocytosis (bacitracin, colchicine and monensin), metabolic inhibitors (2,4-dinitrophenol and NaF), lysosomotropic agents (chloroquine and NH4Cl) and an inhibitor of cysteine-proteinases (leupeptin) decreased the rate of uptake of polyvinylpyrrolidone and denatured albumin very similarly, but only bacitracin had an effect on the processing of insulin. Chloroquine, NH4Cl and leupeptin strongly inhibited the digestion of denatured albumin, but not of insulin. The different responses to the modifiers, with polyvinylpyrrolidone and denatured albumin on the one hand and insulin on the other, suggest that insulin degradation can occur by a non-lysosomal pathway. The very strong inhibitory effect of bacitracin on insulin processing by fibroblasts may point to an important role of plasma membrane proteinases in insulin degradation.     

Glycoprotein catabolism in rat liver. Lysosomal digestion of iodinated asialo-fetuin

(125)I-labelled asialo-fetuin, administered intravenously, rapidly accumulates in rat liver and the radioactivity is subsequently cleared from the liver within 60min. Plasma radioactivity reaches a minimum between 10 and 15 min after injection and rises slightly during the period of liver clearance. Free iodide is the only radioactive compound found in plasma during this latter period. Fractionation of rat liver at 5 and 13min after injection of (125)I-labelled asialo-fetuin supports the hypothesis that asialo-glycoprotein is taken into liver by pinocytosis after binding to the plasma membrane and is then hydrolysed by lysosomal enzymes. At 5min, radioactivity was concentrated 23-fold in a membrane fraction similarly enriched in phosphodiesterase I, a plasma-membrane marker enzyme, whereas at 13min the radioactivity appeared to be localized within lysosomes. Separation of three liver fractions (heavy mitochondrial, light mitochondrial and microsomal) on sucrose gradients revealed the presence of two populations of radioactive particles. One population banded in a region coincident with a lysosomal marker enzyme. The other, more abundant, population of radioactive particles had a density of 1.13 and contained some phosphodiesterase, but very little lysosomal enzyme. These latter particles appear to be pinocytotic vesicles produced after uptake of the asialo-fetuin bound by the plasma membrane. Lysosomal extracts extensively hydrolyse asialo-fetuin during incubation in vitro at pH4.7 and iodotyrosine is completely released from the iodinated glycoprotein. Protein digestion within lysosomes was demonstrated by incubating intact lysosomes containing (125)I-labelled asialo-fetuin in iso-osmotic sucrose, pH7.2. The radioactive hydrolysis product, iodotyrosine, readily passed through the lysosomal membrane and was found in the external medium. These results are not sufficient to account for the presence of free iodide in plasma, but this was explained by the observation that iodotyrosines are deiodinated by microsomal enzymes in the presence of NADPH.     

HDL3 degradation by proteases in isolated rat intestinal mucosal cells

Human 125I-labelled HDL3 is degraded by isolated rat intestinal mucosal cells. In our experimental conditions, lipoprotein degradation occurred by two different mechanisms. In one, lipoprotein was degraded within the cell, following binding and internalisation. In the other, degradation occurred in the medium, which seemed to contain protease activity released from cells during incubation. Though lipoprotein-deficient serum apparently interfered with degradation in the medium, bovine serum albumin had no such effect. The lysosomal inhibitor, chloroquine, reduced degradation by 60% without inhibiting HDL binding. Intestinal cell extracts contained at least two different proteases, with pH optima of 4.5 and 8.0, respectively. Comparing HDL and LDL degradation on a molar basis, more HDL particles were degraded by the cell-free extracts at pH 4.5. This degradation was activated by dithiothreitol and was inhibited by iodoacetic acid. From these observations we conclude that HDL3 is taken up by the rat intestinal mucosal cell through a specific binding site and subsequently degraded by a thiol-dependent protease in the lysosome.     

Lactate, alanine and glutamine metabolism in isolated canine pup liver cells

125I-Labelled alpha 2-macroglobulin-trypsin complex (125I-labelled alpha 2-macroglobulin X trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37 degrees C. The association of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin X trypsin was inhibited by unlabelled alpha 2-macroglobulin X trypsin with a half-inhibition constant of about 8 micrograms/ml (11 nM). 125I-Labelled alpha 2-macroglobulin became cell-associated to a smaller extent (10-40% of that of alpha 2-macroglobulin X trypsin) and the half-inhibition constant was about 35 micrograms/ml in adipocytes. The cell association of 125I-labelled alpha 2-macroglobulin X trypsin was markedly inhibited by dansylcadaverine, bacitracin, omission of Ca2+ from the medium or pretreatment of the cells with trypsin. After incubation for 180 min more than 60% of the cell-associated 125I-labelled alpha 2-macroglobulin X trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized material. 125I-Labelled alpha 2-macroglobulin X trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 micrograms/ml 125I-labelled alpha 2-macroglobulin was approx. 40% of that of 125I-labelled alpha 2-macroglobulin X trypsin. Degradation of 125I-labelled alpha 2-macroglobulin X trypsin was abolished by a high concentration (0.5 mg/ml) of alpha 2-macroglobulin X trypsin. It is concluded that alpha 2-macroglobulin X trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.