Cellular uptake of soluble and aggregated ferritin: distinction between pinocytosis and phagocytosis.

Cellular uptake of ferritin amounting to 0-5 mug/mg cell protein or more can be measured colorimetrically on the basis of ferritin-iron content. 131I-serum albumin, soluble ferritin and aggregated ferritin used in equimolar concentrations are taken up differently by Sarcoma SI80 cells in culture. The net uptakes in 2 h at 37 degrees C are 0-065, 4-3 and 24-7 mug/mg cell protein or 0-93, 8-0 and 45-7 mumol, respectively. Albumin uptake is not inhibited by a 26-fold molar ferritin excess but is significantly inhibited by a 43-fold excess. The transport mechanism of the ferritins differs from that of albumin in that it is significantly inhibitable by 2 times 10(-4) M monoiodoacetate. Soluble ferritin contains small aggregates which are removed by filtration through Millipore membranes of 0-05, 0-1 and 0-22 mum. When the 0-1-mum filtrate is re-examined, uptake is no longer inhibited by iodoacetate. Since it can be inferred from other work that albumin is taken up by pinocytosis and ferritin aggregates by phagocytosis, the difference in susceptibility to inhibition is proposed as a way to distinguish pinocytosis from phagocytosis. Ferritin may form larger visible aggregates in culture medium. The transport mechanism of this aggregated ferritin differs from that of soluble unfiltered ferritin in that it causes concomitant enhancement of albumin uptake. Albumin transported by virtue of this effect becomes partially susceptible to iodoacetate. Thus, in addition to a distinction between pinocytosis and phagocytosis, our data single out 2 forms of albumin transport and 3 forms of ferritin transport.     

Protein transport: A selective membrane mechanism

Proteins are selectively sequestered by a number of cell types. However, only in oocytes is the process sufficiently aggravated and specific to be readily studied. In these cells certain serum proteins are taken up in proportions different from those found in the serum. In vitro incubations of hormonally stimulated and synchronous mosquito oocytes show that the only protein capable of initiating the transport process is the female specific yolk protein. Heterologous proteins such as IgG, bovine serum albumin, cytochrome C, and ferritin are inactive. The female specific protein is a phosphoglycolipoprotein. It is synthesized in the fat body, a liver analog in the insect, and passed into the serum before being transported into the oocytes. Preliminary kinetic analysis shows the uptake process to be specific with an apparent K_m of about 10^?7 M. Glycolytic inhibitors stop protein uptake. The receptor-mediated binding steps in the transport process are most easily studied in the chicken because of the enormous amount of oocyte membrane available from a given oocyte and because up to 1 gm of protein is normally transported per day per oocyte. IgG and the hen specific phosvitin lipovitellin are two of the physiologically important proteins that are transported intact into the chicken oocytes. The uptake appears selective as shown by studies with iodinated proteins. Ferritin conjugated to IgG is shown by electron microscopy to bind to isolated plasma membranes only where coated pits have formed, whereas ferritin alone is not seen localized on any membrane surface. These very specialized regions of the membrane are similar to micropinocytotic pits but, in addition, possess on their cytoplasmic side dense ridges that form the coat. Transport involves binding to the coated pits, the pinching off of the pits, and the subsequent movement of the coated vesicles in the cytoplasm.     

Endocytosis of native and cationized ferritin by intralobular duct cells of the rat parotid gland

Summary The ability of the intralobular ducts of the rat parotid gland to take up protein from the lumen was examined after retrograde infusion of native and cationized ferritin. At high concentrations (3–10 mg/ml), cells of both intercalated- and striated ducts avidly internalized the tracers. No differences were noted in the mode of uptake or fate of native or cationized ferritin. Large, apical ferritin-containing vacuoles up to 5 m in size were present in cells of the intercalated ducts after infusion for 15 min. Small, smooth-surfaced spherical or flattened vesicles and tubules containing ferritin were also observed, often in association with the large vacuoles. Ferritin uptake increased with increasing infusion time, up to 1 h. Uptake by the striated ducts was less consistent than by the intercalated ducts, and occurred mainly in small vesicles and tubules. Secondary lysosomes became labeled with ferritin in both cell types. Ferritin was not observed in the Golgi saccules, nor was it discharged from the cells at the basolateral surfaces. At low concentrations (0.3–1 mg/ml), uptake was reduced, especially by cells of intercalated ducts, and differences were noted in the behavior of the two tracers. Cationized ferritin was internalized mainly into vesicles and tubules of cells of striated ducts; little uptake of native ferritin occurred at low concentrations. These results demonstrate that the ductal cells of the salivary glands are capable of luminal endocytosis of foreign proteins. They also suggest that in addition to modifying the primary saliva by electrolyte reabsorption and secretion, and secretion of various glycoproteins, the ductal cells are able to reabsorb proteins secreted by the acinar cells.     

Protein Uptake and Digestion in Bloodstream and Culture Forms of Trypanosoma brucei*

SYNOPSIS. The mechanisms of ferritin uptake and digestion differ in bloodstream and culture forms of Trypanosoma brucei. Ferritin enters bloodstream forms from the flagellar pocket by pinocytosis in large spiny-coated vesicles. These vesicles become continuous with straight tubular extensions of a complex, mostly tubular, collecting membrane system where ferritin is concentrated. From the collecting membrane system the tracer enters large digestive vacuoles. Small spiny-coated vesicles, which never contain ferritin, are found in the Golgi region, fusing with the collecting membrane system, and around the flagellar pocket. Acid phosphatase activity is present in some small spiny-coated vesicles which may represent primary lysosomes. This enzymic activity is also found in the flagellar pocket, pinocytotic vesicles, the collecting membrane system, the Golgi (mature face), and digestive vacuoles of bloodstream forms. About 50% of the acid phosphatase activity of blood forms is latent. The remaining nonlatent activity is firmly cell-associated and probably represents activity in the flagellar pocket. The structures involved in ferritin uptake and digestion are larger and more active in the short stumpy than in the long slender bloodstream forms. The short stumpy forms also have more autophagic vacuoles. No pinocytotic large, spiny-coated vesicles or Golgi-derived, small spiny-coated vesicles are seen in culture forms. Ferritin leaves the flagellar pocket of these forms and enters small smooth cisternae located just beneath bulges in the pocket membrane. The tracer then passes through a cisternal collecting membrane network, where it is concentrated, and then into multivesicular bodies. In the culture forms, acid phosphatase activity is localized in the cisternal system, multivesicular bodies, the Golgi (mature face), and small vesicles in the Golgi and cisternal regions. The flagellar pocket has no acid phosphatase activity, and almost all the activity is latent in these forms. The culture forms do not release acid phosphatase into culture medium during 4 days growth. Uptake of ferritin by all forms is almost completely inhibited by low temperature. These differences among the long slender and short stumpy bloodstream forms and culture forms are undoubtedly adaptive and reflect different needs of the parasite in different life cycle stages.     

Uptake of long-chain fatty acid methyl esters by mammalian cells

Albumin-bound long-chain fatty acid methyl esters (ME) were taken up and utilized by Ehrlich ascites tumor cells and slices of rat heart, liver, and kidney. Much more ME than albumin was taken up by the tumor cells, indicating that ME dissociated from the carrier protein during their uptake. 70-80% of the radioactivity associated with the cells after 1 min of incubation at 37 degrees C remained as ME. The results of studies with metabolic inhibitors and glucose suggest that uptake of ME is an energy-independent process. Changes in incubation medium pH between 7.8 and 6.5 did not markedly alter uptake of ME. Cells incubated with FFA and methanol did not synthesize ME. These findings indicate that ME are taken up intact, and they suggest that the presence of an anionic carboxyl group is not essential for the binding of a long-chain aliphatic hydrocarbon to a mammalian cell. When incubation with labeled ME was continued for 1 hr, increasing amounts of radioactivity were recovered in FFA, phospholipids, neutral lipid esters, and CO(2). ME radioactivity associated with the cells after a brief initial incubation was released in the form of ME and FFA when the cells were incubated subsequently in a medium containing albumin. If the second incubation medium contained no albumin, most of the ME radioactivity initially associated with the cells was incorporated into phospholipids, neutral lipid esters, and CO(2). These results suggest that much of the ME which is taken up, is hydrolyzed to FFA, and that the fatty acids derived from ME are available for further metabolism.     

Uptake of free hemoglobin by rat liver parenchymal cells

Parenchymal cells isolated from rat liver are capable of taking up free hemoglobin. Uptake was saturable with a concentration for half-maximal velocity of 1.35 mg/ml (1.99 X 10(-5) M) hemoglobin. At a concentration of 0.088 mg/ml, the endocytic index for hemoglobin uptake was 4.5 microliters/h per mg of cell protein. This may be compared with the rate of fluid pinocytosis by these cells of 0.025 microliter/h per mg of cell protein (determined with yeast invertase as the marker). Free beta globin chains were also taken up with an endocytic index of 26.7 microliters/h per mg of cell protein at a beta chain concentration of 0.075 mg/ml. Hemoglobin inhibited the uptake of labeled beta globin. Hemoglobin-haptoglobin complex at a concentration of 0.12 mg/ml (as hemoglobin) was cleared at a rate of 0.89 microliter/h per mg cell protein and its uptake was also inhibited by free hemoglobin. We conclude that haptoglobin serves to conserve the iron of hemoglobin by preventing its renal clearance and not by promoting its hepatic uptake.     

Effect of chloroquine on cultured fibroblasts: release of lysosomal hydrolases and inhibition of their uptake.

Incubation of normal human fibroblasts with 1–5 μM chloroquine at physiological pH for 8 hr produces granular cytoplasmic inclusions, release of lysosomal enzymes into the medium and decrease of intracellular lysosomal enzyme activities. The effects are dose dependent and reversible. The uptake of arylsulfatase A into fibroblasts genetically deficient in arylsulfatase A (grown from skin biopsies of patients with metachromatic leukodystrophy) is completely inhibited by pretreating the cells with 5 μM chloroquine. Arylsulfatase A, which has been taken up as exogenous enzyme from the medium into the cells, is partially released into the culture medium upon incubation with chloroquine. The data suggest that chloroquine competes with the binding of lysosomal enzymes to the cell membrane and to the membranes of pinocytotic vacuoles and causes release of previously internalized exogenous enzyme.     

Synthesis of ferritin by neuroblastoma

Ferritin is an iron-containing protein which is a normal component of serum. The levels of ferritin are increased in the sera of some children with neuroblastoma, and this increase appears to be a potent indicator of prognosis. To determine whether synthesis of ferritin by the tumor cells contributes to these increased serum levels, we examined incorporation of radiolabeled leucine by CHP 126, a neuroblastoma derived cell line, into ferritin. Using sequential immunoprecipitation and gel electrophoresis of sonicates from cells maintained in medium containing iron in amounts standard for tissue culture, incorporation of label into ferritin was 0.04% of that into total protein synthesized over the same time period. Addition of up to 40 micrograms of iron as ferric ammonium citrate increased ferritin synthesis to a maximum of 0.16% without altering synthesis of total protein. The pattern of iron-induced enhancement in the neuroblastoma cells was similar to that which was seen using Chang liver cells, a cell line well known to be capable of ferritin synthesis. These results confirm that neuroblastoma cells can synthesize ferritin and that synthesis is regulated by exogenous iron.     

Uptake and transport of intact macromolecules in the intestinal epithelium of carp (Cyprinus carpio L.) and the possible immunological implications

Summary Two protein antigens, horseradish peroxidase (HRP) and ferritin, have been administered to the digestive tract of carp. Electron-microscopical observations reveal considerable absorption of both antigens in the second segment of the gut (from 70 to 95% of the total length) and also, although to a lesser extent, in the first segment (from 0 to 70% of the total length). Even when administered physiologically with food, a large amount of ferritin is absorbed by enterocytes in the second gut segment.HRP and ferritin are processed by enterocytes in different ways. HRP seems to adhere to the apical cell membrane, probably by binding to receptors, and is transported in vesicles to branched endings of lamellar infoldings of the lateral and basal cell membrane. Consequently, most of the HRP is released in the intercellular space where it contacts intra-epithelial lymphoid cells. Only small amounts of HRP become localized in secondary lysosomes of enterocytes. Ferritin does not bind to the apical cell membrane; after uptake by pinocytosis, it is present in small vesicles or vacuoles that appear to fuse with lysosome-like-bodies. In the second segment, intact ferritin ends up in the large supranuclear vacuoles (after 8 h), where it is digested slowly. Although no ferritin is found in the intercellular space, ferritin-containing macrophages are present between the epithelial cells, in the lamina propria and also to a small extent in the spleen. The transport of antigens from the intestinal lumen, through enterocytes, to intra-epithelial lymphoid cells or macrophages may have immunological implications, such as induction of a local immune response and prospectives for oral vaccination.     

Characterization of [3H]Dopamine Uptake Sites and [3H]Cocaine Recognition Sites in Primary Cultures of Mesencephalic Neurons During In Vitro Development

[3H]Dopamine uptake and [3H]cocaine binding sites were studied in primary cultures of ventral mesencephalon from 14-day-old rat embryos. Specific binding sites for [3H]cocaine and [3H]mazindol were detected only in intact cell cultures of ventral mesencephalon, and were absent in sonicated, washed membranes prepared from these cell cultures. [3H]Cocaine was not taken up by the cells through an active transport process because [3H]cocaine binding occurred also at 4 degrees C. Moreover, the possibility of [3H]cocaine entering the cells by passive diffusion and ion trapping was also excluded because extensive washing failed to remove [3H]cocaine from the cells. [3H]Cocaine binding was reduced to 6% of control when cells were permeabilized with streptolysin O (0.2 U/ml, 5 min). Taken together, these results suggest that in cultured mesencephalic neurons, [3H]cocaine may enter the cell by passive diffusion and then be sequestered by a cytosolic compartment that is lost in the process of permeabilization or sonication and washing of membrane preparations. Permeabilization of cultured neurons failed to alter the storage of [3H]dopamine. When cells were permeabilized with streptolysin O (0.2 U/ml; 5 min) after [3H]dopamine was taken up, [3H]dopamine was retained by the cells and did not leak into the incubation medium, indicating that [3H]dopamine was stored in sites that could not pass through the perforated membranes. In contrast, [3H]dopamine uptake into already permeabilized cells was reduced by 33%, suggesting that a cytosolic protein that had leaked out may play a functional role in the uptake process. In contrast to striatal membrane preparations of adult rats, [3H]cocaine binding in intact mesencephalic cell cultures was Na+ independent.(ABSTRACT TRUNCATED AT 250 WORDS)     

High-density lipoprotein particle uptake and selective uptake of high-density lipoprotein-associated cholesteryl esters by J774 macrophages

High-density lipoprotein (HDL) cholesteryl esters are taken up by fibroblasts via HDL particle uptake and via selective uptake, i.e., cholesteryl ester uptake independent of HDL particle uptake. In the present study we investigated HDL selective uptake and HDL particle uptake by J774 macrophages. HDL3 (d = 1.125-1.21 g/ml) was labeled with intracellularly trapped tracers: 125I-labeled N-methyltyramine-cellobiose-apo A-I (125I-NMTC-apo A-I) to trace apolipoprotein A-I (apo A-I) and [3H]cholesteryl oleyl ether to trace cholesteryl esters. J774 macrophages, incubated at 37 degrees C in medium containing doubly labeled HDL3, took up 125I-NMTC-apo A-I, indicating HDL3 particle uptake (102.7 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein). Apparent HDL3 uptake according to the uptake of [3H]cholesteryl oleyl ether (470.4 ng HDL3 protein/mg cell protein per 4 h at 20 micrograms/ml HDL3 protein) was in significant excess on 125I-NMTC-apo A-I uptake, i.e., J774 macrophages demonstrated selective uptake of HDL3 cholesteryl esters. To investigate regulation of HDL3 uptake, cell cholesterol was modified by preincubation with low-density lipoprotein (LDL) or acetylated LDL (acetyl-LDL). Afterwards, uptake of doubly labeled HDL3, LDL (apo B,E) receptor activity or cholesterol mass were determined. Preincubation with LDL or acetyl-LDL increased cell cholesterol up to approx. 3.5-fold over basal levels. Increased cell cholesterol had no effect on HDL3 particle uptake. In contrast, LDL- and acetyl-LDL-loading decreased selective uptake (apparent uptake 606 vs. 366 ng HDL3 protein/mg cell protein per 4 h in unloaded versus acetyl-LDL-loaded cells at 20 micrograms HDL3 protein/ml). In parallel with decreased selective uptake, specific 125I-LDL degradation was down-regulated. Using heparin as well as excess unlabeled LDL, it was shown that HDL3 uptake is independent of LDL (apo B,E) receptors. In summary, J774 macrophages take up HDL3 particles. In addition, J774 cells also selectively take up HDL3-associated cholesteryl esters. HDL3 selective uptake, but not HDL3 particle uptake, can be regulated.     

Uptake of ferritin and iron bound to ferritin by rat hepatocytes: modulation by apotransferrin, iron chelators and chloroquine

Rat liver ferritin is an effective donor of iron to rat hepatocytes. Uptake of iron from ferritin by the cells is partially inhibited by including apotransferrin in the culture medium, but not by inclusion of diferric transferrin. This inhibition is dependent on the concentration of apotransferrin, with a 30% depression in iron incorporation in the cells detected at apotransferrin concentrations above 40 micrograms/ml. However, apotransferrin does not interfere with uptake of 125I-labeled ferritin, suggesting that apotransferrin decreases retention of iron taken up from ferritin by hepatocytes by sequestering a portion of released iron before it has entered the metabolic pathway of the cells. The iron chelators desferrioxamine (100 microM), citrate (10 mM) and diethylenetriaminepentaacetate (100 microM) reduce iron uptake by the cells by 35, 25 and 8%, respectively. In contrast, 1 mM ascorbate increases iron accumulation by 20%. At a subtoxic concentration of 100 microM, chloroquine depresses ferritin and iron uptake by hepatocytes by more than 50% after 3 h incubation. Chloroquine presumably acts by retarding lysosomal degradation of ferritin and recycling of ferritin receptors.     

Concanavalin A-Induced Morphological Changes and Its Binding Sites in Starfish Follicle Cells as Revealed by Electron Microscopy

Concanavalin A (Con A) stimulates the production in starfish follicle cells of 1-methyladenine, a hormone which induces oocyte maturation. We have therefore investigated Con A-induced morphological changes and Con A-binding sites in the follicle cell using native Con A and horseradish peroxidase- or ferritin-labeled Con A (HRP-Con A, Fer-Con A). After isolated follicle cells were incubated with Con A (1 mg/ml), vacuoles, the Golgi complex and multivesicular body-like organelles (MVBs) became prominent in most of the cells. After follicle cells were prefixed and then incubated with Fer-Con A for 60 min, tagged ferritin was diffusely and randomly distributed as single or small clustered particles on the cell surface. The incubation of isolated follicle cells with Fer-Con A for 10 min before fixation resulted in numerous ferritin particles localized along the internalized membrane, and also in vacuoles, MVBs and small lysosome-like structures. After 60 min incubation with Fer-Con A, ferritin was further located in large lysosome-like structures and in vesicles near and in the Golgi area as well as in the organelles described above. HRP-Con A binding sites were also observed in vacuoles and MVBs of the intact cells.
These results suggest that Con A binds at first to the cell surface and causes rapid internalization and that membrane-bound Con A is easily endocytosed into vacuoles, MVBs and lysosome-like structures, and is later incorporated in some vesicles in the Golgi area.     

Segregation of Ferritin in Glomerular Protein Absorption Droplets

Ferritin was used as a tracer to study the mechanism by which proteins are segregated into droplets by the visceral epithelium of glomerular capillaries. In glomeruli from both normal and aminonucleoside-nephrotic rats ferritin molecules introduced into the general circulation penetrated the endothelial openings and were seen at various levels in the basement membrane. Striking differences between nephrotic and controls were seen only in the amount of ferritin incorporated into the epithelium. In normal animals, a few ferritin molecules were seen in small invaginations of the cell membrane limiting the foot processes, within minute vesicles in the epithelium, or within occasional large vacuoles and dense bodies. In nephrotics, epithelial pinocytosis was marked, and numerous ferritin molecules were seen within membrane invaginations and in small cytoplasmic vesicles at all time points. After longer intervals, the concentration of ferritin increased in vacuoles and particularly within the dense bodies or within structures with a morphology intermediate between that of vacuoles and dense bodies. In nephrotic animals cleft-like cavities or sinuses were frequently encountered along the epithelial cell surface facing the urinary spaces. Some of these sinuses contained material resembling that filling the dense bodies except that it appeared less compact. The findings suggest that ferritin molecules—and presumably other proteins which penetrate the basement membrane—are picked up by the epithelium in pinocytotic vesicles and transported via the small vesicles to larger vacuoles which are subsequently transformed into dense bodies by progressive condensation. The content of the dense bodies may then undergo partial digestion and be extruded into the urinary spaces where it disperses. The activity of the glomerular epithelium in the incorporation and segregation of protein is similar in normal and nephrotic animals, except that the rate is considerably higher in nephrosis where the permeability of the glomerular basement membrane is greatly increased.     

Uptake of foreign ferritin in platy Xiphophorus maculatus (Poeciliidae: Teleostei)

The ability and capacity of various tissues in platy Xiphophorus maculatus L. to take up horse-spleen ferritin injected into the blood stream are described. Ferritin was injected intraperioneally, and the cellular uptake was demonstrated as Prussian blue precipitations in tissues treated with acid ferrocyanide solutions. Ferritin was detected within the heart endocardial cells and macrophages in the trunk kidney and spleen, 1/4 h after the injection, i.e. foreign ferritin was taken up very rapidly by these cells. When the time elapsed between the ferritin injection and sacrifice exceeded 6 h, these cells, and also macrophages in the gill and intestine, were almost completely filled with ferritin. At these stages, however, the amounts of Prussian blue precipitations per volume unit of the tissue were much larger in the heart than in the other organs studied in the present work, i.e. the endocardial tissue seems to play an important role in the clearance of the blood circulation in this species. We suggest that this tissue in platy is specialized to endocytose waste and foreign macromolecules, including pathogenic particles, from the blood stream. The eosinophilic and neutrophilic granulocytes do not appear to take up foreign ferritin, i.e. these cells may play no endocytotic role in the clearance of foreign macromolecules in platy.     

Bile acid uptake by isolated intestinal mucosa cells of guinea pig

Isolated intestinal mucosa cells of the guinea pig were employed to study intestinal transport of bile acids. Chenodeoxycholate and lithocholate were rapidly taken up into jejunal and ileal cells by diffusion. Taurocholate and cholate however showed only a minor diffusion rate and were preferentially taken up by the ileal bile acid carrier. This uptake was saturable with an apparent K_m of 231 μM and V of 7 nmol/mg protein per min for taurocholate; this bile acid was accumulated 90-fold. Its uptake was strongly inhibited by antimycin A, FCCP, ouabain or Na⁺-deficiency in the medium. Sugars or amino acids did not interfere with uptake. Experimental conditions were optimized with regard to incubation medium, cell amount, cell age and length of preincubation. It is concluded that ileal cells of the guinea pig are superior to other experimental models for characterizing the ileal bile acid carrier, because they allow us to determine initial rates of uptake and have a very efficient energetic coupling.     

Association of ferritin with liver cell membrane fractions.

Following intraperitoneal injection of rats with a large dose of ferric ammonium citrate containing ⁵⁹Fe, some 35 to 50% of the dose was deposited in the liver within the first 1 to 4 h. Almost all of the deposited iron could be precipitated with a ferritin antiserum from a homogenate of liver heated to 75 °C, but only half of this was precipitable when the unheated homogenate was treated with antiserum. The remainder of the ferritin iron was made available to the antiserum by treatment with deoxycholate, and was therefore presumed to be associated with membranous components of the cell.Subcellular fractionation of the liver following administration of ⁵⁹Fe-labeled ferric ammonium citrate showed that most of the radioactivity deposited within the first 4 h was equally divided between the cell sap and a light microsome (membrane-rich) fraction. Ferritin in this latter fraction was made available to antibody following deoxycholate treatment. The liver microsome fraction of the young rat contains little unavailable ferritin, but with aging there is an accumulation of ferritin in the microsomal fraction which is unavailable to antibody until the membrane is removed.It is suggested that at least part of the injected iron salt is taken up by pinocytotic vesicles and transferred to ferritin within this fraction, possibly followed by release of some of this ferritin into the cell sap.     

Transient holes in the erythrocyte membrane during hypotonic hemolysis and stable holes in the membrane after lysis by saponin and lysolecithin

Ferritin and colloidal gold were found to permeate human erythrocytes during rapid or gradual hypotonic hemolysis. Only hemolysed cells contained these particles; adjacent intact cells did not contain the tracers. Ferritin or gold added 3 min after the onset of hypotonic hemolysis did not permeate the ghost cells which had, therefore, become transiently permeable. By adding ferritin at various times after the onset of hemolysis, it was determined that for the majority of the cells the permeable state (or interval between the time of development and closure of membrane holes) existed only from about 15 to 25 sec after the onset of hemolysis. It was possible to fix the transient "holes" in the open position by adding glutaraldehyde only between 10 and 20 sec after the onset of hemolysis. The existence of such fixed holes was shown by the cell entry of ferritin and gold which were added to these prefixed cells. Membrane defects or discontinuities (of the order of 200–500 A wide) were observed only in prefixed cells which were permeated by ferritin subsequently added. Adjacent prefixed cells which did not become permeated by added ferritin did not reveal any membrane discontinuities. Glutaraldehyde does not per se induce or create such membrane defects since cells which had been fixed by glutaraldehyde before the 10-sec time point or after the 180-sec time point were never permeable to added ferritin, and the cell membranes never contained any defects. It was also observed that early in hemolysis (7–12 sec) a small bulge in one zone of the membrane often occurred. Ghost cells produced by holothurin A (a saponin) and fixed by glutaraldehyde became permeated by ferritin subsequently added, but no membrane discontinuities were seen. Ghosts produced by lysolecithin and fixed by glutaraldehyde also became permeated by subsequently added ferritin, and many membrane defects were seen here (about 300 A wide).     

THE DISTRIBUTION OF EXOGENOUS FERRITIN IN TOAD SPINAL GANGLIA AND THE MECHANISM OF ITS UPTAKE BY NEURONS

Toad spinal ganglion cells are individually enclosed in sheaths consisting of one or more attenuated layers of satellite cell cytoplasm surrounded externally by a basement membrane. Narrow (~150 A) extracellular channels separate these layers from one another and from the underlying neuron. In both in vivo and in vitro experiments it was found that molecules of ferritin, a water-soluble protein, are to some extent able to pass across the basement membrane and through these channels to reach the neuronal plasma membrane. Ferritin particles arriving at the neuronal surface are engulfed by the neuron in 0.1 to 0.2 µ "coated" vesicles. The concentration of ferritin in these vesicles is higher than in the perineuronal space. The ferritin incorporated into the neuron is segregated, apparently intact, in multivesicular bodies. It is inferred that the 150A channels in the satellite cell sheath are patent, aqueous spaces through which molecules with a diameter as large as 95 A are able to pass, and that these neurons are capable of taking up whole protein from their immediate environment by the process of pinocytosis.     

Biosynthesis of ferritin in rat hepatoma cells and rat livers. II. Binding of iron by ferritin protein.

Ferritin and its protein subunits in rat hepatoma cell clone M-5123-C1 were biosynthetically labeled with [14C]leucine and 59Fe. Radioimmunoassays of ferritin/apoferritin and of protein subunits in the free polyribosome, membrane-bound polyribosome, smooth membrane, and cytosol fractions were done with ferritin-specific and subunit-specific rabbit IgG antibodies at various time intervals after pulsing. Much more 59Fe was bound by ferritin/apoferritin than by subunits in all of the cell fractions. Binding of iron to subunits may have been a random process. When hepatoma cells were simultaneously pulse-labeled with 59Fe and [14C]leucine, uptake of much of the 59Fe by ferritin occurred relatively early, in comparison to incorporation of [14C]leucine, in all of the cell fractions examined. Thus, 59Fe was readily incorporated into pre-existing ferritin. We conclude that most, if not nearly all, of the iron is incorporated after assembly of protein subunits.