Concentration of amylase along its secretory pathway in the pancreatic acinar cell as revealed by high resolution immunocytochemistry

Summary The modified protein A-gold immunocytochemical technique was applied to the localization of amylase in rat pancreatic acinar cells. Due to the good ultrastructural preservation of the cellular organelles obtained on glutaraldehyde-fixed, osmium tetroxide-postfixed tissue, the labelling was detected with high resolution over the cisternae of the rough endoplasmic reticulum (RER), the Golgi apparatus, the condensing vacuoles, the immature pre-zymogen granules, and the mature zymogen granules. Over the Golgi area, the labelling was present over the transitional elements of the endoplasmic reticulum, some of the smooth vesicular structures at thecis- andtrans-faces and all the different Golgi cisternae. The acid phosphatase-positive rigidtrans-cisternae as well as the coated vesicles were either negative or weakly labelled. Quantitative evaluations of the degree of labelling demonstrated an increasing intensity which progresses from the RER, through the Golgi, to the zymogen granules and have identified the sites where protein concentration occurs. The results obtained have thus demonstrated that amylase is processed through the conventional RER-Golgi-granule secretory pathway in the pancreatic acinar cells. In addition a concomitance has been found between some sites where protein concentration occurs: thetrans-most Golgi cisternae, the condensing vacuoles, the pre- and the mature zymogen granules, and the presence of actin at the level of the limiting membranes of these same organelles as reported previously (Bendayan, 1983). This suggests that beside their possible role in transport and release of secretory products, contractile proteins may also be involved in the process of protein concentration.     

Characterization of pancreatic-type tissue in the liver of rat induced by polychlorinated biphenyls

Pancreatic-type tissue induced in the livers of rats treated with polychlorinated biphenyls was characterized by transmission electron microscopy and high-resolution immunocytochemistry. The cells of pancreatic-type tissue were arranged as acini and in small groups. By electron microscopy the pancreatic-type tissue showed features very similar to normal pancreatic acinar tissue, such as well developed rough endoplasmic reticulum (RER), large numbers of mature zymogen granules, and a basally located nucleus. Protein A-gold imunocytochemical technique showed localization of amylase and trypsinogen over the zymogen granules and RER. These findings confirm that this tissue in the liver is morphologically and functionally identical to pancreatic acinar tissue.     

Chromogranin B (secretogranin I), a neuroendocrine-regulated secretory protein, is sorted to exocrine secretory granules in transgenic mice.

Chromogranin B (CgB, secretogranin I) is a secretory granule matrix protein expressed in a wide variety of endocrine cells and neurons. Here we generated transgenic mice expressing CgB under the control of the human cytomegalovirus promoter. Northern and immunoblot analyses, in situ hybridization and immunocytochemistry revealed that the exocrine pancreas was the tissue with the highest level of ectopic CgB expression. Upon subcellular fractionation of the exocrine pancreas, the distribution of CgB in the various fractions was indistinguishable from that of amylase, an endogenous constituent of zymogen granules. Immunogold electron microscopy of pancreatic acinar cells showed co-localization of CgB with zymogens in Golgi cisternae, condensing vacuoles/immature granules and mature zymogen granules; the ratio of immunoreactivity of CgB to zymogens being highest in condensing vacuoles/immature granules. CgB isolated from zymogen granules of the pancreas of the transgenic mice aggregated in a mildly acidic (pH 5.5) milieu in vitro, suggesting that low pH-induced aggregation contributed to the observed concentration of CgB in condensing vacuoles. Our results show that a neuroendocrine-regulated secretory protein can be sorted to exocrine secretory granules in vivo, and imply that a key feature of CgB sorting in the trans-Golgi network of neuroendocrine cells, i.e. its aggregation-mediated concentration in the course of immature secretory granule formation, also occurs in exocrine cells although secretory protein sorting in these cells is thought to occur largely in the course of secretory granule maturation.     

Binding sites of Ulex europaeus-lectin I in human parotid gland

Summary Twenty non-neoplastic parotid glands (removed during neck dissection for regional tumours) were examined for cellular and subcellular binding sites of Ulex europaeus-lectin I (UEA-I), a lectin reported to be specific for -L-fucose. For light microscopy, an extended peroxidase-antiperoxidase method was applied; for the evaluation of the subcellular localization of bound lectin, three of these glands were examined following immunocryoultramicrotomy and staining by the protein A-gold technique.In addition to the known cytoplasmic affinity of UEA-I for capillary endothelium, acinar cells bound the lectin within the cytoplasmic compartment; the number and distribution of stained acinar cells varied among individuals. Furthermore, cytomembrane-bound labelling that occurred most markedly at the luminar surface was observed in striated-duct epithelium.Using the electron microscope, protein A-gold particles were seen in zymogen granules and in Golgi cisternae of serous acinar cells; primary saliva secreted in the lumina exhibited strong labelling; serous acinar cells had binding sites on their cell membranes, striated-duct epithelium had binding sites on its surface membrane and in the vicinity of apical vesicles. Our results show that UEA-I is a useful tool for the study of the structure and functional states of the parotid gland epithelium and its associated pathological alterations.Dedicated to Prof. Dr. med. G. Seifert on the occasion of his 65th birthday     

Ultrastructural, morphometrical and immunocytochemical analyses of the exocrine pancreas in a hibernating dormouse

Pancreatic acinar cells of euthermic, hibernating and arousing individuals of the hazel dormouse Muscardinus avellanarius (Gliridae) have been observed at the electron-microscopic level and analysed by means of ultrastructural morphometry and immunocytochemistry in order to investigate possible fine structural changes of cellular components during periods of strikingly different degrees of metabolic activity. During hibernation, the cisternae of the rough endoplasmic reticulum (RER) flatten assuming a parallel pattern, the Golgi apparatus is extremely reduced and the mitochondria contain many electron-dense particles. The cell nuclei appear irregularly shaped, with deep indentations containing small zymogen granules. They also contain abundant coiled bodies and unusual constituents, such as amorphous bodies and dense granular bodies. Large numbers of zymogen granules occur in all animals. However, the acinar lumina are open and filled with zymogen only in euthermic animals, whereas, in hibernating and arousing individuals, they appear to be closed. Morphometrical analyses indicate that, in pancreatic acinar cells, nuclei and zymogen granules significantly decrease in size from euthermia to hibernation, probably reflecting a drastic decrease of metabolic activities, mainly protein synthesis and processing. In all the studied animals, immunocytochemistry with specific antibodies has revealed an increasing gradient in α-amylase content along the RER-Golgi-zymogen granule pathway, reflecting the protein concentration along the secretory pathway. Moreover, during deep hibernation, significantly larger amounts of α-amylase accumulate in RER and zymogen granules in comparison to the other seasonal phases analysed. Upon arousal, all cytoplasmic and nuclear constituents restore their euthermic aspect and all morphometrical and immunocytochemical parameters exhibit the euthermic values, thereby indicating a rapid resumption of metabolic activities.     

Quantitation of ultrastructural changes in mouse pancreatic acinar cells caused by foreign serum

Quantitative changes in the pancreatic acinar cell organelles were studied in BALB/c mice injected with 1.0 ml fresh rabbit serum intraperitoneally. Groups of 5 mice were killed at 0, 1, 3, 6 and 12 h after the serum injection. Pancreatic tissue was processed for electron microscopy by glutaraldehyde and osmium tetroxide fixation and Epon embedding. The proportions of acinar cell cytoplasm (volume fractions) occupied by zymogen granules, granular endoplasmic reticulum, Golgi apparatus, mitochondria and lysosomes (including autophagosomes) were determined by the point counting method from electron micrographs. The volume fraction of lysosomes increased during the first 3 h and remained markedly elevated up to 12 h. The volume fractions of zymogen granules increased from 12 to 28% in 12 h. It was concluded that the secretory mechanism of pancreatic acinar cells was injured by the foreign serum. The injury caused accumulation of zymogen granules and increased autophagic activity in the acinar cells.     

Immunocytochemical localization of exocrine enzymes in rat pancreatic acinar carcinoma

Ten pancreatic secretory proteins have been demonstrated in differentiated pancreatic acinar carcinoma cells by the protein A-gold immunocytochemical approach. The high resolution of the technique has allowed for the localization of the different proteins in the cellular compartments involved in protein secretion: RER, Golgi and secretory granules. The quantitative evaluation of the labeling for amylase has demonstrated the presence of an increasing gradient in the intensity from the RER to the Golgi and to the secretory granules which may reflect the process of protein concentration along the secretory pathway. These results, together with those obtained using the pulse-labeling autoradiographic approach, demonstrate that differentiated acinar carcinoma cells are capable of processing secretory proteins. When intensities of labeling obtained for different proteins on acinar carcinoma cells were compared to those obtained on normal pancreatic acinar cells, major differences were observed for some proteins. In addition, studies performed on the pancreatic tissue of the tumor-bearing animals have shown the presence of morphological alterations in the acinar cells.     

Stereological analysis of the guinea pig pancreas. I. Analytical model and quantitative description of nonstimulated pancreatic exocrine cells

A stereological model which provides detailed quantitative information on the structure of the fasted, nonstimulated gland has been developed for the guinea pig pancreas. The model consists of morphologically defined space and membrane compartments which were used to describe the general composition of the tissue and the specific components of exocrine cells. The results are presented, where appropriate, relative to a cubic centimeter of pancreas, a cubic centimeter of exocrine cell cytoplasm, and to the volume of an average exocrine cell. The exocrine cells, accounting for 82% of the pancreas volume, consisted of 54% cytoplasmic matrix, 22% rough-surfaced endoplasmic reticulum (RER), 8.3% nuclei, 8.1% mitochondria, 6.4% zymogen granules, and 0.7% condensing vacuoles. Their total membrane surface area was distributed as follows: 60% RER, 21% mitochondria, 9.9% Golgi apparatus, 4.8% plasma membranes, 2.6% zymogen granules, 1.8% plasma membrane vesicles, and 0.4% condensing vacuoles. The application of this model to the study of membrane movements associated with the secretory process is discussed within the framework of an analytical approach.     

Topographical and planar distribution of Helix pomatia lectin-binding glycoconjugates in secretory granules and plasma membrane of pancreatic exocrine acinar cells of the rat: demonstration of membrane heterogeneity

The lectin-gold technique was used to detect Helix pomatia lectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze-fractured preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen-granule content, labeling of the acinar lumen was intense. Fracture-label preparations revealed preferential partition of HPL-binding sites to the exoplasmic half of the zymogen-granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture-face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin-section fracture-label approach. In addition, favorable profiles of thin sections of freeze-fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture-face. We conclude that 1) zymogen granules contain little HPL-binding glycoconjugates, 2) HPL-binding sites are preferentially associated with the exoplasmic half of the zymogen-granule and plasma membranes, and 3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL-binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory-granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become soluble and secreted with the content.     

Glycoprotein synthesis in the adult rat pancreas: II. Characterization of Golgi-rich fractions

Golgi-rich fractions were prepared from homogenates of adult rat pancreas by discontinuous gradient centrifugation. These fractions were characterized by stacks of cisternae associated with large, irregular vesicles and were relatively free of rough microsomes, mitochondria, and zymogen granules. The Golgi-rich fractions contained 50% of the UDP-galactose: glycoprotein galactosyltransferase activity; the specific activity was 12-fold greater than the homogenate. Such fractions represented < 19% of thiamine pyrophosphatase, uridine diphosphatase, adenosine diphosphatase, and Mg²⁺-adenosine triphosphatase. Zymogen granules and the Golgi-rich fractions were extracted with 0.2 m NaHCO3, pH 8.2, and the membranes were isolated by centrifugation. The glycoprotein galactosyltransferase could not be detected in granule membranes, while the specific activity in Golgi membranes was 25-fold greater than the homogenate.At least 35 polypeptide species were detected in Golgi membranes by polyacrylamide gel electrophoresis in 1% sodium dodecylsulfate. These ranged in molecular weight from 12,000 to <160,000. There were only minor differences between Golgi membranes and smooth microsomal membrane. In contrast, zymogen granule membranes contained fewer polypeptides. A major polypeptide, which represented 30–40% of the granule membrane profile, accounted for less than 3% of the polypeptides of Golgi membranes or smooth microsomal membranes.     

Effects of hypothyroidism on the ultrastructure of rat pancreatic acinar cells: a stereological analysis.

The morphological and stereological characteristics of the exocrine pancreas subcellular organelles from healthy and thyroidectomized rats have been studied. The acinar tissue from hypothyroid rats showed an interstitial edema and evidence of degenerative processes. Stereological parameters of zymogen granules were significantly reduced in thyroidectomized rats. The hypothyroidism induced degenerative changes in the pancreatic acinar cells as well as a decrease in the number and size of the zymogen granules. These modifications probably cause functional alterations.     

Cellular and subcellular expression of Golf/Gs and Gq/G11 alpha-subunits in rat pancreatic endocrine cells.

We studied the cellular and subcellular localization of Galpha-subunits in pancreas by immunocytochemistry. Golfalpha and G11alpha were specifically localized in islet insulin B-cells and glucagon A-cells, respectively. Gsalpha and Gqalpha labeling was more abundant in B-cells. The presence of Golfalpha in B-cells was confirmed by in situ hybridization. In B-cells, Golfalpha and Gsalpha were found in the Golgi apparatus, plasma membrane (PM) and, remarkably, in mature and immature insulin secretory granules, mainly at the periphery of the insulin grains. Gqalpha was detected on the rough endoplasmic reticulum (RER) near the Golgi apparatus. In A-cells, the Galpha-subunits were mostly within the glucagon granules: G11alpha gave the strongest signal, Gsalpha less strong, Gq was scarce, and Golf was practically absent. Gqalpha and Gsalpha immunoreactivity was detected in acinar cells, although it was much weaker than that in islet cells. The cell-dependent distribution of the Galpha-subunits indicates that the stimulatory pathways for pancreatic function differ in acinar and in islet B- and A-cells. Furthermore, the G-protein subunits in islet cell secretory granules might be functional and participate in granule trafficking and hormone secretion.     

A cytochemical study on the pancreas of the guinea pig. I. Isolation and enzymatic activities of cell fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

Oxidative injury to isolated rat pancreatic acinar cells vs. isolated zymogen granules

This study compares the susceptibility of pancreatic acinar cells and zymogen granules against oxidative injury and analyzes the mechanisms involved. Zymogen granules and acinar cells, isolated from rat pancreas, were exposed to a reaction mixture containing xanthine oxidase, hypoxanthine, and chelated iron. Cell function and viability were assessed by various techniques. Trypsin activation was quantified by an Elisa for trypsinogen activating peptide. Integrity of granules was determined by release of amylase. The reaction mixture rapidly generated radicals as assessed by deoxyribose and luminol assays. This oxidative stress caused lysis of granules in a matter of minutes but significant cell death only after some hours. Nevertheless, radicals initiated intracellular vacuolization, morphological damage to zymogen granules and mitochondria, increase in trypsinogen activating peptide, and decrease in ATP already after 5–30 min. Supramaximal caerulein concentrations also caused rapid trypsin activation. Addition of cells but not of granules reduced deoxyribose oxidation, suggesting that intact cells act as scavengers. Caerulein pretreatment only slightly increased the susceptibility of cells but markedly that of granules. In conclusion, isolated zymogen granules are markedly more susceptible to oxidative injury than intact acinar cells, in particular, in early stages of caerulein pancreatitis. The results show that oxidative stress causes a rapid trypsin activation that may contribute to cell damage by triggering autodigestion. Zymogen granules and mitochondria appear to be important targets of oxidative damage inside acinar cells. The series of intracellular events initiated by oxidative stress was similar to changes seen in early stages of pancreatitis.     

Morphometry and elemental analysis of rat exocrine pancreas following administration of trypsin inhibitor

The morphological responses of the exocrine pancreas of the adult male rat to soybean trypsin inhibitor (STI) were studied by ultrastructural morphometry and electron probe X-ray microanalysis. STI administered orally in drinking water for 14 days resulted in a 72% increase in the wet weight of the pancreas. This enlargement was due, largely, to an increase in acinar cell mass. Volume increases in the acinar cell mass and extra-acinar cell compartment were 72 and 30%, respectively. The estimated total number of acinar cells in the mean exocrine pancreas was 500 million in the control and 630 million in the experimental group, representing an increase of 27%. Acinar cell volume was 1,790 microns 3 for the control and 2,457 microns 3 for the STI group. The pronounced morphometric changes of the organelles in the STI group were: the mean nucleolar volume increased by 56%; the volume of zymogen granular mass per cell increased by 93%; the volume of the Golgi complex and the condensing vacuoles per cell increased by 52 and 100%, respectively, whereas the membrane area of the Golgi complex and the condensing vacuoles increased by 98 and 47%, respectively. Spectral analysis of seven elements (Na, Mg, P, S, Cl, K and Ca) showed significant changes for nuclei, zymogen granules and mitochondria following STI: nuclei showed Na, P, K increased; zymogen granules showed Na, P, S, K increased, Cl decreased; mitochondrial particles showed Mg, P, Cl, Ca increased, and the mitochondrial matrix showed S decreased. The persistent uptake of STI probably resulted in a continual release of a trophic hormone acting on pancreatic tissue components, consequently causing hyperplasia and hypertrophy of the exocrine pancreas to accommodate a heightened demand for synthesis of exportable proteins.     

Localization of mitochondrial 60-kD heat shock chaperonin protein (Hsp60) in pituitary growth hormone secretory granules and pancreatic zymogen granules.

We used quantitative immunogold electron microscopy and biochemical analysis to evaluate the subcellular distribution of Hsp60 in rat tissues. Western blot analysis, employing both monoclonal and polyclonal antibodies raised against mammalian Hsp60, shows that only a single 60-kD protein is reactive with the antibodies in brain, heart, kidney, liver, pancreas, pituitary, spleen, skeletal muscle, and adrenal gland. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows strong labeling of mitochondria in all tissues. However, in the anterior pitutary and in pancreatic acinar cells, Hsp60 also localizes in secretory granules. The labeled granules in the pituitary and pancreas were determined to be growth hormone granules and zymogen granules, respectively, using antibodies to growth hormone and carboxypeptidase A. Immunogold labeling of Hsp60 in all compartments was prevented by preadsorption of the antibodies with recombinant Hsp60. Biochemically purified zymogen granules free of mitochondrial contamination are shown by Western blot analysis to contain Hsp60, confirming the morphological localization results in pancreatic acinar cells. In kidney distal tubule cells, low Hsp60 reactivity is associated with infoldings of the basal plasma membrane. In comparison, the plasma membrane in kidney proximal tubule cells and in other tissues examined showed only background labeling. These findings raise interesting questions concerning translocation mechanisms and the cellular roles of Hsp60.     

Regulated apical secretion of zymogens in rat pancreas. Involvement of the glycosylphosphatidylinositol-anchored glycoprotein GP-2, the lectin ZG16p, and cholesterol-glycosphingolipid-enriched microdomains

We examined the role of glycosphingolipid- and cholesterol-enriched microdomains, or rafts, in the sorting of digestive enzymes into zymogen granules destined for apical secretion and in granule formation. Isolated membranes of zymogen granules from pancreatic acinar cells showed an enrichment in cholesterol and sphingomyelin and formed detergent-insoluble glycolipid-enriched complexes. These complexes floated to the lighter fractions of sucrose density gradients and contained the glycosylphosphatidylinositol (GPI)-anchored glycoprotein GP-2, the lectin ZG16p, and sulfated matrix proteoglycans. Morphological and pulse-chase studies with isolated pancreatic lobules revealed that after inhibition of GPI-anchor biosynthesis by mannosamine or the fungal metabolite YW 3548, granule formation was impaired leading to an accumulation of newly synthesized proteins in the Golgi apparatus and the rough endoplasmic reticulum. Furthermore, the membrane attachment of matrix proteoglycans was diminished. After cholesterol depletion or inhibition of glycosphingolipid synthesis by fumonisin B1, the formation of zymogen granules as well as the formation of detergent-insoluble complexes was reduced. In addition, cholesterol depletion led to constitutive secretion of newly synthesized proteins, e.g. amylase, indicating that zymogens were missorted. Together, these data provide first evidence that in polarized acinar cells of the exocrine pancreas GPI-anchored proteins, e.g. GP-2, and cholesterol-sphingolipid-enriched microdomains are required for granule formation as well as for regulated secretion of zymogens and may function as sorting platforms for secretory proteins destined for apical delivery.     

A Cytochemical Study on the Pancreas of the Guinea Pig : I. Isolation and Enzymatic Activities of Cell Fractions

Pancreatic tissue, (guinea pig) homogenized in 0.88 M sucrose, was fractionated by differential centrifugation into a nuclear, zymogen, mitochondrial, microsomal, and final supernatant fraction. The components of the particulate fractions were identified with well known intracellular structures by electron microscopy. The fractions were analyzed for protein-N and RNA, and were assayed for RNase and trypsin-activatable proteolytic (TAPase) activity. The zymogen fraction accounted for 30 to 40 per cent of the total TAPase and RNase activities, and its specific enzymatic activities were 4 to 10 times higher than those of any other cell fraction. The zymogen fraction was cytologically heterogeneous; zymogen granules and mitochondria represented its main components. More homogeneous zymogen fractions, obtained by successive washing or by separation in a discontinuous density-gradient, had specific activities 2 to 4 times greater than the crude zymogen fractions. Chymotrypsinogen was isolated by column chromatography from pancreas homogenates and derived cell fractions. The largest amount was recovered in the zymogen fraction. The final supernatant had properties similar to those of the trypsin inhibitor described by Kunitz and Northrop.     

Cytochrome-C localizes in secretory granules in pancreas and anterior pituitary

We used quantitative immunogold electron microscopy to evaluate the subcellular distribution of cytochrome-c in normal rat tissues, employing a wide variety of monoclonal and polyclonal antibodies against mammalian cytochrome-c. Immunogold labeling of tissues embedded in the acrylic resin LR Gold shows highly specific labeling of mitochondria in all tissues examined, including adrenal gland, cerebellum, cerebral cortex, heart, kidney, liver, pituitary, pancreas, skeletal muscle, spleen and thyroid. In pancreatic acinar cells and anterior pituitary, however, there was also strong cytochrome-c reactivity in zymogen granules and growth hormone granules, respectively. In the pancreas, strong immunoreactivity is also detected in condensing vacuoles and in the acinar lumen. Immunocytochemical controls included (i) use of monoclonal antibodies to horse cytochrome-c which recognize an epitope not present in rat cytochrome-c, (ii) preadsorption of antibodies with purified cytochrome-c, and (iii) omission of the primary antibody. The indicated presence of cytochrome-c outside mitochondria in certain tissues under normal physiological conditions raises interesting questions concerning translocation mechanisms and the cellular functions of cytochrome-c.