The human secretory immunoglobulin system: immunohistoligical localization of gamma A, secretory "piece," and lactoferrin in normal human tissues.

The immunohistological localization of gammaA, secretory "piece" (SP), and lactoferrin (LF) in the mucosae of a variety of normal human tissues was investigated using specific fluoresceinated antisera. gammaA staining was localized in the apical portion of the mucosal epithelium, intercellular spaces, basement membrane area, and plasma cells of the interstitium or lamina propria of a number of normal human tissues. SP was ubiquitous in the mucosal epithelium of all tissues studied which included parotid and submaxillary glands, bronchi, pancreas, GI tract, sweat glands, kidney, and gall bladder. In addition, SP staining was localized in the intercellular spaces and on the surface of the epithelial cells lining the lumen of the secretory glands. No SP staining was observed in the plasma cells of the interstitium or lamina propria surrounding the secretory glands in these tissues, and no SP staining was observed in sections of normal spleen or lymph node tissue. SP staining was observed in the sweat glands, pancreas, and kidney in the absence of gammaA staining. LF was much less ubiquitous in the epithelial cells of the various tissues studied and appeared to be restricted primarily to the acinar epithelium of the bronchial mucosae, parotid, and submaxillary salivary glands, and was also found in renal tubular cells. A hypothetical model for the transport of gammaA and SP across mucosal membrane epithelium is presented.

Localization of an endogenous lectin in chicken liver, intestine, and pancreas.

Extracts of adult chicken liver, pancreas, and intestine contain high levels of a lectin which appears to be identical to one previously purified from embryonic chick muscle. This lectin is virtually absent from adult muscle, but is highly concentrated in cells lining liver sinusoids, intestinal goblet cells, and the extracellular spaces surrounding pancreatic acini. These findings suggest that the lectin may play different roles in different tissues and at different times in the life of a chicken.

Rapid release of the zymogen granule protein by osmium tetroxide and its retention during fixation by glutaraldehyde.

Fixation by osmium tetroxide and glutaraldehyde of zymogen granules isolated from rat parotid and pancreas was investigated. Protein determinations showed that osmium tetroxide caused rapid release of most of the soluble protein of the granule during fixation in buffered isotonic sucrose. Such granules when examined in the electron microscope after shadow casting appeared quite flat, indicating that most of the contents had indeed been removed. Numerous damaged membranes of the granules were also observed. In contrast, zymogen granules fixed by glutaraldehyde and shadow cast essentially retained the spherical shape and the protein contents. The application of the shadow-casting technique in quantitative studies on the protein content of zymogen granules is discussed.

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.

Freeze-fracture cytochemistry: localization of wheat-germ agglutinin and concanavalin A binding sites on freeze-fractured pancreatic cells.

The combined application of thin-section and critical-point-drying "fracture-label" is used to determine the pattern of distribution and partition of wheat-germ agglutinin and concanavalin A binding sites on the membrane faces of freeze-fractured exocrine and endocrine rat pancreatic cells. Whereas the exoplasmic face of plasma membrane is preferentially labeled by both lectins, the endoplasmic reticulum and nuclear envelope are strongly and uniformly labeled by concanavalin A but not by wheat-germ agglutinin. The results support current views in the glycosylation of membrane proteins and do not support the backflow of sialidated glycoproteins to the endoplasmic reticulum.

Use of colloidal gold particles in double-labeling immunoelectron microscopy of ultrathin frozen tissue sections.

Complexes of protein-A with 5 and 16 nm colloidal gold particles (PA/Au5 and PA/Au16) are presented as sensitive and clean immunoprobes for ultrathin frozen sections of slightly fixed tissue. The probes are suitable for indirect labeling and offer the opportunity to mark multiple sites. The best procedure for double labeling was to use the smaller probe first, i.e., antibody 1 - PA/Au5 - antibody 2 - PA/Au16. When this was done, no significant interference between PA/Au5 and PA/Au16 occurred. Using this double-labeling procedure we made an accurate comparison between the subcellular distributions of amylase as a typical secretory protein and of GP-2 a glycoprotein, characteristic for zymogen granule membrane (ZGM) preparations. We prepared two rabbit antibodies against GP-2. One antibody (R x ZGM) was obtained by immunizing with native membrane material. The specificity of R x ZGM was achieved by adsorption with the zymogen granule content subfraction. The other, R x GP-2, was raised against the GP-2 band of the SDS polyacrylamide profile of ZGM. We found that the carbohydrate moiety of GP-2 was involved in the antigenic determinant for R x ZGM, while R x GP-2 was most likely directed against GP-2 polypeptide backbone. THe immunocytochemical observations showed that GP-2, on the one hand, exhibited the characteristics of a membrane protein by its occurrence in the cell membrane, the Golgi membranes, and its association with the membranes of the zymogen granules. On the other hand, GP-2 was present in the contents of the zymogen granules and in the acinar and ductal lumina. Also, a GP-2-like glycoprotein was found in the cannulated pancreatic secretion (Scheffer et al., 1980, Eur. J. Cell Biol. 23:122-128). Hence, GP-2 should be considered as a membrane-associated secretory protein of the rat pancreas.

Distribution of cell surface saccharides on pancreatic cells. II. Lectin-labeling patterns on mature guinea pig and rat pancreatic cells.

The surface saccharide composition of collagenase-dispersed pancreatic cells from adult guinea pig and rat glands was examined by using eight lectins and their ferritin conjugates: Concanavalin A (ConA); Lens culinaris (LCL); Lotus tetragonolobus (LTL); Ricinus communis agglutinins I and II (RCA I, RCA II); Soybean agglutinin (SBA); Ulex europeus lectin (UEL); and wheat germ agglutinin (WGA). Binding studies of iodinated lectins and lectin-ferritin conjugates both revealed one population of saturable, high-affinity receptor sites on the total cell population (approximately 95% acinar cells). Electron microscopy, however, revealed differences in lectin-ferritin binding to the plasmalemma of acinar, centroacinar, and endocrine cells. Whereas acinar cells bound heavily all lectin conjugates, endocrine and centroacinar cells were densely labeled only by ConA, LCL, WGA, and RCA I, and possessed few receptors for LTL, UEL, and SBA. Endocrine and centroacinar cells could be differentiated from each other by using RCA II, which binds to centroacinar cells but not to endocrine cells. Some RCA II receptors appeared to be glycolipids because they were extracted by ethanol and chloroform-methanol in contrast to WGA receptors which resisted solvent treatment but were partly removed by papain digestion. RCA I receptors were affected by neither treatment. The apparent absence of receptors for SBA on endocrine and centroacinar cells, and for RCA II on endocrine cells, was reversed by neuraminidase digestion, which suggested masking of lectin receptors by sialic acid. The absence of LTL and UEL receptors on endocrine and centroacinar cells was not reversed by neuraminidase. We suggest that the differential lectin-binding patterns observed on acinar, centroacinar, and endocrine cells from the adult pancreas surface-carbohydrate-developmental programs expressed during morphogenesis and cytodifferentiation of the gland.

Calmodulin-stimulated protein kinase activity from rat pancreas.

Previous work from our laboratory has demonstrated that neurohumoral stimulation of the exocrine pancreas is associated with the phosphorylation of the Mr 29,000 ribosomal protein S6. In a cell-free system using pancreatic postmicrosomal supernatant as the kinase donor, we found that the following co-factors stimulate the phosphorylation of the Mr 29,000 ribosomal protein: calcium with calmodulin, calcium with phosphatidyl serine, and cAMP. These findings suggest that the pancreas contains a calcium-calmodulin-dependent protein kinase (CaM-PK) that can phosphorylate the Mr 29,000 ribosomal protein. A CaM-PK activity was partially purified sequentially by ion exchange, gel filtration, and calmodulin-affinity chromatography. Phosphorylation of the Mr 29,000 ribosomal protein by the partially purified CaM-PK was dependent on the presence of both calcium and calmodulin and not on the other co-factors. The CaM-PK fraction contained a phosphoprotein of Mr 51,000 whose phosphorylation was also dependent on calcium and calmodulin. When 125I-calmodulin-binding proteins from the CaM-PK fraction were identified using electrophoretic transfers of SDS-polyacrylamide gels, a single Mr 51,000 protein was labeled. The preparation enriched in CaM-PK activity contained an Mr 51,000 protein that underwent phosphorylation in a calcium-calmodulin-dependent manner and an Mr 51,000 calmodulin-binding protein. It is therefore possible that the CaM-PK may comprise a calmodulin-binding phosphoprotein component of Mr 51,000.

Subcellular distribution of signal recognition particle and 7SL-RNA determined with polypeptide-specific antibodies and complementary DNA probe.

Signal recognition particle (SRP) is a ribonucleoprotein consisting of six distinct polypeptides and one molecule of small cytoplasmic 7SL-RNA. The particle was previously shown to function in protein translocation across and protein integration into the endoplasmic reticulum membrane. Polypeptide specific antibodies were raised in rabbits against the 72,000-, 68,000-, and 54,000-mol-wt polypeptide of SRP. All three antibodies are shown to neutralize SRP activity in vitro. A solid phase radioimmune assay is described and used to follow SRP in various cell fractions. The partitioning of SRP is shown to be dependent on the ionic conditions of the fractionation. Under conditions approximating physiological ionic strength, SRP is found to be about equally distributed between a membrane associated (38%) and a free (15%) or ribosome associated (47%) state. Furthermore, it is shown that greater than 75% of the total cellular 7SL-RNA is associated with SRP polypeptide in these fractions. Thus it is likely that the major--if not the only--cellular function of 7SL-RNA is as a part of SRP.

Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.

Calcium and pancreatic secretion. I. Subcellular distribution of calcium and magnesium in the exocrine pancreas of the guinea pig.

The distribution of calcium and magnesium has been studied in the acinar cells of the pancreas of the guinea pig. Most of the magnesium was found to be associated with the rough microsomes (probably bound to the ribosomes) and with the postmicrosomal supernate. In contrast, calcium was distributed among all the particulate fractions, primarily the mitochondria, microsomes (especially smooth surfaced), zymogen granules, and the plasmalemma, and was low in the postmicrosomal supernate. Most of the calcium recovered in the particulate fractions was found to be membrane bound. The highest concentrations were found in the membranes of the zymogen granules and in the plasmalemma. By means of control experiments using -45Ca as the tracer, it was established that a considerable redistribution of calcium occurs during homogenization and cell fractionation. At least some of the resulting artifacts were estimated quantitatively and the data were corrected accordingly. The biochemical results were confirmed with the cytochemical antimonate technique carried out on the tissue as well as on isolated fractions. The role of calcium associated with the zymogen granules and with their limiting membranes is discussed in relation to the architecture of the granule and to the functionality of the pancreatic juice.

Calcium phosphate granules in the hepatopancreas of the blue crab Callinectes sapidus.

The hepatopancreas of the adult male blue crab Callinectes sapidus in intermolt was found to contain substantial amounts of calcium, magnesium, and inorganic phosphorus, averaging about 260, 20, and 250 microg-atoms per g wet tissue, respectively, accounting for over 10% of the tissue dry weight. Electron microscopy of the intact tissue showed three qualitatively different granular structures having electron densities suggestive of high mineral content. After fractionation of the tissue using centrifugal techniques, almost 95% of the total mineral was found to reside in a heavy, nonmitochondrial particulate fraction(s). The bulk of the low-speed pellet consisted of relatively dense, roughly spherical granules 1-5 microm in diameter, which could be considerably purified by repeated suspension in water and low-speed sedimentation. In the electron microscope the isolated granules appeared basically similar to one of the three characteristic types of electron-dense granules seen in the intact tissue. Although the freshly isolated granules lost approximately 50% of their wet weight when dried at 105 degrees C, only 10% more was lost upon dry ashing at 450 degrees C, suggesting a fairly low content of organic material. Chemical analysis revealed calcium, magnesium, and inorganic phosphate at 5.7, 2.1, and 4.4 microg-atoms per mg dried granules, respectively, accounting for 69% of the dry weight of the fraction. By specific enzymatic assays, the freshly isolated granules were found to contain ATP, ADP, and AMP at levels of 0.13, 0.03, and 0.01 micromol/mg, or 8% of their total dry weight. The remainder of the total phosphorus contributed an additional 3%, whereas carbonate, citrate, oxalate, and protein each constituted no more than 1%. The mineral granules of the crab hepatopancreas appear to function as storage forms of calcium and phosphate during the intermolt period. This tissue appears promising as a model for study of the cellular events associated with biological calcification, since conventional biochemical techniques can be employed. Furthermore, the major mineralized component of the tissue can be obtained in large amounts for direct study by a simple fractionation procedure.

Hormone-induced protein phosphorylation. II. Localization to the ribosomal fraction from rat exocrine pancreas and parotid of a 29,000-dalton protein phosphorylated in situ in response to secretagogues.

In the preceding paper, we demonstrated that the endogenous phosphorylation of a protein with a molecular weight of 29,000 was enhanced by various secretagogues in rat pancreatic and parotid lobules, the phosphorylation of this protein correlating both temporally and in a dose-dependent fashion with secretory protein discharge. In the present study, we established a specific methodology to characterize this phosphoprotein. Once established, this 29,000-dalton phosphoprotein was then followed selectively and quantitatively throughout subcellular fractionation procedures. Analysis of two-dimensional polyacrylamide gels demonstrated that proteins with similar mobilities (Mr 29,000; pl greater than 8.4) were affected by cholecystokinin octapeptide and isoproterenol in rat pancreatic and parotid lobules, respectively, suggesting that the same 29,000-dalton phosphoprotein was covalently modified in both tissues. Cellular fractionation studies using differential velocity and sucrose density gradient centrifugation revealed that the 29,000-dalton phosphoprotein copurified with the rough microsomal fraction of pancreas and was highly enriched in ribosomal fractions of both pancreas and parotid. Electrophoresis in two dimensions confirmed that the 29,000-dalton polypeptide that was resolved directly from stimulated cells and from ribosomal fractions exhibited a common mobility, and apparent identity of the species was strongly suggested when the 29,000-dalton polypeptides from both sources were compared by peptide mapping following limited digestion with Staphylococcus aureus V8 protease. This phosphoprotein was tentatively identified as ribosomal protein S6 after analysis by pH 8.6/4.2 two-dimensional PAGE.

Composition of cellular membranes in the pancreas of the guinea pig. IV. Polyacrylamide gel electrophoresis and amino acid composition of membrane proteins.

TWO METHODS OF POLYACRYLAMIDE GEL ELECTROPHORESIS (THE ACID METHOD OF EYTAN AND OHAD AND THE NA DODECYLSULFATE (SDS) DISC METHOD OF MAIZEL) HAVE BEEN USED FOR ANALYZING THE PROTEINS OF GEL FRACTIONS ISOLATED FROM THE GUINEA PIG PANCREATIC EXOCRINE CELLS AND IN PARTICULAR THE PROTEINS BOUND TO THE MEMBRANES INVOLVED IN THE SYNTHESIS, INTRACELLULAR TRANSPORT, AND DISCHARGE OF SECRETORY ENZYMES: rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM). Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained. The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands. In contrast, with highly purified membrane fractions different patterns were obtained: RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between approximately 150,000 and 15,000 daltons. The pattern of ZG membranes was greatly different in the two systems: only two bands were separated by the acid method and as many as 23 by the SDS method. PM gave a rather complex pattern in either system. Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins. Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity. By amino acid analysis we found only slight differences: relative to the other fractions: RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine. Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products.

Detection of complex carbohydrates in the Golgi apparatus of rat cells.

Two methods used for the electron microscopic detection of glycoproteins were applied to a variety of cell types in the rat; one involved successive treatment of sections with periodic acid, chromic acid, and silver methenamine; and the other, a brief treatment with a chromic acid-phosphotungstic acid mixture. The results obtained with the two methods were identical and, whenever the comparison was possible, similar to those obtained with the periodic acid-Schiff technique of light microscopy. In secretory as well as in nonsecretory cells, parts of the Golgi apparatus are stained. The last saccule on one side of each Golgi stack is strongly reactive (mature face), and the last saccule on the other side shows little or no reactivity (immature face); a gradient of reactivity occurs in between these saccules. The more likely explanation of the increase in staining intensity is that carbohydrate is synthesized and accumulates in saccules as they migrate toward the mature face. In many secretory cells, the mature face is associated with strongly stained secretory granules. Other structures stained are: (1) small vesicles, dense and multivesicular bodies, at least some of which are presumed to be lysosomal in nature; (2) cell coat; and (3) basement membrane. The evidence suggests that the Golgi saccules provide glycoproteins not only for secretion, but also for the needs of the lysosomal system as well as for incorporation into the cell coat and perhaps basement membrane.

Morphological and biochemical studies of B cells of fetal rat endocrine pancreas in organ culture. Evidence for (pro) insulin biosynthesis.

Fetal rat pancreases explanted on the 18th day of gestation and maintained in organ culture for 1-10 days were utilized for this series of studies. Ultrastructurally, at the time of explantation, the majority of fetal B cells was sparsely granulated and characterized by numerous free ribosomes and undeveloped rough endoplasmic reticulum (RER) and Golgi complexes. During the culture period, extensive development of the RER and Golgi complexes preceded an increasing accumulation of beta-granules. This later increase in the number of beta-granules and in the concentration of immunoreactive insulin was paralleled by a reduction of RER and Golgi complex activity. High resolution radioautographic studies of pulse-chase experiment over a 1 hr period demonstrated the shift of silver grains from the elements of the RER, through the Golgi region, and finally to the beta-granules. Incubation with (14)C-labeled leucine demonstrated the incorporation of radioactivity into molecules possessing the immunological and electrophoretic properties of insulin. These studies indicate that de novo synthesis of (pro)insulin occurs also during culture of fetal rat pancreas explanted relatively late in gestation.

The development of the dorsal and ventral mammalian pancreas in vivo and in vitro.

The origin, morphogenesis, and biochemical differentiation of the dorsal and ventral pancreas of the rat embryo have been investigated in order to ascertain the similarities and dissimilarities between the two lobes. We have utilized a culture system in which the primitive gut gives rise to a number of differentiated organs, including the dorsal and ventral pancreas. The two pancreases do not undergo fusion in these cultures, thus allowing independent analyses of the two lobes for comparison with in vivo results. The dorsal pancreas first appeared at the 23-25 somite stage while the ventral pancreas appeared approximately 12 hr later at the 29-30 somite stage. Guts from embryos as young as 12 somites were capable of developing both pancreases in vitro. In spite of the 12 hr difference between the times of their appearance, the dorsal and ventral pancreases exhibited identical patterns of morphological and biochemical differentiation. The two lobes contained the same exocrine enzymes and hormones, at similar levels, differing only in their glucagon content, the dorsal pancreas possessing a fivefold higher glucagon specific activity. The implications of these results are discussed.

Composition of cellular membranes in the pancreas of the guinea pig. I. Isolation of membrane fractions.

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.

Composition of cellular membranes in the pancreas of the guinea pig. II. Lipids.

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to approximately 20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.

Isolation, characterization, and distribution of an unusual pancreatic human secretory protein.

An unusual protein was isolated from acid extracts of normal human pancreas and pancreatic secretion in the form of uniform 7-10-nm long single threads without visible axial periodicity or other structure, as seen in the electron microscope. It accounts for as much as 300 micrograms/ml in some pancreatic secretions as measured by specific radioimmunoassay. The protein undergoes a freely reversible, pH dependent, globule-fibril transformation, being stable in the fibril form between pH 5.4 and 9.2. The monomer at acid pH has an apparent molecular weight of approximately 14,000 and consists of a single polypeptide chain, the amino acid composition of which is rich in aromatic amino acids and lacks carbohydrate, fatty acid, and phosphate. The amino acid sequence of 45 residues from the amino terminus shows no homology with any other reported protein sequences other than that of the A chain of the bovine pancreas thread protein (reported elsewhere). A sensitive radioimmunoassay employing monoclonal antibodies against human pancreatic thread protein failed to detect the antigen in a wide range of human tissues other than pancreas, nor was the antigen measurable in normal human sera. Immunohistochemistry utilizing these antibodies revealed the antigen as a component of the cytoplasm of some but not all the pancreatic acinar cells. A physiologic function has not yet been determined for this protein.