Myelin-like structures as a possible source of the smooth endoplasmic reticulum in early amphibian oocytes

A comparative study of amphibian oocyte ultrastructural organization has shown a significant accumulation of elements of the smooth endoplasmic reticulum in the oocyte cytoplasm at the third stage of development. The analysis of oocytes of two frog species, Xenopus laevis and Rana temporaria, at the first and second stages of their development enabled us to recognize in the cytoplasm of the oocyte some myelin-like structures (MLs) made of 30-40 densely packaged membranous layers and shaped as dense bodies. MLs are also present in the adjacent follicular cells and in the intercellular space. In the oocyte cytoplasm these structures are located near the nuclear envelope and other intracellular organelles. At the third stage of oogenesis, which is characterized by a high functional activity of the cells, MLs are seen to unwrap sequentially into double-layer membranes similar to the smooth endoplasmic reticulum cisternae. Intermediate steps of this process being also observed. It is supposed that MLs may play the role of membrane stocks to be used eventually for the formation of nascent endoplasmic membranes in the amphibian oocytes.     

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

Changes in the cell surface coat during the development ofXenopus laevis embryos,detected by lectins

Summary The composition of the surface coat in embryonic cells ofXenopus laevis was examined by agglutination and fluorescent staining with lectins.Cells of early and mid gastrula stages were agglutinated by lectins specific for D-mannose, D-galactose, L-fucose, N-acetyl-D-glucosamine and N-acetyl-D-galactosamine. No differences in agglutinability among ectoderm, mesoderm and endoderm cells were observed with lectins specific for D-mannose, D-galactose and N-acetyl-D-galactosamine, though agglutination of gastrula cells with fluorescent lectins revealed considerable differences in the intensity of lectin binding among cells within an aggregate. These differences in amount of lectin bound were not related to cell size or morphology. Patches of fluorescent material formed on the cells, suggesting that lectin receptors are mobile in the plane of the plasma membrane.In the early cleavage stages intensive lectin binding occurs only at the boundary between preexisting and nascent plasma membranes. The external surface of the embryo has few lectin receptors up to the late gastrula stage. The unpigmented nascent plasma membranes, when exposed to fluorescent lectins, do not assume any fluorescence distinguishable from the background autofluorescence of yolk, in stages up to the mid-blastula. From this stage onwards lectin binding was observed on the membranes of the reverse side of surface layer cells and on the membranes of deep layer cells. During gastrulation there is an accumulation of lectin-binding material on surfaces involved in intercellular contacts.The significance of lectin binding material for morphogenesis is discussed.     

A study of intracellular transport of secretory glycoproteins in rat liver

To study the transport of secretory glycoproteins in the endoplasmic reticulum of rat liver, the distribution of nascent glycoproteins in the membrane and luminal fraction of rough and smooth microsomes has been examined after a short-time incorporation of radioactive glucosamine in vivo. 50--60% of the radioactivity was associated with the membranes of rough and smooth microsomes, whereas about 10% of the serum albumin was found in the same fractions. The relative amount of radioactivity in the membranes was the same whether the luminal content of the microsomal vesicles was released by sonication, French press, Triton X-100, Brij 35 or sodium deoxycholate. The distribution of labeled glycoproteins between the membrane and luminal fraction of rough and smooth microsomes did not change during the time interval of 15--120 min after administration of the isotope. The similarity of the labeling patterns obtained after sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis indicated that the same set of glycoproteins were located in the lumen and the membrane of rough and smooth microsomes. A specific precipitation of nascent glycoproteins from both the membrane and luminal fractions of rough and smooth microsomes were obtained with rabbit antiserum against rat serum. The nascent glycoproteins associated with the membranes were not released by high ionic strength or treatment with mercaptoethanol. A slow exchange between [14C]glucosamine-labeled glycoproteins in the lumen and membrane fraction was, however, found.     

Studies on the mechanisms of autophagy: formation of the autophagic vacuole

Autophagic vacuoles form within 15 min of perfusing a liver with amino acid-depleted medium. These vacuoles are bound by a "smooth" double membrane and do not contain acid phosphatase activity. In an attempt to identify the membrane source of these vacuoles, I have used morphological techniques combined with immunological probes to localize specific membrane antigens to the limiting membranes of newly formed or nascent autophagic vacuoles. Antibodies to three integral membrane proteins of the plasma membrane (CE9, HA4, and epidermal growth factor receptor) and one of the Golgi apparatus (sialyltransferase) did not label these vacuoles. Internalized epidermal growth factor and its membrane receptor were not found in nascent autophagic vacuoles but were present in lysosome-like degradative autophagic vacuoles. All these results suggested that autophagic vacuoles were not formed from plasma membrane, Golgi apparatus, or endosome constituents. Antisera prepared against integral membrane proteins (14, 25, and 40 kD) of the RER was found to label the inner and outer limiting membranes of almost all nascent autophagic vacuoles. In addition, ribophorin II was identified at the limiting membranes of many nascent autophagic vacuoles. Finally, secretory proteins, rat serum albumin and alpha 2u- globulin, were localized to the lumen of the RER and to the intramembrane space between the inner and outer membranes of some of these vacuoles. The results were consistent with the formation of autophagic vacuoles from ribosome-free regions of the RER.     

Resonance energy transfer imaging of phospholipid vesicle interaction with a planar phospholipid membrane: undulations and attachment sites in the region of calcium-mediated membrane--membrane adhesion

Membrane fusion of a phospholipid vesicle with a planar lipid bilayer is preceded by an initial prefusion stage in which a region of the vesicle membrane adheres to the planar membrane. A resonance energy transfer (RET) imaging microscope, with measured spectral transfer functions and a pair of radiometrically calibrated video cameras, was used to determine both the area of the contact region and the distances between the membranes within this zone. Large vesicles (5-20 microns diam) were labeled with the donor fluorophore coumarin- phosphatidylethanolamine (PE), while the planar membrane was labeled with the acceptor rhodamine-PE. The donor was excited with 390 nm light, and separate images of donor and acceptor emission were formed by the microscope. Distances between the membranes at each location in the image were determined from the RET rate constant (kt) computed from the acceptor:donor emission intensity ratio. In the absence of an osmotic gradient, the vesicles stably adhered to the planar membrane, and the dyes did not migrate between membranes. The region of contact was detected as an area of planar membrane, coincident with the vesicle image, over which rhodamine fluorescence was sensitized by RET. The total area of the contact region depended biphasically on the Ca2+ concentration, but the distance between the bilayers in this zone decreased with increasing [Ca2+]. The changes in area and separation were probably related to divalent cation effects on electrostatic screening and binding to charged membranes. At each [Ca2+], the intermembrane separation varied between 1 and 6 nm within each contact region, indicating membrane undulation prior to adhesion. Intermembrane separation distances < or = 2 nm were localized to discrete sites that formed in an ordered arrangement throughout the contact region. The area of the contact region occupied by these punctate attachment sites was increased at high [Ca2+]. Membrane fusion may be initiated at these sites of closest membrane apposition.     

Immunocytochemical studies of cardiac myofibrillogenesis in early chick embryos. III. Generation of fasciae adherentes and costameres

To study whether the first myofibrils are separate from or firmly bound to the myocytic cell membranes, whole mount preparations of 6-12-somite-stage chick embryonic hearts were examined by fluorescence microscopy after double labeling with antibodies to vinculin (fluorescein-conjugated) and rhodamine-phalloidin, or with antibodies to titin (rhodamine-conjugated) and nitrobenz-oxadiazole-phallacidin. When a small number of myofibrils appeared for the first time at the nine somite stage, most of them were already bound to the cell membranes through zonulae adherentes, fasciae adherentes, or costameres. In the outer of the two myocardial cell layers, in which the myocytes were closely in contact with each other along polygonal boundaries, fasciae adherentes and costameres developed at the boundaries, apparently by conversion of preexisting zonulae adherentes. On the other hand, in the inner cell layer, in which myocytes were more loosely associated with each other, both costameres and fasciae adherentes appeared to develop de novo, the former in association with the inner surface of the myocardial wall and the latter at the intercellular boundaries. The myofibrillar tracks in the inner layer followed long and smooth courses and were as a whole aligned in the circumferential direction of the tubular heart wall from the earliest stage of myofibril formation. Those in the outer layer were arranged in a pattern of two- or three-dimensional networks in the 9-10 somite stage, although many myofibrils were also circumferentially directed. The fact that the majority of the first myofibrils were already bound to the cell membranes in a directed manner suggests that myocytes at the earliest stage of myofibril formation are endowed with spatial information that directs the organization of nascent myofibrils. It is proposed that the myocyte cell membranes perform an essential role in cardiac myofibrillogenesis.     

Binding of ribosomes to the rough endoplasmic reticulum mediated by the Sec61p-complex

The cotranslational translocation of proteins across the ER membrane involves the tight binding of translating ribosomes to the membrane, presumably to ribosome receptors. The identity of the latter has been controversial. One putative receptor candidate is Sec61 alpha, a multi- spanning membrane protein that is associated with two additional membrane proteins (Sec61 beta and gamma) to form the Sec61p-complex. Other receptors of 34 and 180 kD have also been proposed on the basis of their ability to bind at low salt concentration ribosomes lacking nascent chains. We now show that the Sec61p-complex has also binding activity but that, at low salt conditions, it accounts for only one third of the total binding sites in proteoliposomes reconstituted from a detergent extract of ER membranes. Under these conditions, the assay has also limited specificity with respect to ribosomes. However, if the ribosome-binding assay is performed at physiological salt concentration, most of the unspecific binding is lost; the Sec61p- complex then accounts for the majority of specific ribosome-binding sites in reconstituted ER membranes. To study the membrane interaction of ribosomes participating in protein translocation, native rough microsomes were treated with proteases. The amount of membrane-bound ribosomes is only slightly reduced by protease treatment, consistent with the protease-resistance of Sec61 alpha which is shielded by these ribosomes. In contrast, p34 and p180 can be readily degraded, indicating that they are not essential for the membrane anchoring of ribosomes in protease-treated microsomes. These data provide further evidence that the Sec61p-complex is responsible for the membrane- anchoring of ribosomes during translocation and make it unlikely that p34 or p180 are essential for this process.     

Temperature Dependence of Membrane Lipid Composition in Early Blastula Embryos of Lytechinus pictus: Selective Sorting of Phospholipids into Nascent Plasma Membranes

Lytechinus pictus eggs were fertilized and incubated at 10, 16, and 23°C until the early blastula stage of embryonic development. The phospholipid composition of the embryos and control unfertilized eggs remain identical and unchanged as incubating temperatures are varied; thus, neither incubating temperature, fertilization nor membrane assembly affect their total phospholipid composition. This result agrees with metabolic studies by others, using only a single incubation temperature, and indicates that embryonic development to the early blastula stage occurs with little, if any, de novo phospholipid biosynthesis. However, as in all poikilotherms, the phospholipid composition of the nascent plasma membranes varies with the incubation temperature. Thus, until the blastula stage of embryonic development, the lipids of these newly formed plasma membranes are derived from lipid pools within the embryo whose phospholipid composition is static. The variation of plasma membrane composition is primarily reflected in an increase in the phosphatidylethanolamine (PE): phosphatidylcholine (PC) ratio as incubating temperatures decrease; this is achieved by an exchange of PE for PC. Several mechanisms are considered for the specificity of the selective sorting and assembly of these phospholipids into the nascent plasma membranes. Received: 16 March 1999/Revised: 15 May 1999     

Autophagy in the epithelial cells of murine seminal vesicle in vitro

The spontaneous autophagic activity in epithelial cells of isolated tissue slices of murine seminal vesicle is strongly enhanced by short (5 min) pretreatment in a medium containing 0.03% Triton X-100. In addition to the significant increase in the cytoplasmic volume fraction and the mean size of autophagic vacuoles, the appearance of shorter or longer smooth membrane pairs located between cisterns of rough endoplasmic reticulum (RER) and in the vicinity of nucleus is also greatly stimulated. Their morphological features observed after application of various fixation methods, freeze-substitution and freeze-fracture techniques show that they are unclosed nascent isolation membranes, representing a unique class of intracellular membranes. They may grow around the nucleus, leading to its complete autophagic sequestration and degradation, which is observed here for the first time. Treatment with 3-methyladenine or wortmannin inhibits the formation of autophagosomes, leading to their regression with a halving time of 7 min. In contrast, these inhibitors cause extremely fast shrinking of nascent isolating membranes, leading to their complete disappearance within 7 min. We propose that the early events of autophagy involve three main steps: initiation, growth and closure, and suggest that the growth of nascent isolation membranes is reversible i.e. the membranes may be subject to disassembly before their closure is completed. Bending and closure of the isolation membrane and the stability of neighbouring cellular structures appear as important determinants of size regulation. These early steps of autophagy are good candidates for very fast accommodation to changing conditions and subtle regulation by phosphoinositide kinases as indicated by wortmannin sensitivity.     

Immunocytochemical evidence for the presence of two domains in the plasma membrane of sea urchin blastomeres

Summary The blastomeres of sea urchin embryos have two surface regions with different properties. Numerous microvilli are present in the apical surface region, while the baso-lateral surface region, either on adjoining adjacent cells or facing the blastocoel, is smooth. When blastomeres are isolated from embryos and stained with fluorescein-isothiocyanate-labelled anti-(egg surface) antibody (anti-ES) prepared against membranes isolated from fertilized eggs, the apical microvillous region fluoresces while the smooth region does not [Yazaki I (1984) Acta Embryol Morphol Exp 5∶3–22]. In order to study quantitatively the ‘bindability’ of the membrane in the two regions to anti-ES, immunoelectron microscopy was used. Blastomeres isolated from embryos ofHemicentrotus pulcherrimus at the eight-cell stage were treated with rabbit anti-ES serum or pre-immune serum and then with ferritin-conjugated goat anti-(rabbit IgG) for 10 min at 0°C, mainly before fixation. About 10 times (maximally 45 times) more ferritin particles were counted per contour length in the microvillous surface region than in the smooth surface region. These results suggest that the membrane of the blastomeres of sea urchin embryos is a mosaic of two different membrane territories: one represented by the microvillous surface originating from the unfertilized egg, which binds anti-ES, the other by the smooth surface newly organized after the first cleavage, which does not react with anti-ES. The mechanism of segregation of the membrane into these two regions is discussed.     

The Escherichia coli SRP and SecB targeting pathways converge at the translocon.

Two distinct protein targeting pathways can direct proteins to the Escherichia coli inner membrane. The Sec pathway involves the cytosolic chaperone SecB that binds to the mature region of pre-proteins. SecB targets the pre-protein to SecA that mediates pre-protein translocation through the SecYEG translocon. The SRP pathway is probably used primarily for the targeting and assembly of inner membrane proteins. It involves the signal recognition particle (SRP) that interacts with the hydrophobic targeting signal of nascent proteins. By using a protein cross-linking approach, we demonstrate here that the SRP pathway delivers nascent inner membrane proteins at the membrane. The SRP receptor FtsY, GTP and inner membranes are required for release of the nascent proteins from the SRP. Upon release of the SRP at the membrane, the targeted nascent proteins insert into a translocon that contains at least SecA, SecY and SecG. Hence, as appears to be the case for several other translocation systems, multiple targeting mechanisms deliver a variety of precursor proteins to a common membrane translocation complex of the E.coli inner membrane.     

Differential phosphorylation of band 3 and glycophorin in intact and extracted erythrocyte membranes

This report presents an analysis of the phosphorylation of human and rabbit erythrocyte membrane proteins which migrate in NaDodSO₄-polyacrylamide gels in the area of the Coomassie Blue-stained proteins generally known as band 3. The phosphorylation of these proteins is of interest as band 3 has been implicated in transport processes. This study shows that there are at least three distinct phosphoproteins associated with the band 3 region of human erythrocyte membranes. These are band 2.9, the major band 3, and PAS-1. The phosphorylation of these proteins is differentially catalyzed by solubilized membrane and cytoplasmic cyclic AMP-dependent and -independent erythrocyte protein kinases. Band 2.9 is present and phosphorylated in unfractionated human and rabbit erythrocyte ghosts but not in NaI- or dimethylmaleic anhydride (DMMA)-extracted membranes. These latter membrane preparations are enriched in band 3 and in sialoglycoproteins. The NaI-extracted ghosts contain residual protein kinase activity which can catalyze the autophosphorylation of band 3 whereas the DMMA-extracted ghosts are usually devoid of any kinase activity. However, both NaI- and DMMA-extracted ghosts, as well as Triton X-100 extracts of the DMMA-extracted ghosts, can be phosphorylated by various erythrocyte protein kinases. The kinases which preferentially phosphorylate the major band 3 protein are inactive towards PAS-1 while the kinases active towards PAS-1 are less active towards band 3. The band 3 protein in the DMMA-extracted ghosts can be cross-linked with the Cu²⁺ -σ-phenanthroline complex. The cross-linking of band 3 does not affect its capacity to serve as a phosphoryl acceptor nor does phosphorylation affect the capacity of band 3 to form cross-links. In addition to band 2.9, the major band 3 and PAS-1, another minor protein component appears to be present in the band 3 region in human erythrocyte membranes. This protein is specifically phosphorylated by the cyclic AMP-dependent protein kinases isolated from the cytoplasm of rabbit erythrocytes. The rabbit erythrocyte membranes lack PAS-1 and the cyclic AMP-dependent protein kinase substrate.     

A signal sequence for the insertion of a transmembrane glycoprotein. Similarities to the signals of secretory proteins in primary structure and function.

The biosynthesis of a secretory protein and a transmembrane viral glycoprotein are compared by two different experimental approaches. (a) NH2-terminal sequence analysis has been performed on various forms of the transmembrane glycoprotein of vesicular stomatitis virus synthesized in cell-free systems. The sequence data presented demonstrate that the nascent precursor of the glycoprotein contains a "signal sequence" of 16 amino acids at the NH2 terminus, whose sequence is Met-Lys-Cys-Leu-Leu-Tyr-Leu-Ala-Phe-Leu-Phe-Ile-(His-Val-Asn)-Cys. This signal sequence is proteolytically cleaved during the process of insertion into microsomal membranes prior to chain completion. The new NH2 terminus of the inserted, cleaved, and glycosylated membrane protein is located within the lumen of the microsomal vesicles and is identical to that of the authentic glycoprotein from virions. (b) Nascent chain competition experiments were performed between this glycoprotein, bovine pituitary prolactin (a secretory protein), and rabbit globin (a cytosolic protein). It was found that the nascent membrane glycoprotein, but not nascent globin, competed with nascent prolactin for membrane sites involved in the early biosynthetic event of transfer across membranes. These data suggest that an initially common pathway is involved in the biogenesis of secretory proteins and at least one class of integral membrane proteins.     

Nascent ribosomal and messenger RNA in DNA-membrane-complexes of Escherichia coli

Summary From Escherichia coli, DNA-membrane-complexes have been isolated which contain about 40% of the ribosomes, about 95% of the DNA and nearly all of the nascent RNA. The kinetic data on pulse labeled RNA show an average time of turnover of about 60 sec both for nascent messenger- and nascent ribosomal RNA. A proportion of the polysomes with nascent messenger RNA as well as most of the nascent ribosomal RNA is found in association with membranes, as has been shown by subfractionations of the DNA-membrane-complex involving treatment with DNase and desoxycholate. In this early transient stage, ribosomal precursor RNA already acquires some ribosomal proteins, as has been shown by arginine pulse label. Data on partial release of DNA from the DNA-membrane-complex by treatment with extremely low doses of DNase indicate that messenger RNA synthesis occurs in clusters on the DNA.The results support models in which, at any given time, RNA synthesis proceeds mainly in sections of the DNA close to the membrane. Thereby the DNA is linked to the membrane via nascent RNA contained in ribosomal precursors as well as via nascent messenger RNA on membrane-bound polysomes.     

Nascent chicken ovalbumin contains the functional equivalent of a signal sequence

Highly purified mRNA for chicken ovalbumin has been translated in a cell-free protein synthesizing system from rabbit reticulocytes in the presence or absence of EDTA-stripped microsomal membranes from dog pancreas. Nascent--but not completed--ovalbumin was transferred across the microsomal membrane, as demonstrated by cotranslational core glycosylation of ovalbumin nascent chains, by resistance to posttranslational proteolysis of only the glycosylated ovalbumin chains, and by cosedimentation with the membrane of exclusively the glycosylated form. Furthermore, nascent chains of bovine prolactin were observed to compete with nascent ovalbumin for transfer across the microsomal membrane. However, no competition for membrane sites was observed between nascent chains of rabbit globin and either nascent ovalbumin or prolactin. We interpret these results to suggest that nascent ovalbumin contains the functional equivalent of a signal sequence for transfer across membranes, and that membrane components involved in the segregation of secretory proteins with cleaved signal sequences also function in the segregation of ovalbumin.     

The fine structure of the gamont of Pterospora floridiensis (Apicomplexa: Eugregarinida)

Transmission electron microscopy of the gamont stage of Pterospora floridiensis has revealed a number of features. The gamont's surface varies from smooth to crenulate, with numerous pockets and folds. The pellicle is composed of an outer membrane, a middle lucent region, and an inner dense layer comprised of two tightly appressed membranes. Short ridges on the pellicle are 200-300+ nm long, 75-100 nm wide, and have a height of approximately 50 nm. The thickness of the pellicle is 100 nm when measured from the inner membrane to the top of a ridge. The ridges are formed by the plasma membrane and an underlying structure that is circular in cross-section. The surface folds and the pellicular ridges are distributed over the soma and the cell's unusual branching arms, though both are reduced near the junction between two gamonts in syzygy, and are absent at the central area of the junctional site. The cell has numerous active Golgi complexes associated with vesicles, as well as scattered dense mitochondria, lipid droplets, and paraglycogen granules. The nucleus has a large (13 microm) endosome, eccentrically located, and peripheral chromatin along the inner nuclear membrane.     

Patterns of expression of a 15K beta-D-galactoside-specific lectin during early development of the avian embryo

We have determined, by immunohistochemical and biochemical techniques, the distribution of an endogenous beta-D-galactoside-binding lectin between the early primitive streak stage and the 5th day of embryonic development of the chick.The lectin, which was purified from the pectoral muscle of 16-day-old chick embryos, migrates on SDS-PAGE as a single polypeptide of relative molecular mass 15 x 10(3). Antibodies to this pure lectin interact with the 15K (K = 10(3) M(r)) polypeptide as well as with a 6.5K polypeptide; this second component appears to be antigenically related to the 15K lectin, as antibodies affinity purified on the 15K band recognize both polypeptides. In early stages of development, lectin immunoreactivity was present in most cells of the epiblast and hypoblast in the region of the primitive streak, while towards the edge of the area pellucida the epiblast was stained less intensely. During gastrulation, strong immunoreactivity was present also in migrating cells and in the mesoblast, while at the margin of the area pellucida the epiblast was negative. Up to the 10-somite stage, lectin immunoreactivity was present in the somites, neural tube and presumptive cardiac region; the non-neural ectoderm and the extracellular matrix were not labeled; the predominant immunoreactive component at this stage of development was the 6.5K polypeptide. Later in development, the lectin immunoreactivity gradually disappeared from the dermamyotome and nervous system to reappear conspicuously as soon as a differentiated myotome could be detected. Immunoreactivity was very high in the myotome, skeletal and cardiac muscles and transient in smooth muscles. The only region of the nervous system that continued to express the lectin throughout development was the trigeminal (semilunar) ganglion; in all regions of the nervous system, the lectin immunoreactivity disappeared early in development to be re-expressed only much later. The lining epithelium of the digestive tract and other endodermal derivatives expressed the lectin transiently. In the extraembryonic membranes, immunoreactivity to the lectin was observed in the yolk sac and in both layers of the amnion. The striking regulation of the expression of this endogenous lectin suggests that its functions are linked to cell proliferation and/or to the selective expression of a developmentally-timed cell phenotype.     

Amino acid sequences of transport peptides associated with canine exocrine pancreatic proteins

Using microsequencing techniques and proteins labeled in vitro with tritiated amino acids we have obtained the following NH2-terminal sequences for six canine pancreatic presecretory proteins: pretrypsinogen 1, pretrypsinogen 2+3, prechymotrypsinogen 2, preproelastase1, preporcarboxypeptidase A1, preamylase. Points of cleavage by the transport peptidase, indicated by the vertical arrows, were located from sequences of authentic products synthesized in the presence of membranes of the rough endoplasmic reticulum. All of the identified residues in the pancreatic transport peptides are hydrophobic. Predictions of secondary structure were calculated for each of the transport peptides. The data indicated neither a common primary of secondary structure which could be interpreted as the signal for functional binding of the nascent presecretory protein to the rough endoplasmic reticulum membrane. These findings suggest that the initial interaction with the membrane or membrane receptor may depend in part, on the hydrophobic nature of the transport peptides. Five of the presecretory proteins showed a region with a high probability of forming a beta-turn immediately following the cleavage point. This feature may give the nascent peptide a region of flexibility that would facilitate both its insertion as a loop structure into the membrane and its cleavage by the transport peptidase. The sequences of authentic secretory products derived from a variety of pancreatic tissues suggest that hydrophilic residues are required immediately following the cleavage point in order to allow translocation of the nascent polypeptide chains across the membrane.     

Characterization and regulation by protein kinase C of renal glomerular atrial natriuretic peptide receptor-coupled guanylate cyclase

The nature and regulation of atrial natriuretic peptide (ANP)-sensitive guanylate cyclase in rat renal glomerular membranes was examined. By affinity crosslinking techniques, three bands with apparent molecular masses of 180, 130 and 64 kDa were specifically labeled with [125I]ANP. A specific antibody to the 180 kDa membrane guanylate cyclase of rat adrenocortical carcinoma recognized a 180 kDa band on Western blot analysis of solubilized, GTP-affinity purified glomerular membrane proteins. The same antibody completely inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. Partially purified protein kinase C inhibited ANP-stimulated guanylate cyclase activity in glomerular membrane fractions. It is concluded that a 180 kDa ANP-sensitive guanylate cyclase is present in glomerular membranes, and that this enzyme is inhibited directly by protein kinase C.