Developmental and strain-specific heterogeneity of rat adrenal chromaffin cells recognized by a monoclonal antibody against intact chromogranin B.

We have raised a monoclonal antibody (MAB-1E10) reactive with the intact forms but not the processing products of the chromaffin cell vesicle protein chromogranin B (CgB). The antibody recognizes rat and human, but not bovine and chick adrenal chromaffin cells. In addition, MAB-1E10 immunoreactivity was detected in rat PC 12 pheochromocytoma cells and in pituitaries. Several other tissues, including pancreas, small intestine and superior cervical ganglia, which are known to contain CgB in endocrine cells or neurons, respectively, were found not to be reactive with MAB-1E10. Using short-term cultures of dissociated adrenal chromaffin cells from Hannover-Wistar rats, we found that the expression of intact CgB is developmentally regulated. Between embryonic day 19 and postnatal day 40, about 80% of adrenal chromaffin cells--identified by their reactivity with an antibody against the enzyme dopamine-beta-hydroxylase--were found to be reactive with MAB-1E10. The proportion of positive cells subsequently decreased to about 5% at postnatal day 90. In the presence of glucocorticoids, this decrease was reduced to about 45% CgB-positive cells at postnatal day 90. In another rat strain, Sprague-Dawley rats, the proportion of MAB-1E10-immunoreactive chromaffin cells (about 50%) remained constant from birth to adulthood. Our results indicate that CgB is differentially expressed and/or processed in different rat tissues, strains and during development, and furthermore, that expression or processing in rat chromaffin cells might be regulated by glucocorticoids. Intact CgB appears to be a marker for a subpopulation of chromaffin cells, but its function(s) remains to be clarified.

Calcium-dependence of chromogranin A-catecholamine interaction.

Major components of the secretory organelle of bovine adrenal medullary cells, the chromaffin vesicles, are the acidic protein chromogranin A, catecholamines and Ca2+. The binding of Ca2+ to chromogranin A has been established. To study the interaction between chromogranin A and catecholamines and its dependence on Ca2+ we immobilized chromogranin A to a newly raised monoclonal antibody. It is shown that chromogranin A can bind (i) about 0.5 mol catecholamines per mol in a non-calcium-dependent manner and (ii) about 5 mol per mol in the presence of calcium. These results further support the notion that chromogranin A may act as a secretory granule-condensing protein.

Polyclonal and monoclonal antibodies against chicken gizzard 5'-nucleotidase inhibit the spreading process of chicken embryonic fibroblasts on laminin substratum.

Polyclonal and monoclonal antibodies raised against chicken gizzard 5'-nucleotidase were tested in adhesion assays of embryonic chicken fibroblasts (CEF) for their ability to interfere with the adhesion process of these cells on either laminin or fibronectin substrata. The initial attachment process of CEF on fibronectin and laminin substrata was not influenced by preincubating these cells with antibodies against chicken gizzard 5'-nucleotidase. However, the subsequent spreading process of these cells was found to be inhibited for at least 2 h on a laminin substratum. This effect was obtained with a polyclonal antibody as well as with one from 12 monoclonal antibodies raised against the native enzyme purified from chicken gizzard. In vitro assays demonstrated a competition of laminin and this monoclonal antibody for the binding site on purified 5'-nucleotidase. Spreading-arrested and rounded CEF do not develop prominent intracellular stress-fibers like control cells, instead they seem to concentrate their available actin in areas of presumptive initial contact with the laminin substratum.

Immunochemical characterization of a band 3-like anion exchanger in collecting duct of human kidney.

Poly- and monoclonal antibodies have been prepared against the cytoplasmic domain (43 kDa) and the 17-, 20-, and 35-kDa fragments of the membrane-spanning domain of the human erythrocyte anion exchanger, band 3. The antibodies were used to localize and further characterize analogues of band 3 in the human kidney. We report here that the basolateral membrane of intercalated cells of the connecting tubules and collecting ducts contains an analogue of band 3 that appears to be highly homologous to the erythrocyte anion exchanger. This band 3-like protein is probably important for reabsorption of bicarbonate in the collecting duct system and thus for acidification of the forming urine. The band 3-like protein of the intercalated cells contain immunoreactive sites of both the cytoplasmic domain and the three major fragments of the membrane-spanning domain of erythrocyte band 3. Although no immunological differences were detected between the membrane-spanning domains of band 3 in erythrocytes and intercalated cells, there are at least three sites along the cytoplasmic domain of kidney band 3 that differ from erythrocyte band 3 in either amino acid composition or posttranslational modifications. The main kidney analogue of band 3 that contains epitopes of the cytoplasmic domain as well as the 17- and 35-kDa membrane-spanning domain of erythroid band 3 is a polypeptide with an apparent molecular mass of 100-110 kDa. Further immunoreactive polypeptides at approximately 180, approximately 140, approximately 38, approximately 25-30 kDa that were detected at lower stringency and higher sensitivity of the immunoblotting procedure may be members of a multigene family that encodes a series of related proteins.

Reconstitution of purified chicken gizzard 5'-nucleotidase in phospholipid vesicles. Evidence for its transmembraneous character and the existence of functional domains on both sides of the phospholipid bilayer.

5'-Nucleotidase, purified to homogeneity from chicken gizzard using published procedures [Dieckhoff, J., Knebel, H., Heidemann, M. and Mannherz, H. G. (1985) Eur. J. Biochem. 151, 377-383] was incorporated into artificial phospholipid vesicles after prolonged dialysis against detergent-free buffer or by a gel filtration procedure. After dialysis the obtained liposomes exhibit a mean diameter of 80 nm and contain 5'-nucleotidase at random orientation, demonstrated by finding up to 50% of the total liposome-incorporated AMPase activity to be cryptic, i.e. could only be measured after their permeabilization by addition of detergent. By affinity chromatography a phospholipid vesicle fraction could be obtained containing almost exclusively cryptic AMPase activity, thus representing the inside-out orientation of 5'-nucleotidase. Comparative analysis of physiochemical and enzymatic properties of 5'-nucleotidase reveals differences between the detergent-solubilized and the liposome-incorporated 5'-nucleotidase including a changed accessibility of the enzyme to polyclonal and monoclonal antibodies. Binding and AMPase inhibition studies with different polyclonal antibodies strongly indicate to the existence of a cytoplasmic domain of chicken gizzard 5'-nucleotidase. F-actin appears preferentially to interact with the cytoplasmic domain of liposome-incorporated 5'-nucleotidase.

Monoclonal antibodies against 5'-nucleotidase from chicken gizzard. Evidence for species and tissue specific differences of 5'-nucleotidase.

5'-Nucleotidase from chicken gizzard smooth muscle was purified to homogeneity and used as immunogen for generating monoclonal antibodies. From about 150 positive clones nine IgG producing hybridoma cell lines have been selected for further characterization and antibody preparation. The resulting antibodies bind 5'-nucleotidase from chicken smooth muscle, chicken skeletal muscle, and chicken heart muscle but not the enzyme from chicken liver or rat liver. It could clearly be demonstrated that the nine antibodies recognize different antigenic determinants. Four of these antibodies are strong inhibitors of the AMPase activity of 5'-nucleotidase. One antibody is a weak inhibitor and four other antibodies have no effect on its enzymic activity. One of the monoclonal antibodies was used for immunoaffinity purification of 5'-nucleotidase from chicken heart muscle and chicken skeletal muscle. Pure and active enzymes could be isolated from detergent extracts in one step with a 10 to 20-fold higher yield compared to classical purification procedures. The subcellular distribution of 5'-nucleotidase in chicken gizzard was investigated using indirect immunofluorescence. We found a staining of the plasma membrane of smooth muscle cells and endothelial cells by all of the nine antibodies with variations in the staining intensity.

A statistical approach to determine monoclonality after limiting cell plating of a hybridoma clone.

One of the standard methods to isolate a hybridoma clone producing a monoclonal antibody requires successive steps of limiting dilution. The probability of obtaining a monoclonal antibody increases with the number of limiting dilution steps. However, without meticulous visual screening monoclonality is hard to prove. Here we describe a statistical analysis, based on Poisson's approximation, which allows one to calculate the number of hybridoma cells at a given plating efficiency so that when seeded a predictable number of mono-, bi-, etc.-clonal cultures are obtained after the first step of limiting cell plating.

Tetanus toxin binding to different morphological phenotypes of cultured rat and bovine adrenal medullary cells.

Tetanus toxin (TT) binding to cultured rat and bovine adrenal medullary cells has been investigated using indirect immunofluorescence and anti-dopamine-beta-hydroxylase (DBH) antibodies as a probe to identify catecholaminergic cells. TT binds to all rat adrenal medullary cells which display a neuronal phenotype induced by treatment with nerve growth factor and/or medium conditioned by C6 glioma cells. In contrast, 90-95% of the rounded DBH-positive cells are TT-negative, suggesting that in vitro-transdifferentiation of rat chromaffin cells alternates the expression of membrane properties. Cultured bovine chromaffin cells have no TT binding sites independent of their morphological phenotype.

Shape determinations of ribosomal proteins in situ.

Ribosomes are heterogeneous for neutrons because RNA and proteins have different neutron-scattering-length densities. This heterogeneity is an obstacle to the shape determination of single ribosomal components within the ribosome. Therefore, we homogenized (matched) the neutron-scattering-length densities of RNA and proteins. 23S and 5S RNA from the large ribosomal subunit were isolated from cells grown in a medium containing 76% 2H2O. The total protein fraction of the large ribosomal subunit was isolated from cells grown in a medium containing 84% 2H2O. When these constituents were used for total reconstitution of 50S subunits, neutron scattering measurements of the reconstituted particles revealed excellent matching near 100% 2H2O. A three-step reconstitution procedure was developed that allowed the reconstitution of 50S subunits from deuterated RNA, deuterated total (i.e., unfractionated) proteins, and single protonated proteins. The reconstituted particles contain one protonated protein or two in a matched ribosomal matrix and were used for shape determination or distance measurement of mass centers of gravity, respectively. The signal/noise ratio is high enough to allow measurement in solutions containing nearly 100% 2H2O at concentrations of only 300-500 A260 nm units/ml. Our experiments have proved the feasibility of our biochemical strategy. The shape determinations of ribosomal proteins in situ gave radii of gyration for L1, L3, L4, and L23 of 26 +/- 2, 22 +/- 2, 20 +/- 2, and 13 +/- 2 A, respectively.