Max Zollinger

Maturation and subcellular compartmentation of potato starch phosphorylase.

The subcellular localization and maturation of starch phosphorylase (EC 2.4.1.1) was studied in developing potato tubers. The enzyme is localized inside the stroma of amyloplasts in young tubers, whereas in mature tubers it is found within the cytoplasm in the immediate vicinity of the plastids. A phosphorylase cDNA clone was isolated and used in RNA gel blot experiments to demonstrate that phosphorylase mRNAs are of the same size and abundance in both young and mature tubers. In vitro translation of mRNAs followed by immunoprecipitation with a phosphorylase antiserum indicates that the enzyme is synthesized as a higher molecular weight precursor in both young and mature tubers. The presence of a transit peptide at the N terminus of the protein was confirmed by the sequencing of the phosphorylase cDNA clone. The transit peptide has several structural features common to transit peptides of chloroplast proteins but contains a surprisingly large number of histidine residues. The mature form of the enzyme is present in both young and mature tubers, suggesting that a similar processing of the transit peptide may take place in two different subcellular locations.

Investigation of a possible role of the amino-terminal pro-region of proopiomelanocortin in its processing and targeting to secretory granules

Proopiomelanocortin (POMC) is a polyprotein which is targeted to the regulated secretory pathway of neuroendocrine cells where it undergoes tissue-specific proteolysis to yield peptides such as adrenocorticotropic hormone, beta-lipotropin and beta-endorphin. The pro-region of POMC is 49 amino acid long with two disulfide bonds between cysteine residues 2 and 24 and 8 and 20. These cysteine residues are conserved across the species. The pro-region contains no known hormonal sequence. Sorting to the regulated secretory pathway is thought to involve targeting signals encoded in the structure of secretory proteins. In the present study, we have examined the possibility that the disulfide bridges located in the NH2-terminal portion of the pro-region of POMC are essential for maintaining a determinant involved in the sorting of POMC to the regulated secretory pathway. Using site-directed and deletion mutagenesis of the porcine POMC cDNA, we created mutants in which one or both disulfide bridges were disrupted or in which the first 26 amino acid residues of the pro-region were deleted. Recombinant retroviruses carrying the mutated POMC cDNAs were used to infect Neuro2A cells. Immunofluorescence and immunoelectron microscopy studies performed on infected cells revealed that the unmutated and mutated POMC-immunoreactive peptides were localized in dense-core vesicles at the tips of cellular extensions. Analysis of the POMC-immunoreactive peptides extracted from the infected Neuro2A cells indicated that the mutated precursors in which one disulfide bridge was disrupted (POMC-S2 or POMC-S8) were stored and processed as efficiently as the unmutated POMC. By contrast, the mutated precursor in which both disulfide bridges were disrupted (POMC-S2,8) did not accumulate in intracellular compartments to the same extent as unmutated POMC. Moreover, this mutant was very inefficiently processed and no release could be observed upon stimulation of the cells with K+/Ca2+. These results suggest that POMC-S2,8 entered the regulated secretory pathway less efficiently than the unmutated precursor. However, when both disulfide bridges were removed from the precursor from the precursor by deletion of the first 26 amino acid residues of POMC, the truncated precursor (POMC delta 1-26) behaved as the unmutated POMC. Taken together our results indicate that the NH2-terminal portion of the pro-region including both disulfide bridges can be deleted without affecting the targeting of the molecule to secretory granules. However, when the entire POMC sequence is expressed in Neuro2A cells, the proper folding of the NH2-terminal region might be important for efficient processing and targeting.

Expression of porcine pro-opiomelanocortin in mouse neuroblastoma (Neuro2A) cells: Targeting of the foreign neuropeptide to dense-core vesicles

Pro-opiomelanocortin (POMC) is the precursor to several pituitary hormones and neuropeptides including adrenocorticotropic hormone (ACTH) and beta-endorphin (beta-END). In neuroendocrine cells, peptide hormones and neuropeptides are targeted to the dense-core vesicles of the regulated secretory pathway. These vesicles are transported to the ends of cellular extensions where they are stored until they release their content upon external stimulation of the cell. In order to study the cellular mechanisms involved in targeting of neuropeptides, we have expressed POMC in Neuro2A cells, a cell line of neural origin. Using immunofluorescence labeling and immunoelectron microscopy we show that in Neuro2A cells POMC is packaged in dense-core vesicles which accumulate at the tips of cellular processes. Intracellular accumulation of POMC was not observed in NIH 3T3 fibroblasts. When a soluble form of an integral membrane protein, neutral endopeptidase (E.C. 3.4.24.11) (secNEP), was expressed in Neuro2A cells, the protein was found to be constitutively secreted without prior accumulation in dense-core vesicles. Our results suggest that in Neuro2A cells, targeting to the regulated secretory pathway is restricted to peptide hormones and neuropeptides and establish this cell line as a valid model for studying the molecular events involved in neuropeptide sorting into the regulated secretory pathway.

A new electron microscopic method for studying protein-nucleic acid interactions.

Abstract A new method for preparation of nucleic acid specimens for electron microscopy has been adapted to study the interaction of proteins with DNA. Both a detergent and a basic protein are added to the DNA-protein solution before spreading on a hypophase containing 0.2 m ammonium acetate. This method has been tested using T7 DNA and Escherichia coli RNA polymerase. Specifically bound enzyme molecules were clearly visible on the well extended DNA molecules; the binding sites were located at 0.59, 1.24, 1.57, and 1.86% of the total length of T7 DNA. Under carefully controlled conditions, 40–85% of the DNA molecules specifically bound at least one enzyme molecule.

L5‐67 and LUQ‐1 peptide precursors of Aplysia californica: Distribution and localization of immunoreactivity in the central nervous system and in peripheral tissues

Two genes (L5‐67 and LUQ‐1) that encode neuropeptide precursors have recently been shown to be expressed in a distinct and non‐overlapping manner in the five left upper quadrant (LUQ) cells of the abdominal ganglion of Aplysia (Landry et al. [1992], J. Neurobiol. 23:89–101). By using wholemount immunohistochemistry and radioimmunoassay (RIA), the pattern of expression of these two genes was assessed at the protein level throughout the central nervous system (CNS) and in peripheral tissues of Aplysia californica. The distribution of LUQ‐1 precursor‐like immunoreactivity was fairly limited, occurring in the ventral LUQ cell (L5) and in a total of approximately 20 additional neurons in the abdominal and cerebral ganglia. L5‐67 precursor‐like immunoreactive material was more prevalent, appearing in a total of approximately 100 neurons distributed among each of the central ganglia. Identified L5‐67‐immunoreactive neurons included the four dorsal LUQ cells (L2‐4 and L6) and two giant neurons (R2 and LPI1). In one group of cells, the H cluster of the cerebral ganglion, L5‐67 immunofluorescence was substantially more intense in larger versus smaller animals, suggesting that this peptide precursor is subject to developmental regulation in certain neurons. Immunoelectron microscopic examination of the subcellular localization of L5‐67 immunoreactivity in LUQ cell somata and axons revealed its association with dense‐core vesicles (approximately 114 nm in diameter). In the periphery, L5‐67‐immunoreactive fibers were detected in specific regions of the circulatory system (auricle, ventricle, cristae aorta, anterior aorta) and the reproductive system (genital ganglion, large hermaphroditic duct, small hermaphroditic duct, ovotestis. The kidney and the intestine, two tissues in which considerable secretion and absorption occur, contained material immunoreactive to both L5–67 and LUQ‐1 antisera. The localization of the two peptide precursors in these tissues differed substantially, with L5‐67 occurring in widely ramifying varicose fibers, whereas LUQ‐1 was found in restricted foci of fibers and in small spherical cells that appeared to lack processes. These results support previous findings concerning the heterogeneity of neurotransmitter phenotypes in the LUQ cells. Furthermore, they are indicative of a fairly broad role for the L5‐67‐derived neuropeptides, and a more limited role for the LUQ‐1‐derived neuropeptides, in the regulation of the visceral organ systems of Aplysia.

Gene products from LUQ neurons in the abdominal ganglion are present at the renal pore of Aplysia californica

The L2-4,6 and L5 cells located in the left upper quadrant of the abdominal ganglion of Aplysia californica express the L5-67 and LUQ-1 genes, respectively, in a nonoverlapping manner. These cells send major neurites to the kidney and at least some of them were shown to innervate the renal pore closer muscle, and thereby control its function. By using in-situ hybridization and immunofluorescence, the presence of L5-67 and LUQ-1 mRNAs and peptides was studied in the kidney, with emphasis on the region of the renal pore. We detected immunoreactive materials in many small varicose nerve fibers running along the central epithelium in the inner parts of the kidney, and in neurites located within a large nerve associated with muscles inside the renal pore. Our observations represent the first direct evidence of the presence of gene products from LUQ cells at the renal pore, suggesting that they may be responsible for mediating LUQ cell signals. Furthermore, mRNAs coding for the L5-67 and LUQ-1 peptides were also found in the nerve structure inside the renal pore. Our report documents a striking example of neuropeptide mRNA targeting nerve terminals that are very distant from their cell bodies.

Characterization of morphogenetic intermediates and progeny of normal and alkylated bacteriophage T7

Analysis of thin sections of Escherichia coli B cells infected by normal (nonalkylated) or alkylated bacteriophage T7 showed that alkylation altered phage morphogenesis. To understand these morphogenetic alterations, we have isolated phage-related particles from infected-cell lysates by differential and sucrose gradient centrifugation. Cells infected by normal and by alkylated phage produced mature phage particles, empty heads, and proheads; however, production of proheads and mature phage particles was less in the case of alkylated phage. These lysates also contained sedimentable material which migrated more slowly than empty heads on sucrose gradients. In the case of alkylated phage, this peak contained radioactive material in amounts nearly equal to that in either proheads or empty heads; for normal phage, this peak represented a smaller fraction of the total radioactivity. Examination of the gradient fractions by electron microscopy revealed appreciable quantities of phage tails and tail-related particles. The same gradient fractions contained phage tail proteins: gene products (gps) 11, 12, and 17 as well as smaller amounts of gp 8, the head-tail connector. In addition, these fractions contained two other proteins which we believe to be of bacterial origin. These proteins may be related to tail formation or function as part of the phage receptor. On the basis of our data, we propose an alternative morphogenetic pathway for T7 tail formation, a pathway which would involve formation of a complex of tail proteins prior to association with the phage head.

Analysis of membrane fractions from Mycoplasma gallisepticum

Membrane fractions have been isolated from Mycoplasma gallisepticum following a procedure derived from that described by Maniloff, J. and Quinlan, D.C. (J. Bacteriol. (1974) 120, 495-501). A light fraction F1 was obtained which contained structures resembling the bleb-infrableb apparatus characteristic of M. gallisepticum. It was enriched in DNA and had an electrophoretic profile different from that of unfractionated membranes. Cholesterol-to-phospholipid ratios higher than two and elevated values of the ratio of saturated to unsaturated fatty acids were other characteristics of this fraction. The two other fractions isolated (FII and FIV) also differed from intact membranes by their cholesterol and phospholipid content as well as by their saturation ratios. The membrane fluidity of FII and FIV, estimated by fluorescence polarization, was similar to that of unfractionated membranes while a slight but significant difference was recorded for the light fraction. Possible relationships between the lateral heterogeneity of the M. gallisepticum membrane and the obtainment of fractions are discussed.

Treatment of ultracentrifuge tubes in a glow discharge makes their surfaces wettable

After exposure to a glow discharge, Ultra-Clear ultracentrifuge tubes become wettable and hence suitable for use with conventional gradient formers. Tubes treated by this method can thus replace the formerly widely used cellulose nitrate tubes which are no longer available.

Molecular Structure of Neutral Endopeptidase 24.11 (Enkephalinase)

Whereas termination of the action of classical neurotransmitters is known to occur either by reuptake or degradation, inactivation has been proposed to be a major mechanism for terminating peptidergic signals. When incubated in vitro with crude brain extracts, the neuropeptides Leu- and Met-enkephalins are rapidly hydrolysed to inactive metabolites2,3. In the perfused brain in vivo, the tripeptide Tyr-Gly-Gly has been found to be a major metabolite of the enkephalins arising by hydrolysis of the Gly3-Phe4 bond. Preparations from the striatum, a brain region rich in peptidergic nerve terminals, contain a membrane-bound enzyme that is able to hydrolyse the enkephalins at the same position4. This enzyme termed “enkephalinase” was detected in other tissues and was shown to be particularly enriched in the kidney cortex. Its identity with Neutral Endopeptidase (NEP: E.C.3.4.24.11), has been established using specificity, sensitivity to inhibitors and immunological criteria5. Both renal and brain enzymes hydrolyse peptides on the amino-terminal side of a hydrophobic residue. The enzyme has therefore the potential to hydrolyse a wide range of regulatory peptides6. Although the enkephalins may be important physiological substrates for the enzyme, evidence is accumulating that the tachykinin peptides such as substance P may also be inactivated by this enzyme in the brain7,8. There is little to support the concept of specific peptide hydrolases tailored for individual neuropeptides9.