Cross-Reaction of Anti-Rat B-50: Characterization and Isolation of a "B-50 Phosphoprotein" from Bovine Brain

Abstract: Antibodies to the phosphoprotein B-50 of rat brain were used to trace cross-reacting brain proteins of vertebrates. With the SDS-gel-immunoperoxidase method, a cross-reacting protein (CP) of apparent M_r 53,000 was demonstrated in the homogenate and the synaptic plasma membrane fraction of bovine brain. Sequence 1–24 of adrenocorticotropin (ACTH_1-24) (10⁻⁵ M and 10⁻⁴ M ) inhibited endogenous phosphorylation of CP in synaptic plasma membranes. The protein was partially characterized and purified to homogeneity from bovine brain by procedures previously described for rat B-50. CP was enriched in ammonium sulfate precipitated protein (ASP) fractions and phosphorylated by an endogenous protein kinase. Two-dimensional gel analysis of bovine and rat ASP showed that the cross-reacting protein had an isoelectric point less acidic than B-50. Limited proteolysis by Staphylococcus aureus protease yielded a "peptide map" analogous to B-50. Two major fragments of M_r 30,000 and 17,000 were produced. In addition, CP exhibited other similarities to rat B-50: phosphorylation by rat brain protein kinase C, microheterogeneity observed after isoelectric focusing, and possibly degradation by endogenous proteolysis. Cross-reaction of proteins in brain homogenates of other mammalian species and of chicken was demonstrated: the M_r of the proteins ranged from 47,000 to 53,000. We conclude that (1) the cross-reacting bovine protein is a "B-50 protein," and (2) the M _r of the "B-50 protein" varies from species to species.     

Phosphorylation and nucleotidylation of proteins in Rhodocyclus gelatinosus

Abstract Cells of Rhodocyclus gelatinosus were radioactively labeled by addition of [³²P]orthophosphate, [¹⁴C]inosine or [¹⁴C]orotic acid during anaerobic growth on citrate in the light. Protein analysis by two-dimensional gel electrophoresis and autoradiography of the gels revealed the presence of several radioactively labeled protein species in this organism. The molecular mass and the isoelectric point of all these proteins were determined. Treatment of the ³²P-labeled protein fractions with acid and alkaline phosphatase clearly showed that at least 8 protein species were modified by phosphorylation. The experiments conducted with the ¹⁴C-labeled precursors of purines and pyrimidines indicated the presence of 4 protein species which were modified by a compound containing a purine and phosphate, and a single protein simultaneously being labeled with pyrimidine and phosphate.     

A mesoderm-inducing factor from a Xenopus laevis cell line

Summary We have compared the chemical properties and biological activities of the mesoderm-inducing factor that is secreted by the Xenopus XTC cell line with the vegetalizing factor from chicken embryos. The inducing activity of the factors was tested in different concentrations on totipotent ectoderm either by implantation into early gastrulae of Triturm alpestris or by application of solutions to isolated ectoderm of early gastrulae of Xenopus laevis. Both factors have similar properties. They are not irreversibly inactivated after treatment with 6 M urea or with phenol at 60° C. Reduction with thioglycolic acid inactivates the factors completely. The inducing activity of XTC-conditioned medium decreases only slightly after treatment with 50% formic acid. The apparent molecular mass and the isoelectric point of the factors are similar. The XTC factor was partially purified by size-exclusion and reversed-phase high-pressure liquid chromatography and by isoelectric focusing. The possible relationship of these factors to transforming growth factor is discussed.Dedicated to Prof. Dr. Sulo Toivonen on the occasion of his 80th birthday     

Solubility of phenothiazines in red blood cell membranes as evidenced by photoaffinity labeling

Radioactively labeled 7-azido-fluphenazine and 7-azido-triflupromazine methiodide have been synthesized and their binding to membranes of intact red blood cells and to ghosts was compared after irradiation. The results indicated that tertiary phenothiazines react with integral membrane components. We conclude from the results that amphiphilic substances solubilize in biological membranes. This is in contradiction to the proposal that these compounds are excluded from the hydrophobic core of biological membranes (Conrad & Singer (1979) Proc.Natl.Acad.Sci.U.S.A. 76, 5202-5206 and (1981) Biochemistry 20, 808-818).     

A rational approach for the development of reduced-size analogues of neuropeptide Y with high affinity to the Y1 receptor

Four sets of centrally truncated analogues of neuropeptide Y have been synthesized. In each series the N-terminal part was constant, while the C-terminal segment was systematically varied in length. The C- and N-terminal parts were linked by 6-aminohexanoic acid. The affinity to the Y₁ receptor was investigated on human neuroblastoma cells SK-N-MC. Significant differences were found between the series of peptides as well as within each set. Remarkably, the affinity did not solely depend on the length of the segment, and with increasing numbers of residues the IC₅₀ values were not always decreased. With a given N-terminal segment, only one optimal length of the C-terminal segment was found, which suggests that it is not the amino acids themselves but their 3D arrangement and orientation that is important for high receptor affinity.     

N-Terminal-Specific Anti-B-50 (GAP-43) Antibodies Inhibit Ca2+-Induced Noradrenaline Release, B-50 Phosphorylation and Dephosphorylation, and Calmodulin Binding

Abstract: B-50 (GAP-43) is a presynaptic protein kinase C (PKC) substrate implicated in the molecular mechanism of noradrenaline release. To evaluate the importance of the PKC phosphorylation site and calmodulin-binding domain of B-50 in the regulation of neurotransmitter release, we introduced two monoclonal antibodies to B-50 into streptolysin O-permeated synaptosomes isolated from rat cerebral cortex. NM2 antibodies directed to the N-terminal residues 39–43 of rat B-50 dose-dependently inhibited Ca²⁺-induced radiolabeled and endogenous noradrenaline release from permeated synaptosomes. NM6 C-terminal-directed (residues 132–213) anti-B-50 antibodies were without effect in the same dose range. NM2 inhibited PKC-mediated B-50 phosphorylation at Ser⁴¹ in synaptosomal plasma membranes and permeated synaptosomes, inhibited ³²P-B-50 dephosphorylation by endogenous synaptosomal phosphatases, and inhibited the binding of calmodulin to synaptosomal B-50 in the absence of Ca²⁺. Similar concentrations of NM6 did not affect B-50 phosphorylation or dephosphorylation or B-50/calmodulin binding. We conclude that the N-terminal residues 39–43 of the rat B-50 protein play an important role in the process of Ca²⁺-induced noradrenaline release, presumably by serving as a local calmodulin store that is regulated in a Ca²⁺- and phosphorylation-dependent fashion.     

Functional anatomy of the mechanoreceptor cells in tendrils of Bryonia dioica Jacq.

The ultrastructure of epidermal cells of tendrils of Bryonia dioica Jacq. (Cucurbitaceae) was determined using microscopic, histochemical and immunochemical techniques with focus on the tactile blep, the mechano-receptor of these cells. Tactile bleps resemble bordered pits in structure and probably in formation. They contain cytoplasm rich in endoplasmic reticulum, dictyosomes, mitochondria and microbodies. The cytoplasm is highly vesiculated and usually contains lipid-body-like structures. Cytoskeletal elements (microtubules, actin filaments) are uniquely arranged in the tactile blep, and chlorotetracycline-fluorescence analysis shows large amounts of membrane-associated calcium within the tactile blep. We propose a physically interconnected cytoskeleton-membrane device as the immediate force sensor and transducer which creates a primary intracellular signal, for which calcium is a likely candidate.Abbreviations CTC chlorotetracycline - ER endoplasmic reticulum - MT microtubule This work was funded by grants of the Deutsche Forschungsgemeinschaft to E.W.W. and G.W. (SFB 1462) and by the Fonds der Chemischen Industrie (literature provision).     

SV2 and o-rab3 Remain Associated with Recycling Synaptic Vesicles

Abstract: o-rab3 is an electric ray homologue of low molecular weight GTP-binding proteins thought to be involved in targeting of secretory vesicles to sites of exocytosis. The stimulation-dependent association of o-rab3 with synaptic vesicles was compared with that of the membrane-integral synaptic vesicle protein 2 (SV2). On application of immunoelectron microscopy and the colloidal gold technique, antibodies against either protein labeled the synaptic vesicle membrane compartment. Synaptic vesicles recycled under conditions of low frequency stimulation (0.1 Hz) retained their complement of both SV2 and o-rab3. Isolation of synaptic vesicles by density-gradient centrifugation and subsequent column chromatography yielded no indication of a stimulation-dependent release of o-rab3 from synaptic vesicles. In contrast, multivesicular bodies and vacuoles occasionally observed in the nerve terminals contained SV2 but little if any o-rab3. It is concluded that o-rab3 remains associated with the synaptic vesicle membrane compartment during stimulation-induced cycles of repeated exo- and endocytosis. o-rab3 may be lost once the vesicle enters the prelysosomal pathway.     

Cloning,sequencing, and characterization of a gene (narT) encoding a transport protein involved in dissimilatory nitrate reduction inStaphylococcus carnosus

A Tn917 mutant ofStaphylococcus carnosus TM300, nrIII, was isolated and characterized. Mutant nrIII did not take up nitrate or accumulate nitrite when grown in B-medium supplemented with up to 10 mM nitrate under anoxic conditions; however, it displayed wild-type levels of benzyl viologen-linked nitrate reductase activity. Cultivated in B-medium with nitrate under oxic conditions, mutant nrIII accumulated fivefold less nitrite than the wild-type. The mutation inS. carnosus nrIII could be complemented with a 2-kb chromosomalEcoRI-HpaI fragment from the wild-type. The gene affected by transposon insertion in mutant nrIII was cloned and sequenced. Analysis of the deduced amino acid sequence revealed that this gene, designatednarT, encodes a highly hydrophobic 42-kDa transmembrane protein of 388 amino acids and shows similarities to transport proteins that play a role in nitrate import or nitrite export. The inability of nrIII to take up nitrate under anoxic conditions and its ability to take up and accumulate nitrite in the presence of benzyl viologen, a nitrate ionophore, under the same conditions suggest that NarT represents a transport protein required for nitrate uptake under anoxic conditions inS. carnosus.