Characterization of a Nerve Growth Factor-Stimulated Protein Kinase in PC12 Cells Which Phosphorylates Microtubule-Associated Protein 2 and pp250

Treatment of PC12 cells with nerve growth factor (NGF) resulted in the rapid, but transient, activation of a protein kinase which specifically phosphorylated an endogenous 250-kDa cytoskeletal protein (pp250). We report that the microtubule-associated protein, MAP2, is an alternative substrate for the NGF-activated kinase. NGF treatment maximally activated the kinase within 5 min; however, the activity declined with longer exposure to NGF. The enzyme was localized predominantly in microsomal and soluble fractions and phosphorylated MAP2 on serine and threonine residues. The soluble enzyme was fractionated by DEAE chromatography and gel filtration and had an apparent Mr of 45,000. The enzyme was purified to near homogeneity by chromatofocussing and had a pI of 4.9. Kinetic analysis revealed that NGF treatment caused a sevenfold increase in Vmax for MAP2. The Km with respect to the MAP2 substrate was approximately 50 nM and was not altered by NGF treatment. A novel feature of the NGF-stimulated enzyme was its sharp dependence on Mn2+ concentration. The active enzyme is likely to be phosphorylated, because inclusion of phosphatase inhibitors was required for recovery of optimal activity and the activity was lost on treatment of the enzyme with alkaline phosphatase. Histones, tubulin, casein, bovine serum albumin, and the ribosomal subunit protein S-6 were not phosphorylated by this enzyme. The NGF-stimulated kinase was distinct from A kinase, C kinase, or other NGF-stimulated kinases. The rapid and transient activation of the protein kinase upon NGF treatment suggests that the enzyme may play a role in signal transduction in PC12 cells.     

Location in Muscarinic Acetylcholine Receptors of Sites for [3H]Propylbenzilcholine Mustard Binding and for Phosphorylation with Protein Kinase C

Muscarinic acetylcholine receptors purified from porcine cerebra or atria were covalently labeled with [3H]propylbenzilylcholine mustard ([3H]PrBCM), and then the labeled receptors were subjected to limited hydrolysis with trypsin, V8 protease, and lysyl endopeptidase, followed by analysis involving sodium dodecyl sulfate-polyacrylamide gel electrophoresis, fluorography, autoradiography, or immunostaining. The labeled peptides were located on the basis of their reactivity with antibodies raised against three synthetic peptides with partial sequences of the m1 or m2 receptor, and of their sensitivity to endoglycosidase F, which was taken as evidence that they contain glycosylation sites near the N terminus. The [3H]PrBCM-binding site in both cerebral and atrial receptors was found to be located between the N terminus and the second intracellular loop, because the size of the smallest deglycosylated peptide that contained both the [3H]PrBCM-binding and glycosylation sites was approximately 16 kDa. Cerebral receptors were 32P-phosphorylated with protein kinase C, and the major phosphorylation sites in cerebral muscarinic receptors were found to be located in a C-terminal segment including a part of the third intracellular loop, because a 32P-labeled peptide of 12-14 kDa reacted with anti-(m1 C-terminal peptide) antiserum. The presence of an intramolecular disulfide bond, probably between Cys 98 and Cys 178 in the first and second extracellular loops, respectively, was suggested by the finding that a peptide of approximately 17 kDa containing the [3H]PrBCM-binding site, but not the glycosylation sites, was partly converted to a peptide of approximately 12 kDa on treatment with beta-mercaptoethanol.     

Stimulation of Muscarinic Acetylcholine Receptors Increases Synaptosomal Free Calcium Concentration by Protein Kinase-Dependent Opening of L-Type Calcium Channels

In synaptosomes prepared from rat cerebral cortex, free cytosolic calcium concentration ([Ca2+]i) was measured using the fluorescent dye fura-2. Incubation of fura-2-loaded synaptosomes with carbachol increased [Ca2+]i in a dose-dependent manner (1-1,000 microM), with a maximum response of 22 +/- 2% at approximately 100 microM and an EC50 (calculated concentration producing 50% of the maximum response) of 30 microM. The effect of carbachol (100 microM) on [Ca2+]i was antagonised by atropine, but not by hexamethonium (10 microM). The calculated concentration of atropine needed for 50% inhibition (IC50) was 260 nM. The rise in [Ca2+]i produced by carbachol was reduced in the absence of extrasynaptosomal Ca2+ and effectively blocked by the L-type calcium channel blocker nifedipine (with an IC50 of 29 nM). The response to carbachol was reduced if the synaptosomes were preincubated with the protein kinase inhibitors H7 [1-(5-isoquinolinylsulfonyl)-2- methylpiperazine] (from 17% in the solvent control to 4%) and staurosporine (from 20% in the solvent control to 3%). These results show that stimulation of muscarinic acetylcholine receptors in synaptosomes increases [Ca2+]i by protein kinase-dependent activation of 1,4-dihydropyridine-sensitive calcium channels.     

Molecular evolution of enzyme structure: Construction of a hybrid hamster/Escherichia coli aspartate transcarbamoylase

Summary Aspartate transcarbamoylase (ATCase, EC 2.1.3.2) is the first unique enzyme common to de novo pyrimidine biosynthesis and is involved in a variety of structural patterns in different organisms. InEscherichia coli, ATCase is a functionally independent, oligomeric enzyme; in hamster, it is part of a trifunctional protein complex, designated CAD, that includes the preceding and subsequent enzymes of the biosynthetic pathway (carbamoyl phosphate synthetase and dihydroorotase). The complete complementary DNA (cDNA) nucleotide sequence of the ATCase-encoding portion of the hamster CAD gene is reported here. A comparison of the deduced amino acid sequences of the hamster andE. coli catalytic peptides revealed an overall 44% amino acid similarity, substantial conservation of predicted secondary structure, and complete conservation of all the amino acids implicated in the active site of theE. coli enzyme. These observations led to the construction of a functional hybrid ATCase formed by intragenic fusion based on the known tertiary structure of the bacterial enzyme. In this fusion, the amino terminal half (the “polar domain”) of the fusion protein was provided by a hamster ATCase cDNA subclone, and the carboxyl terminal portion (the “equatorial domain”) was derived from a clonedpyrBI operon ofE. coli K-12. The recombinant plasmid bearing the hybrid ATCase was shown to satisfy growth requirements of transformedE. coli pyrB ⁻ cells. The functionality of thisE. coli-hamster hybrid enzyme confirms conservation of essential structure-function relationships between evolutionarily distant and structurally divergent ATCases.     

Uptake of Exogenous PhosphatidyJserine by Human Neuroblastoma Cells Stimulates the Incorporation of [methyl-l4C]Cholne into Phosphatidylcholine

The phosphatidylserine (PtdSer) content of human cholinergic neuroblastoma (LA-N-2) cells was manipulated by exposing the cells to exogenous PtdSer, and the effects on phospholipid content, membrane composition, and incorporation of choline into phosphatidylcholine (PtdCho) were investigated. The presence of liposomes containing PtdSer (10-130 microM) in the medium caused time- and concentration-dependent increases in the PtdSer content of the cells, and smaller and slower increases in the contents of other membrane phospholipids. The PtdSer levels in plasma membrane and mitochondrial fractions prepared by discontinuous sucrose density gradient centrifugation increased by 50 and 100%, respectively, above those in control cells after 24 h of exposure to PtdSer (130 microM). PtdSer caused a concomitant, concentration-dependent increase of up to twofold in the incorporation of [methyl-14C]choline chloride into PtdCho at a choline concentration (8.5 microM) compatible with activation of the CDP-choline pathway, suggesting that the levels of PtdSer in membranes may serve as a stimulus to regulate overall membrane composition. PtdSer caused a mean increase of 41% in PtdCho labeling, but the phorbol ester, phorbol 12-myristate 13-acetate (PMA), which stimulates PtdCho synthesis in a number of cell lines, increased [14C]PtdCho levels by only 14% in LA-N-2 cells, at a concentration (100 nM) which caused complete translocation of the calcium- and phospholipid-dependent enzyme protein kinase C to the membrane. The translocation was inhibited by prior exposure of the cells to PtdSer. Treatment with PMA for 24 h diminished protein kinase C activity by 80%, but increased the labeling of PtdCho in both untreated and PtdSer-treated cells. These data suggest that uptake of PtdSer by LA-N-2 cells alters both the phospholipid composition of the membrane and synthesis of the major membrane phospholipid PtdCho; the latter effect does not involve activation of protein kinase C.     

Effects of a New Calcium Channel Blocker, KB-2796, on Protein Synthesis of the CA1 Pyramidal Cell and Delayed Neuronal Death Following Transient Forebrain Ischemia

The effects of a new calcium channel blocker, 1-[bis(4-fluorophenyl)methyl]-4-(2,3,4-trimethoxybenzyl)-piperazine dihydrochloride (KB-2796), on delayed neuronal death (DND) in the hippocampus were examined in gerbils in comparison with those of pentobarbital and flunarizine. The neuronal density in the hippocampal CA1 subfield was counted on the seventh day of recirculation following 5 min of bilateral carotid occlusion, and protein biosynthesis in the brain was also determined at 1, 2, 4, 24, and 72 h following occlusion. The drugs were intraperitoneally administered after recirculation. KB-2796 (10 mg/kg) significantly prevented DND in the CA1 subfield. Pentobarbital (40 mg/kg), but not flunarizine (3 and 10 mg/kg), inhibited DND. Protein synthetic activity in the CA1 subfield was reduced by ischemia and the reduction was not restored even at 72 h after recirculation. KB-2796 did not ameliorate the reduction of protein synthesis in the CA1 subfield by 24 h after recirculation, but in one of three animals restoration of protein synthesis was observed at 72 h of recirculation. Pentobarbital also restored the reduced protein synthesis in two of three animals at 72 h. These results suggest that calcium influx into neurons participates in the pathogenesis of DND, and also that KB-2796 might prevent both morphological and functional cell damage in CA1 neurons induced by transient ischemia.     

Immunogold localization of chymotrypsin inhibitor-2, a lysine-rich protein,in developing barley endosperm

Chymotrypsin inhibitor-2, a lysine-rich protein in the barley endosperm, has been localized at the ultrastructural level by immunocytochemistry in developing barley endosperm cells 14 days post anthesis. The protein is deposited in the protein bodies. Two morphologically distinct types of protein bodies, small spherical and large irregularly shaped, are present. Golgi-apparatus-derived vesicles whose content is labelled by chymotrypsin inhibitor-2 antibody-gold particles are observed at the Golgi complex and around the vacuoles. These observations indicate that the transport of the protein to the site of deposition is mediated by the Golgi apparatus.Abbreviations CI chymotrypsin inhibitor - DPA days post anthesis - ER endoplasmic reticulum The authors wish to thank Dr. V.R. Franceschi (Department of Botany, Washington State University, Pullman, USA) for many helpful discussions and advice during the work, and the staff at the Electron Microscope Center at Washington State University for technical assistance.     

Effect of NaCl and mannitol on plasma membrane proteins in corn roots

Summary The short-term effects of NaCl and mannitol stress on plasma membrane (PM) polypeptides from corn roots (Zea mays L.) were determined using two-dimensional gel electrophoresis following radiolabeled amino acid incorporation. After 2.5 hours, both stress treatments altered synthesis of several polypeptides. Changes included up-regulation of some polypeptides with concomitant down-regulation of others. Some changes were unique to the stress treatment while others were common to both NaCl and mannitol. No new polypeptides appeared in either case. Pulse-chase experiments following 0.5-hours and 2.5-hours incubation periods with radiolabeled amino acids did not reveal differences in turnover of PM polypeptides. The results support the contention that altered synthesis of PM proteins under stress may contribute to the alteration of membrane function.Abbreviations ER endoplasmic reticulum: GA Golgi - PM plasma membrane - PVPP polyvinylpolypyrrolidone     

Relationship between gene expression and hybrid vigor in primary root tips of young maize (Zea mays L.) plantlets

Summary To provide an insight into the molecular basis of heterosis, we investigated gene expression in primary root tips of a heterotic maize hybrid (B73 × Mo17) and its parental lines (B73 and Mo17). This analysis was carried out (i) by differential plaque hybridization of a recombinant cDNA library made to poly(A) RNA isolated from B73 × Mo17 primary root tips, and (ii) by comparing with two-dimensional gel electrophoresis proteins synthesized in vitro in the rabbit reticulocyte system by poly(A) RNA isolated, at different stages of development, from the three genotypes. The results showed that there are sets of proteins and mRNAs that are differentially synthesized and expressed in the F₁ primary root tips in comparison to the parental lines. Moreover, results from the survey of 21 major in-vitrosynthesized polypeptide variants, from mRNAs of primary root tips of the parental lines and their F₁ hybrid, indicated that in seven instances hybrid proteins translated in vitro were more abundant or possibly new. In most of the remaining cases, hybrid spots were similar in intensity to the same protein produced by one of the two parental lines.