Dysfunctional C1-inhibitor(At), isolated from a type II hereditary-angio-oedema plasma, contains a P1 ''reactive centre'' (Arg444----His) mutation.

Simple rapid procedures for identification and analysis of dysfunctional C1-inhibitor proteins mutated at the reactive-centre P1 residue have been developed and used to define structurally a C1-inhibitor protein, C1-inhibitor(At), isolated from an individual with hereditary angio-oedema. The observed mutation, Arg444----His, is compatible with a single base change in the codon used for Arg444 in the native protein.     

Preliminary crystal structure of Acinetobacter glutaminasificans glutaminase-asparaginase

The preliminary structure of a glutaminase-asparaginase from Acinetobacter glutaminasificans is reported. The structure was determined at 3.0-A resolution with a combination of phase information from multiple isomorphous replacement at 4-5-A resolution and phase improvement and extension by two density modification techniques. The electron density map was fitted by a polypeptide chain that was initially polyalanine. This was subsequently replaced by a polypeptide with an amino acid sequence in agreement with the sizes and shapes of the side chain electron densities. The crystallographic R factor is 0.300 following restrained least squares refinement with data to 2.9-A resolution. The A. glutaminasificans glutaminase-asparaginase subunit folds into two domains: the aminoterminal domain contains a five-stranded beta sheet surrounded by five alpha helices, while the carboxyl-terminal domain contains three alpha helices and less regular structure. The connectivity is not fully determined at present, due in part to the lack of a complete amino acid sequence. The A. glutaminasificans glutaminase-asparaginase structure has been used successfully to determine the relative orientations of the molecules in crystals of Pseudomonas 7A glutaminase-asparaginase, in crystals of Vibrio succinogenes asparaginase, and in a new crystal form of Escherichia coli asparaginase (space group 1222, one subunit per asymmetric unit).     

On the active site of liver acetyl-CoA. Arylamine N-acetyltransferase from rapid acetylator rabbits (III/J)

A covalent, catalytic intermediate of cytosolic liver acetyl coenzyme A: arylamine N-acetyltransferase (EC 2.3.1.5) from rapid acetylator rabbits (III/J) was isolated and chemically characterized. The active site was further studied using two covalent inhibitors, [2-3H]iodoacetic acid and bromoacetanilide. Inhibition experiments with [2-3H]iodoacetic acid at pH 6.9 showed that the incorporation of 0.7 mol of [2-3H]iodoacetic acid/mol of N-acetyltransferase led to rapid, irreversible loss of enzyme activity. Preincubation of the enzyme with acetyl coenzyme A (acetyl-CoA) completely protected against inactivation by [2-3H]iodoacetic acid. After incubating the N-acetyltransferase with [2-3H]acetyl-CoA in the absence of an acceptor amine, an acetyl-cysteinyl-enzyme intermediate was isolated and characterized. Preincubation of N-acetyltransferase with iodoacetic acid prevented the incorporation of the [2-3H]acetyl group into the enzyme. The product analog, bromoacetanilide, caused a rapid irreversible loss of N-acetyltransferase activity. The reaction was pseudo first-order and saturated at high bromoacetanilide concentrations (KI = 0.67 mM; k3 = 1 min-1). Preincubation of the enzyme with acetyl-CoA prevented inactivation by the inhibitor. The acceptor amine 4-ethylaniline did not prevent inhibition. Incorporation of the inhibitor was directly proportional to the loss of activity showing a 1:1 stoichiometry of enzyme to inhibitor. The target amino acid was identified as cysteine by amino acid analysis of inhibitor-treated enzyme.     

Self-assembly of apoferritin from horse spleen after reversible chemical modification with 2,3-dimethylmaleic anhydride

Apoferritin from horse spleen is composed of 24 subunits that undergo partial dissociation after chemical modification with 2,3-dimethylmaleic anhydride (DMMA), yielding dimeric, trimeric, and tetrameric intermediates, stable at pH 8.5 and 0 degrees C. Deacylation at neutral pH and elevated temperature provides a means to initiate reassembly by appropriate shifts of the solvent conditions. In order to monitor the pathway of self-assembly, starting from different intermediates of dissociation, dimers, trimers, and tetramers were isolated and investigated with respect to their capacity to accomplish reassociation. Intrinsic protein fluorescence, gel permeation chromatography, and analytical ultracentrifugation were applied to characterize the intermediate and final stages of association. The assembly of both the dimer and trimer yields greater than 85% of the native tetracosamer; the overall rate, starting from the dimer, exceeds the one starting from the trimer. Under comparable conditions, the tetramer exhibits only partial reassociation via the dimer and monomer; the corresponding dissociation reaction determines the observed slower rate. Significant assembly intermediates are "structured monomers", dimers, trimers, and dodecamers. Polymerization of the dimer via the tetramer, octamer, etc., does not occur on the pathway of assembly. The results confirm the assembly scheme proposed previously on the basis of cross-linking and spectroscopic experiments [Gerl, M., & Jaenicke, R. (1987) Eur. Biophys. J. 15, 103-109]. Comparison of structural models involving the different subunit interactions responsible for the sequential association supports the monomer----dimer----trimer----hexamer----dodecamer----tetracosamer mechanism of apoferritin self-assembly.     

Induction of a substrate for casein kinase II during lymphocyte mitogenesis

Particulate fractions prepared from concanavalin A-activated murine T lymphocytes contain an endogenous protein kinase that phosphorylates an endogenous protein substrate of Mr 112 000. The phosphorylation of 112 kDa protein is greatly reduced or absent in unstimulated T cells. Phosphoamino acid analysis indicates that 112 kDa protein is labeled on a serine. Add-back experiments using purified protein kinases indicate that 112 kDa protein serves as a substrate for casein kinase II. Phosphorylation of 112 kDa protein by the endogenous kinase is inhibited by heparin, a known casein kinase II inhibitor. The site or sites modified by the endogenous kinase and exogenous casein kinase II appear identical by peptide-mapping experiments. A time-course of the appearance of phosphorylated 112 kDa protein following stimulation with concanavalin A, measured in the presence or absence of added casein kinase II, suggests that 112 kDa protein is induced in activated T cells. Subcellular localization studies suggest that 112 kDa protein is a nuclear protein. Silver-binding and purification studies suggest that 112 kDa protein is of the nucleolar organizing region.     

Ontogeny of the smooth muscle response to histamine in rabbit and human small bowel

Histamine is known to stimulate small bowel smooth muscle contraction in adults. We studied the response to histamine of small bowel from 27 day gestation fetal (term = 31 days), 4 days-old neonatal, and weanling rabbits using an isolated muscle strip technique in vitro. Sensitivity to histamine stimulation decreased with increasing age, with a six-fold difference in mean D50 between fetal and weanling bowel. A strong contractile response was also obtained in human fetal bowel between 16 and 24 weeks gestation. The response to histamine was inhibited at all ages in both rabbit and human bowel by specific histamine H1 receptor blockade with diphenhydramine, and not by H2 or cholinergic receptor blockers. We conclude that histamine stimulates rabbit and human fetal small bowel contraction; stimulation specifically occurs via the histamine H1 receptor, and is unrelated to release of acetylcholine; sensitivity to histamine decreases significantly with maturity in the rabbit.     

Amino acid sequence of the bacterioferritin (cytochrome b1) of Escherichia coli-K12

The complete amino acid sequence of bacterioferritin (cytochrome b1) from Escherichia coli-K12 has been derived from the nucleotide sequence of the cloned gene. It comprises 158 amino acid residues giving an Mr of 18,495. The identity of the gene product was confirmed by an 87 residue N-terminal sequence obtained from the purified protein, but it differs significantly from much of the previously published partial amino acid sequence (1). Secondary structure prediction indicates a high alpha-helical content consistent with a 4-helix-bundle conformation. The fully assembled bacterioferritin molecule comprising 24 identical subunits and 12 haem moieties is a tetracosamer with an Mr of approximately 452,000.     

Development of a novel photoreactive calmodulin derivative: cross-linking of purified adenylate cyclase from bovine brain

A novel photoreactive calmodulin (CaM) derivative was developed and used to label the purified CaM-sensitive adenylate cyclase from bovine cortex. 125I-CaM was conjugated with the heterobifunctional cross-linking agent p-nitrophenyl 3-diazopyruvate (DAPpNP). Spectral data indicated that diazopyruvoyl (DAP) groups were incorporated into the CaM molecule. Iodo-CaM-DAPs behaved like native CaM with respect to (1) Ca2+-dependent enhanced mobility on sodium dodecyl sulfate-polyacrylamide gels and (2) Ca2+-dependent stimulation of adenylate cyclase activity. 125I-CaM-DAP photochemically cross-linked to CaM-binding proteins in a manner that was both Ca2+ dependent and CaM specific. Photolysis of forskolin-agarose-purified adenylate cyclase from bovine cortex with 125I-CaM-DAP produced a single cross-linked product which migrates on sodium dodecyl sulfate-polyacrylamide gels with an apparent molecular weight of approximately 140,000.     

Effects of nutrient and light limitation on the biochemical composition of phytoplankton

Three marine phytoplankters (Isochrysis galbana, Chaetoceros calcitrans andThalassiosira pseudonana), commonly used in the culture of bivalve larvae, were grown in batch or semi-continuous cultures. Changes in protein, carbohydrate, lipid and some fatty acids were measured as growth became limited by nitrogen, silicon, phosphorus or light. Under N starvation (2 d) the % lipid remained relatively constant, while% carbohydrate increased and% protein decreased in all 3 species compared to cells growing under no nutrient limitation. Under Si starvation (6 h) there was no change in lipid, protein or carbohydrates. The amount of two fatty acids, 20 : 53 and 22 : 63 remained relatively constant under N, P and Si starvation, exept for a sharp drop in the cells of P-starvedT. pseudonana. However, there were pronounced species differences withI. galbana containing significantly less 20 : 5 3 thanC. calcitrans orT. pseudonana. Under light limitation the amount of lipid per cell showed no consistent trend over a range of irradiances for all 3 species. The amount of N per cell (an index of protein content) as a function of irradiance, was relatively constant forI. galbana andT. pseudonana, while the amount of N per cell was lower under low irradiances forC. calcitrans. These examples of changes in protein, carbohydrate, lipid and certain fatty acids under nutrient (N, Si or P) or light limitation, emphasize the importance of knowing the phase (e.g. logarithmic vs stationary) of the growth curve in batch cultures, since the nutritional value of the phytoplankters could change as cultures become dense and growth is terminated due to nutrient or light limitation.Presented at the XIIIth International Seaweed Symposium, University of British Columbia, Vancouver, Canada, August 1989.     

Gonadotropes in a novel rat pituitary tumor cell line,RC-4B/C. Establishment and partial characterization of the cell line

Summary An epithelial cell line (RC-4B/C) was established from a pituitary adenoma obtained from a 3-yr-old (ACI/fMai × F344/fMai)F1 male rat. Before Year 5 in vitro, RC-4B/C cells could not be viably recovered from cryogenic storage. Recovery of viable cells from cryogenic storage in Year 5 was associated with a more transformed phenotype, including the appearance of endogenous C-type rat retroviral particles. The ultrastructural appearance of the cells was similar to that of differentiated anterior pituitary cell; the cultured cells contained numerous, electron dense, secretory granules, Golgi complexes, and extended arrays of rough endoplasmic reticulum. Immunocytochemical study showed that all cell types present in the rat anterior pituitary gland were present in the cell line. The percentage of luteinizing hormone beta (LHβ) cells in the cell line was higher (19.9%) and that of growth hormone cells was lower (12.2%) than in normal male rat pituitary, whereas the cell line contained a comparable percentage of follicle stimulating hormone beta (FSHβ), prolactin (PRL), ACTH, and thyrotropin beta cells. Radioimmunoassay data demonstrated the PRL content of the cells was comparable to that of normal male rat pituitary gland, whereas the content of LH and FSH was 70- and 800-fold lower, respectively. Assay of specific receptor sites for gonadotropin releasing hormone (GnRH) using Scatchard plots of the data established the RC-4B/C cells contained GnRH receptor sites of the same affinity as in the pituitary gland, but of twofold lower capacity. These data suggest the RC-4B/C cell line warrants further study as a model for the induction and maintenance of the gonadotropic function of the pituitary gland. An abstract of portions of these results was presented at the 8th International Congress of Endocrinology, Kyoto, Japan, 1988. This work was supported in part by grants DK-17631 (E.H.L.), CA-24145 (W.G.B.), CA-31102 (H.G.B.), AG-01753 (D.E.H.) and HD-1778 (M.T.D.) from the National Institutes of Health, Bethesda, MD, and by a grant from the Association pour la Recherche sur le Cancer, France (M.J.). The NIH is not responsible for the contents of this publication nor do the contents necessarily represent the official views of that agency. Jolanta Polkowska was a recipient of a Foundation Simone et Cino del Duca grant.