Role of transcription factors in fetal lung development and surfactant protein gene expression.

Branching morphogenesis of the lung and differentiation of specialized cell populations is dependent upon reciprocal interactions between epithelial cells derived from endoderm of embryonic foregut and surrounding mesenchymal cells. These interactions are mediated by elaboration and concerted actions of a variety of growth and differentiation factors binding to specific receptors. Such factors include members of the fibroblast growth factor family, sonic hedgehog, members of the transforming growth factor-beta family, epidermal growth factor, and members of the platelet-derived growth factor family.

Thr268 in substrate binding and catalysis in P450BM-3.

Members of the gene superfamily of proteins called "P450" catalyze monooxygenation reactions that require an input of two electrons and a molecule of oxygen per catalytic cycle. These proteins are widely distributed among living organisms, from bacteria to human. P450BM-3, a soluble protein isolated from Bacillus megaterium, is self-sufficient, containing P450 and reductase domains on the same polypeptide.

The role of lipid signaling in constitutive membrane traffic.

Several lines of evidence indicate that enzymes that modify membrane lipids function in the regulation of constitutive membrane traffic. Recent evidence suggests that specific phosphatidylinositides may regulate the activity of proteins with diverse functions in membrane transport, such as dynamin, the clathrin-associated AP-2 complex, and proteins that stimulate guanine nucleotide exchange on ADP-ribosylation factors (ARFs). ARF proteins activate a phospholipase D that produces phosphatidic acid from phosphatidylcholine, and this may be essential for the formation of certain types of transport vesicles or may be constitutive vesicular transport to signal transduction pathways.

Reconstitution of the fatty acid hydroxylase activity of cytochrome P450BM-3 utilizing its functional domains.

Cytochrome P450BM-3, a catalytically self-sufficient fatty acid monooxygenase from Bacillus megaterium, is a multidomain protein containing heme, FAD, and FMN. Previous attempts to reconstitute the fatty acid monooxygenase activity of intact P450BM-3 utilizing equimolar concentrations of the separate heme (BMP) and reductase (BMR) domains, have been unsuccessful because two-electron reduced FMN, which rapidly accumulates, is incapable of electron transfer to the heme iron. The present study of the reconstitution of the monooxygenase activity of P450BM-3 utilized combinations of the different functional domains of P450BM-3.

A CRE-like element plays an essential role in cAMP regulation of human SP-A2 gene in alveolar type II cells.

The human has two genes encoding surfactant protein-A (SP-A), termed SP-A1 and SP-A2; the SP-A2 gene is more highly regulated by cAMP and during fetal development than is SP-A1. In this study, by use of primary cultures of human type II cells transfected with fusion genes containing various amounts of SP-A2 5'-flanking DNA linked to human growth hormone (hGH) structural gene, as reporter, we found that -296 bp of SP-A2 upstream sequence is sufficient to direct high basal and cAMP-inducible expression in type II cells, but not in other cell types. By use of competitive EMSA, we observed that nuclear proteins isolated from midtrimester human fetal lung tissue bind specifically to a cAMP response element (CRE)-like sequence, TGACCTTA, at -242 bp, which we have termed CRESP-A2.

Endocytosis of chimeric influenza virus hemagglutinin proteins that lack a cytoplasmic recognition feature for coated pits.

The influenza virus A/Japan/305/57 hemagglutinin (HA) can be converted from a protein that is essentially excluded from coated pits into one that is internalized at approximately the rate of uptake of bulk membrane by replacing the HA transmembrane and cytoplasmic sequences with those of either of two other glycoproteins (Roth et al., 1986. J.

Evidence that phospholipase D mediates ADP ribosylation factor-dependent formation of Golgi coated vesicles.

Formation of coatomer-coated vesicles from Golgi-enriched membranes requires the activation of a small GTP-binding protein, ADP ribosylation factor (ARF). ARF is also an efficacious activator of phospholipase D (PLD), an activity that is relatively abundant on Golgi-enriched membranes. It has been proposed that ARF, which is recruited onto membranes from cytosolic pools, acts directly to promote coatomer binding and is in a 3:1 stoichiometry with coatomer on coated vesicles.

The flavoprotein domain of P450BM-3: expression, purification, and properties of the flavin adenine dinucleotide- and flavin mononucleotide-binding subdomains.

P450BM-3 is a self-sufficient fatty acid monooxygenase that can be expressed in Escherichia coli as either the holoenzyme or as the individual hemo- and flavoprotein domains. The flavoprotein domain (BMR) of P450BM-3 is soluble and contains an equimolar ratio of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) and is functionally analogous to microsomal nicotinamide adenine dinucleotide phosphate (NADPH)-P450 reductases. These reductases have been proposed to have evolved through a fusion of genes encoding simple flavin-containing electron-transport proteins [Porter, T.

Equilibrium and transient state spectrophotometric studies of the mechanism of reduction of the flavoprotein domain of P450BM-3.

The flavoprotein domain of P450BM-3 (BMR), which is functionally analogous to eukaryotic NADPH-P450 oxidoreductases, contains both FAD and FMN. When BMR is titrated with NADPH or sodium dithionite under anaerobic conditions, addition of 2 electron equivalents per mole of BMR results in the reduction of the high potential flavin (FMN) without the accumulation of semiquinone intermediates. Additional sodium dithionite first produces some neutral, blue flavin semiquinone radical and, finally, fully reduced FADH2.

Allosteric enzymes as models for chemomechanical energy transducing assemblies.

In chemomechanical energy transducing assemblies such as muscle and ATP synthase, substrates and macromolecules are locked together as partners where energy available from (or required for) a chemical transformation is exchanged with protein conformational changes. Allosteric binding proteins and enzymes are also chemomechanical energy transducers, using binding energy to generate protein conformational changes, and transduce energy in amounts almost as large as those used to drive muscle contraction and the synthesis of ATP. The recently determined structure of the F1-ATPase reveals a direct correspondence between the types of conformational changes in this transducer and simpler allosteric binding proteins and enzymes.

Characterization of the cyclic adenosine 3',5'-monophosphate response element of the rabbit surfactant protein-A gene: evidence for transactivators distinct from CREB/ATF family members.

Surfactant protein-A (SP-A) gene transcription in fetal lung explants is stimulated by factors that increase intracellular cAMP. In transfected type II cells, expression of fusion genes containing 991 bp of 5'-flanking DNA from the rabbit SP-A gene linked to the human GH gene as reporter is stimulated more than 20-fold by cAMP. Mutagenesis of a putative cAMP responsive element (CRE) located -261 bp upstream of the SP-A transcription initiation site to a sequence known not to bind the transcription factor CRE-binding protein (CREB) caused a marked decrease in basal and cAMP-inducible reporter gene expression.

Domain-domain interaction in cytochrome P450BM-3.

The influence of ionic strength on the interactions between individually expressed functional domains of cytochrome P450BM-3 and the domains in the holoenzyme has been analyzed by spectrophotometric and fluorometric techniques. High ionic strength facilitated electron transfer from NADPH to the FMN moiety of the reductase domain (BMR) of P450BM-3 and did not affect the first electron transfer from FMN to the heme in the holoenzyme. The cytochrome c reductase activity of the holoenzyme was higher than that of BMR within the range of ionic strength tested.

In vivo effects of lipopolysaccharide on hepatic free-NAD(P)(+)-linked redox states and cytosolic phosphorylation potential in 48-hour-fasted rats.

This study was performed to determine the magnitude and time of onset of in vivo changes in hepatic bioenergetics in response to a sublethal dose of lipopolysaccharide (LPS), a bacterial endotoxin. Male rats (48-hour-fasted) were administered an intraperitoneal injection of LPS (5 mg/kg body weight) or vehicle alone, and the livers were freeze-clamped 5, 30, or 180 minutes or 24 hours later. Liver tissue was extracted with perchloric acid, and the metabolites necessary to calculate NAD(+)- and NADP(+)-linked redox states and the cytosolic phosphorylation potential were measured.

Apical and basolateral coated pits of MDCK cells differ in their rates of maturation into coated vesicles, but not in the ability to distinguish between mutant hemagglutinin proteins with different internalization signals.

In polarized epithelial MDCK cells, all known endogenous endocytic receptors are found on the basolateral domain. The influenza virus hemagglutinin (HA) which is normally sorted to the apical plasma membrane, can be converted to a basolateral protein by specific mutations in its short cytoplasmic domain that also create internalization signals. For some of these mutations, sorting to the basolateral surface is incomplete, allowing internalization of two proteins that differ by a single amino acid of the internalization signal to be compared at both the apical and basolateral surfaces of MDCK cells.

NADPH-P-450 reductase: structural and functional comparisons of the eukaryotic and prokaryotic isoforms.

The comparison of the properties of microsomal NADPH-P-450 reductase and the flavoprotein domain of P-450BM-3 (BMR) has revealed a significant difference in the mechanism of reduction of the hemoprotein P-450 by these flavoproteins. Microsomal NADPH-P-450 reductase transfers electrons to the hemoprotein by shuttling between hydroquinone and semiquinone forms of the FMN delivering one electron per cycle. Since the microsomal NADPH-P450 reductase has evolved as a component of multi-enzyme system, this type of mechanism may permit regulation of the steps of the P-450 reaction via variation in the affinity of the reductase for different P-450s, interaction with cytochrome b5, etc.

Methionine enkephalin is hydrolyzed by aminopeptidase N on CD4+ and CD8+ spleen T cells.

Exogenous methionine enkephalin incubated with CD4+ or CD8+ T cells purified from murine spleen is metabolized primarily, if not exclusively, by aminopeptidase N (aminopeptidase M, EC 3.4.11.

Serpins: the uncut version.

The structure of active antithrombin, the first active serpin to be solved, sheds new light on the conformational forms of this important class of inhibitor.

Methionine enkephalin metabolism by murine macrophage ectopeptidase(s).

Ectopeptidases which hydrolyze opioid and other neuropeptides have been identified in brain, kidney and intestine. In this study, identification of the enzymes metabolizing the opioid peptide methionine enkephalin (YGGFM) in murine macrophages was undertaken. Incubation of methionine enkephalin with intact murine peritoneal macrophages results in five products identified as Y, F, FM, GFM and GGFM by amino acid analysis and peptide microsequencing after fractionation by HPLC.

Crystallization and preliminary X-ray studies of extracellular signal-regulated kinase-2/MAP kinase with an incorporated His-tag.

The extracellular signal-regulated kinase ERK2, a member of the protein kinase superfamily, phosphorylates a variety of cellular proteins in response to extracellular signals. ERK2 expressed in Escherichia coli as a fusion protein with the sequence Ala-His6 at the N terminus has low basal activity and very low levels of phosphate incorporation, but can be fully activated. The Ala-His6 ERK2 as expressed in the unphosphorylated form has been crystallized in space group P2(1).