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The kinetics of interdomain electron transfer (IET) between the flavin mononucleotide (FMN) and heme domains in a rat neuronal nitric oxide synthesis (nNOS) holoenzyme in the presence and absence of added calmodulin using laser flash photolysis of CO dissociation in comparative studies on partially reduced NOS and a single domain NOS oxygenase construct is reported. The results attribute the slower IET between FMN and heme in the holoenzyme to the additional step of dissociation of the FMN domain from the reductase complex before reassociation with the oxygenase domain to form the electron-transfer competent output state complex.

The kinetics of intersubunit intraprotein electronic transfer (IET) between the flavin monoclueotide (FMN) and heme domains in the nNOS (neuronal) oxyFMN (flavin mononucleotide) construct in the presence and absence of added calmodulin (CaM) were determined using laser flash photolysis of CO dissociation in comparative studies. The IET rate constant in the presence of CaM [262 [s.sup.-1]] was increased approximately 10-fold compared to that in the absence of CaM [22 [s.sup.-1]].

A novel truncated two-domain oxyFMN construct murine inducible nitric oxide synthase (iNOS) are designed and expressed. Laser flash photolysis is used to investigate the kinetics of the intramolecular electron transfer (IET) between the FMN and the heme domains in a novel two-domain murine inducible iNOS oxyFMN construct in which only the FMN and oxygenase domains are expressed.

Fourier transform infrared (FTIR) spectroscopic studies of the iron ligand CO stretch mode of inducible nitric oxide synthase (iNOS) oxygenase domain have been undertake to look at effects of arginine and tetrahydrobiopterin. The stretch vibration frequencies of the photodissociate and the iron-bound CO have a negative linear relationship. The photodissociated ligand appears to stay close to the heme.

A study was conducted on CO and NO binding to inducible nitric oxide synthase. The CO binding results indicate that L-Arg and H4B have a dramatic effect on the iNOSox distal heme pocket and that L-Arg and H4B have relatively similar capacities to restrict heme pocket access. The NO binding data suggest that NO complex formation controls the rate of NO synthesis during the steady state. Moreover, the high off-rates for NO complexes of iNOSox may be a key component that drives decomposition of the enzyme-NO complex.

The N-terminal amino acid segment in inducible nitric oxide synthase oxygenase (iNOSox) involved in the subunit dimerization and productive H4biopterin interaction was identified through analysis of expressed H4biopterin-free iNOSox in Escherichia coli. It was found that residues 1-117 were critical for iNOSox dimerization and H4biopterin/L-arginine interaction. Radioligand binding revealed that residue 66-114 played an important role in H4biopterin interaction and dimerization.

Fifteen conserved acidic residues in the dihydrofolate reductase (DHFR) module of inducible nitric-oxide synthase (iNOSox) were individually mutated using alanine screening to determine whether any might be involved in binding the basic guanidino or alpha-carbon amino groups of Arginine. Mutagenesis of conserved acidic residues within iNOSox region was found to lead to exclusive defects in dimerization and Arginine binding.

The isolated oxygenase and reductase domains of the hemeprotein, cytokine-inducible NO synthase (iNOS) were examined to study how they form and stabilize the active dimeric enzyme. The dimeric oxygenase broke up into folded monomers with heme when incubated with 2-5 M urea but the reductase domain unfolded and can no longer catalyze NADPH-dependent cytochrome c reduction under similar conditions. The oxygenase domain was found to have all the determinants required for subunit dimerization.

The reductase and oxidase domains of inducible macrophage NO synthase catalyze only the second step of NO synthesis. The interaction involves the transfer of one electron from the reductase to the oxidase domain. These domains can only catalyze the formation of NO from the intermediate N(super omega)-hydroxy-L-arginine. These domains are most active when the amount of oxygenase present is four times that of reductase. The inability of these domains to catalyze the complete NO synthesis is probably due to incomplete electron transfer between these domains.

Limited proteolysis of the dimeric enzyme macrophage NO synthase enables an identification of the domains that facilitate subunit interaction and prosthetic group binding. A structural model, proposed on the basis of the two domains involved, consists of a head-to-head alignment of the subunits. The reductase domains do not interact and remain as independent extensions, while the oxygenase domains interact to form a dimer.

The notable effects of mutations in the adjacent flavin mononucleotide (FMN) domain on the heme structure in a human inducible nitric oxide synthase (iNOS) bidomain oxygenase/FMN construct are seen by using low-temperature magnetic circular dichroism (MCD) spectroscopy. The comparative MCD study of wild-type and mutant proteins has shown that a properly docked FMN domain has contributed to the L-Arg perturbation of the heme MCD spectrum in the wild-type protein.

The paper focuses on investigation of properties of two most widely used self-set sand binder systems APNB and FNB across the Globe, for making molds and cores in foundries to produce castings of different sizes involving wide range of metals and alloys, ferrous and nonferrous. This includes study of compression strength values of samples made out of molding sand at different binder addition level using new, mechanically reclaimed (MR) and thermally reclaimed (TR) sand. Strength values studied include dry strength (at room temperature) at specified intervals simulating different stages of mold handling, namely stripping and pre heating, followed by degraded strength after application of thinner based zircon wash by brush, subsequent lighting of, then checking strength both in warm (degraded strength) & cold (recovered strength) conditions. Throughout the cycle of mold movement from stripping to knock out, strength requirements can be divided into two broad classifications, one from stripping to closing (dry strength) and another from pouring to knock out (hot & retained strength). Although the process for checking of dry strength are well documented, no method using simple equipments for checking hot & retained strength are documented in literature. Attempts have been made in this paper to use some simple methods to standardize process for checking high strength properties using ordinary laboratory equipments. Temperature of 450°C has been chosen by trial & error method to study high temperature properties to get consistent & amplified values. Volume of gases generated for both binders in laboratory at 850°C have also been measured. Nature of gases including harmful BTEX and PAH generated on pyrolysis of FNB and APNB bonded sands are already documented in a publication [1]. This exercise has once again been repeated in same laboratory, AGH University, Poland with latest binder formulations in use in two foundries in India. [ABSTRACT FROM AUTHOR]