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An aminopeptidase with a preference for N-terminal aspartyl and glutamyl residues but distinct from glutamyl aminopeptidase (EC 3.4. 11.7) was purified to near homogeneity from rabbit brain cytosol. Its properties were similar to an enzyme described previously (Kelly, J. A., Neidle, E. L., and Neidle, A. (1983) J. Neurochem. 40, 1727-1734). Aspartyl aminopeptidase had barely detectable activity toward simple aminoacyl-naphthylamide substrates. Its activity was determined with the substrate Asp-Ala-Pro-naphthylamide in the presence of excess dipeptidyl-peptidase IV (EC 3.4.14.5). The native enzyme has a molecular mass of 440 kDa and migrates as a single band of 55 kDa after SDS-polyacrylamide gel electrophoresis. The sequences of three tryptic peptides were used to screen the GenBankTM data base of expressed sequence tags. Human and mouse clones described as "similar to a yeast vacuolar aminopeptidase" and containing full-length cDNAs were identified and sequenced. The human cDNA was expressed in Escherichia coli. The amino acid sequence has significant homology to yeast aminopeptidase I, placing it as the first identified mammalian member of the M18 family of metalloproteinases. Homologous sequences in Caenorhabditis elegans and in prokaryotes revealed three conserved histidines, three conserved glutamates and five conserved aspartates. Aspartyl aminopeptidase is found at relatively high levels in all mammalian tissues examined and is likely to play an important role in intracellular protein and peptide metabolism.

Dynamic theory of volume holography is used to calculate the variations in the thickness direction of the hologram-constituting refractive-index modulation and the externally observable effects of these variations in electrooptic materials. It is shown that thick holographic gratings may exhibit significant amplitude and grating phase variations with thickness including amplitude sign reversal. These nonuniformities strongly affect holographic grating recording and readout characteristics such as maximum possible diffraction efficiency and angular selectivity. Thus a variety of grating applications will be affected by these nonuniformities.

Regulation of basal activities of adenylyl cyclase (AC) 2 and 6, expressed in Sf9 cells by infection with recombinant baculovirus, was studied. An antipeptide antibody that recognizes AC2 and AC6 with equal sensitivity was used to establish that equivalent levels were expressed. Basal activities of AC2 and AC6 were compared at varying concentrations of Mg2+ or Mn2+ ions; AC2 had 15- and 10-fold greater activity than AC6, respectively. At 20 mM Mg2+, the Km values for ATP were 88 and 39 microM for AC2 and AC6, respectively, whereas their Vmax values were 281 and 11 pmol/mg protein.min. With 100 microM forskolin and either Mg2+ or Mn2+, the difference in activities between AC2 and AC6 was reduced to approximately 2-fold. Forskolin stimulated AC6 greater than 40-fold at 0.5-2 mM Mg2+, whereas AC2 was stimulated 4-6-fold. At 20 mM Mg2+, AC2 was stimulated 2-fold by forskolin, whereas AC6 was stimulated 18-fold. With Mg2+ alone, activities of AC2 and AC6 were not saturable up to 20 mM and yielded curvilinear Hofstee transformations. With forskolin, activities of both AC2 and AC6 were saturable by 10 mM Mg2+ and yielded linear Hofstee transformations. These data indicate that there are substantial differences in the basal enzymatic activities of adenylyl cyclase isoforms, due to differential regulation by Mg2+ ions rather than intrinsic catalytic capabilities. Thus the presence and relative abundance of adenylyl cyclase subtypes could greatly affect the resting cellular cAMP levels with consequent effects on important biological functions, such as differentiation and proliferation.

In a previous communication we proposed a reaction scheme to explain our observation that thyroid peroxidase and lactoperoxidase degrade H2O2 catalatically in the presence of low concentrations of iodide. An essential feature of the scheme was the proposal that enzyme-bound hypoiodite, designated [EOI]-, is a common intermediate in various peroxidase-catalyzed reactions involving iodide. In the present investigation, we tested the validity of this scheme by studying the predictions that it makes concerning the formation of OH-, O2, I2, and organically bound iodine. Stoichiometric and kinetic measurements were made to correlate formation of these various products. Three different peroxidase-catalyzed reactions were studied: 1) oxidation of I- to I2; 2) iodide-dependent catalytic degradation of H2O2 to O2; and 3) iodination of tyrosine or thyroglobulin. Reaction 2 was also studied nonenzymatically using I2, for comparison with the enzyme-catalyzed reaction. In all three reactions, both the stoichiometric and kinetic results with thyroid peroxidase agreed closely with the predictions made by the proposed scheme. This was largely the case with lactoperoxidase also. However, in the case of lactoperoxidase-catalyzed iodination of tyrosine or thyroglobulin, we observed a marked discrepancy between initial rates of OH- release and iodination, inconsistent with the mechanism originally proposed for the iodination reaction. As a possible explanation for this kinetic discrepancy, we postulate that lactoperoxidase generates hypoiodous acid and that the latter is the active intermediate in the various reactions involving iodide.

Mechanisms that have been proposed for peroxidase-catalyzed iodination require the utilization of 1 mol of H2O2 for organic binding of 1 mol of iodide. When we measured the stoichiometry of this reaction using thyroid peroxidase or lactoperoxidase at pH 7.0, we consistently obtained a ratio less than 1.0. This was shown to be attributable to catalase-like activity of these enzymes, resulting in unproductive cleavage of H2O2. This catalatic activity was completely iodide-dependent. To elucidate the mechanism of the iodide-dependent catalatic activity, the effects of various agents were investigated. The major observations may be summarized as follows: 1) The catalatic activity was inhibited in the presence of an iodine acceptor such as tyrosine. 2) The pseudohalide, SCN-, could not replace I- as a promoter of catalatic activity. 3) The inhibitory effects of the thioureylene drugs, methimazole and carbimazole, on the iodide-dependent catalatic activity were very similar to those reported previously for thyroid peroxidase-catalyzed iodination. 4) High concentrations of I- inhibited the catalatic activity of thyroid peroxidase and lactoperoxidase in a manner similar to that described previously for peroxidase-catalyzed iodination. On the basis of these observations and other findings, we have proposed a scheme which offers a possible explanation for iodide-dependent catalatic activity of thyroid peroxidase and lactoperoxidase. Compound I of the peroxidases is represented as EO, and oxidation of I- by EO is postulated to form enzyme-bound hypoiodite, represented in our scheme as [EOI]-. We suggest that the latter can react with H2O2 in a catalase-like reaction, with evolution of O2. We postulate further that the same form of oxidized iodine is also involved in iodination of tyrosine, oxidation of thioureylene drugs, and oxidation of I-, and that inhibition of catalatic activity by these agents occurs through competition with H2O2 for oxidized iodine.

The properties of guided-mode resonance reflection filters constructed with multiple thin-film layers are addressed. Greatly improved filter characteristics are shown to follow by the incorporation of multiple homogeneous layers with the spatially modulated layer. Calculated results for single-layer, double-layer, and triple-layer filter structures are presented. Whereas good filter characteristics are obtainable with single layers that are half-resonance-wavelength thick, there remains a residual reflection in the sidebands unless the cover and the substrate permittivities are equal. With double-layer and triple-layer designs, extensive wavelength ranges with low sideband-reflectance values are shown to be possible without requiring equal cover and substrate permittivities. The antireflection properties of the layer stack can be understood if the modulated layer is modeled as a homogeneous layer characterized by its average relative permittivity. However, as the grating-modulation index increases, this approximation deteriorates. In particular it is found that, for a given high modulation index, the double-layer antireflection thin-film approximation fails, whereas for the same modulation in a triple-layer system it holds firmly. Multilayer designs can thus have significantly large filter passbands, as they may contain heavily modulated resonant gratings without corruption of the ideal filter characteristics.

Interferometry based on double exposure Fourier transform holography in photorefractive crystals is applied for visualization of aerodynamic flow fields. The interferograms obtained are of similar quality as those produced using holographic film but with greatly simplified procedures. The results presented are obtained using a high-power cw argon laser and iron doped lithium niobate crystals. The angular characteristics of the Fourier transform data holograms are studied.

Regulation of adenylyl cyclases 1, 2, and 6 by Galphas was studied. All three mammalian adenylyl cyclases were expressed in insect (Sf9 or Hi-5) cells by baculovirus infection. Membranes containing the different adenylyl cyclases were stimulated by varying concentrations of mutant (Q227L) activated Galphas expressed in reticulocyte lysates. Galphas stimulation of AC1 involved a single site and had an apparent Kact of 0.9 nM. Galphas stimulation of AC2 was best explained by a non-interactive two site model with a "high affinity" site at 0.9 nM and a "low affinity" site at 15 nM. Occupancy of the high affinity site appears to be sufficient for Gbetagamma stimulation of AC2. Galphas stimulation of AC6 was also best explained by a two-site model with a high affinity site at 0. 6-0.8 nM and a low affinity site at 8-22 nM; however, in contrast to AC2, only a model that assumed interactions between the two sites best fit the AC6 data. With 100 microM forskolin, Galphas stimulation of all three adenylyl cyclases showed very similar profiles. Galphas stimulation in the presence of forskolin involved a single site with apparent Kact of 0.1-0.4 nM. These observations indicate a conserved mechanism by which forskolin regulates Galphas coupling to the different adenylyl cyclases. However, there are fundamental differences in the mechanism of Galphas stimulation of the different adenylyl cyclases with AC2 and AC6 having multiple interconvertible sites. These mechanistic differences may provide an explanation for the varied responses by different cells and tissues to hormones that elevate cAMP levels.

We have isolated and determined the nucleotide sequence of overlapping cDNA clones, representing the entire structural gene for pig thyroid peroxidase. The protein coding region extends from an ATG residue at base 252 to a termination codon at base 3030, coding for a 100.4-kDa apoprotein of 926 amino acids. The derived amino acid composition agrees well with the experimentally determined amino acid composition of purified pig thyroid peroxidase. Five potential glycosylation sites are present in the protein. Potential membrane spanning regions are present at the amino-terminal end (1-23) and near the carboxyl-terminal end (845-870) of the protein. These data indicate that pig thyroid peroxidase is synthesized as a single polypeptide that is membrane-bound.