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We have obtained the carbon-13 nuclear magnetic resonance spectra of a series of tryptophan-containing peptides and model systems, together with their X-ray crystallographic structures, and used quantum chemical methods to predict the (13)C NMR shifts (or shieldings) of all nonprotonated aromatic carbons (C(gamma), C(delta 2) and C(epsilon 2). Overall, there is generally good accord between theory and experiment. The chemical shifts of Trp C(gamma) in several proteins, hen egg white lysozyme, horse myoglobin, horse heart cytochrome c, and four carbonmonoxyhemoglobins, are also well predicted. The overall Trp C(gamma) shift range seen in the peptides and proteins is 11.4 ppm, and individual shifts (or shieldings) are predicted with an rms error of approximately 1.4 ppm (R value = 0.86). Unlike C(alpha) and N(H) chemical shifts, which are primarily a function of the backbone phi,psi torsion angles, the Trp C(gamma) shifts are shown to be correlated with the side-chain torsion angles chi(1) and chi(2) and appear to arise, at least in part, from gamma-gauche interactions with the backbone C' and N(H) atoms. This work helps solve the problem of the chemical shift nonequivalences of nonprotonated aromatic carbons in proteins first identified over 30 years ago and opens up the possibility of using aromatic carbon chemical shift information in structure determination.

Yu, W.-Z. and R. J. Bodnar. Interactions between Angiotensin Ii and delta opioid receptor subtype agonists upon water intake in rats. Peptides 18(2) 241–245, 1997.—Delta opioid receptor agonists, like those of mu and kappa receptors, stimulate water intake. To assess the relative contributions of delta 1 and delta 2 receptors in the modulation of water intake stimulated by Angiotensin II (AII), the present study examined the respective actions of [D-Pen 2 , D-Pen 5 ]-enkephalin (DPDPE: 5–20 ug, icv) and [D-Ala 2 , Glu 4 ]-Deltorphin (Delt II: 5–20 ug, icv) upon water intake per se, and upon AII (0.02–20 ng, icv)-induced hyperdipsia in rats. Both DPDPE and Delt II dose-dependently stimulated spontaneous water intake. An ineffective (5 ug) dose of DPDPE differentially altered water intake when paired with AII, significantly increasing intake at the 0.02 ng dose, not changing intake at the 0.2 ng dose and significantly decreasing intake at the 2 ng dose. In contrast, pairing ineffective doses of Delt II (5 ug) and AII (0.2 ng) significantly increased water intake that persisted when either the Delt II dose (1 ug) or the AII dose (0.02 ng) was lowered. However, neither delta 1 nor delta 2 opioid agonists significantly altered the ED 50 for AII-induced drinking. Thus, delta 2 opioid agonism appeared more consistent and reliable than delta 1 opioid agonism in stimulating water intake when paired with AII, and these data indicate interactions between delta 1 and delta 2 agonists and AII in mediating water intake.

The blood clam Barbatia lima subsp. from Amami-Oshima, Japan, expresses three types of haemoglobins in erythrocytes: a tetramer (alpha 2 beta 2), a homodimer (delta 2) and a polymer consisting of two kinds of chains, a 34 kDa two-domain (2D) globin and a delta chain. This is in sharp contrast to the congeneric clams B. reeveana (a North American species) and B. lima from Kochi, Japan, each containing a tetramer and a polymer consisting of the 2D globin, but not the delta chain. We have determined the cDNA-derived amino acid sequences of all four chains, alpha (163 residues), beta (155 residues), delta (152 residues) and 2D (308 residues) of B. lima (Amami-Oshima). The alpha chain has an extremely long N-terminal extension of 20 residues that may form a 'pre-A helix', and this makes the alpha chain the longest known globin. B. lima alpha and beta chains show about 50% sequence identity with the alpha and beta chains, respectively, of tetrameric haemoglobin from a related clam, Anadara trapezia. The B. lima homodimeric delta chain shows 71-74% identity with each of the two domains of the 2D chain, but only 39% identity with the homodimeric gamma chain of Anadara. In addition, the alignment of amino acid sequences of the delta chain and the two domains of the 2D chain revealed that the delta chain lacks one amino acid (Lys) at the C-terminus, suggesting that the C-terminal Lys (codon AAA or AAG) of the two domains of 2D chain could result from the stop codon TAA in the delta chain by nucleotide substitutions. These results, together with the fact that the delta and 2D chains form a polymeric haemoglobin, indicates that the delta chain is the ancestral single-domain globin for the 2D globin. The delta chain is expressed only in B. lima (Amami-Oshima), and appears to be a relic of molecular evolution.

1H-NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9-15-ppm region. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A-Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3-7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50-ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. delta-D-gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed. Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the C epsilon 1 and C delta 2 protons of an uncharged imidazole ring. We identify D as arising from the N epsilon 2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two-dimensional NOE spectra have provided additional information. Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered. The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7-10.6-ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.

Mitochondrial delta 3,5, delta 2,4-dienoyl-CoA isomerase, which catalyzes the conversion of 3,5-octadienoyl-CoA to 2,4-octadienoyl-CoA, was purified from rat liver 370-fold at almost 30% yield by a six-step purification procedure. The final preparation appeared to be homogeneous as judged by gel electrophoresis. The molecular weights of the native enzyme and its subunit(s) were estimated to be 126,000 and 32,000, respectively. The purification of delta 3,5, delta 2,4-dienoyl-CoA isomerase completes the characterization of the enzymes functioning in the NADPH-dependent pathway for the beta-oxidation of unsaturated fatty acids with double bonds extending from odd-numbered carbon atoms. This novel pathway may not be operative in peroxisomes because delta 3,5, delta 2,4-dienoyl-CoA isomerase was only detected in mitochondria. Substrates of this pathway are 2,5-dienoyl-CoAs formed from 5-enoyl-CoAs by acyl-CoA dehydrogenase. Two sequential isomerization reactions catalyzed by delta 3, delta 2-enoyl-CoAs isomerase and delta 3,5, delta 2,4-dienoyl-CoA isomerase, respectively, convert 2,5-dienoyl-CoAs to 2,4-dienoyl-CoAs, which are reduced by NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) before reentering the beta-oxidation spiral.

Rearrangements of the T-cell receptor (TCR) delta locus are observed in the majority of human B-cell precursor acute lymphoblastic leukemias (ALL) with a striking predominance of V delta 2(D)D delta 3 recombinations in common ALL (cALL) patients. Recently, we and others showed that almost 20% of cALL cases are characterized by further recombination of V delta 2(D)D delta 3 segments to J alpha elements, thereby deleting the TCR delta locus in analogy to the delta Rec/psi J alpha pathway in differentiating alpha/beta-positive T cells. We report here that two human cALL-derived cell lines, REH and Nalm-6, are competent to recombine the TCR delta/alpha locus under standard tissue culture conditions. Analysis of different REH subclones obtained by limiting dilution of the initial culture showed a biased recombination of V delta 2D delta 3 to distinct J alpha elements. During prolonged tissue culture, a subclone acquired growth advantage and displaced parental cells as well as other subclones. Frequently, the DJ junctions of REH subclones contained extended stretches of palindromic sequences derived from modified D delta 3 coding elements. The other cell line, Nalm-6, started the TCR delta/alpha recombination with an unusual signal joint of a cryptic recombinase signal sequence (RSS) upstream of D delta 3 to the 3′ RSS of D delta 3. The RSS dimer was subsequently rearranged in all investigated subclones to an identical J alpha element. Both cell lines might become valuable tools to unravel the complex regulation of TCR delta/alpha recombination pathways in malignant and normal lymphopoiesis.

NMR studies on δ-decanolide samples obtained from 1 and 5 in reduction experiments with baker's yeast in D 2 O and H 2 O, respectively, show that the double bond saturation involves beta re-face trans formal addition of hydrogen atoms, arising to a large extent from water. The reduction does not depend on the absolute configuration at position 5 and shows a kinetic preference for the ( R ) enantiomer.

[formula: see text] An efficient strategy for the conversion of 1,2-diketones into saturated 1,6-diketones and delta 2,3/delta 3,4-unsaturated congeners thereof is reported.

Before and after bone marrow transplantation (BMT) for hematologic malignancies, peripheral blood mononuclear cells from 10 patients were obtained. The relative and absolute numbers of CD3+ T-cell receptor yS+ (TCRyS+) cells, as defined by the reaction of monoclonal antibodies (MoAbs) directed against CD3 and the TCR yS (anti-TCRyS-1). were determined. Before transplantation, eight of nine patients tested had less than 10% CD3+TCRyS+ cells. Consistent increased numbers of yS cells up to eightfold the pretransplant level can be seen in four of nine patients tested within the first 4 months after BMT. The large majority of early posttransplant yS and 4 T cells express the CD45RO antigen, which is usually expressed on "memory" cells only. The V-region usage of the TCRyS' T cells was analyzed using fresh mononuclear cells and MoAbs against known Vy and V6 regions. For more detailed analysis, CD3'TCRyS + cells were WO DISTINCT FORMS of the T-cell receptor (TCR) T have been described, the UP TCR and the y8 TCR.'32 The y8 TCR is expressed by a minority of CD3+ cells (0.5% to 17%) in peripheral blood (PB) and thymus.',3 The y8 cells form a distinct and functionally heterogeneous T-cell lineage and are evenly distributed throughout the lymphoid ~ystem.~ Monoclonal antibodies (MoAbs) specific for the human y8 TCR as well as for V-regions have been described?-" Although the function of TCRy8 cells remains largely unknown, recent reports indicate that these cells are capable of interacting with a heterogeneous set of ligands, among which are alloantigenic determinants, tetanus toxoid, mycobacterial (heat shock) proteins,''~'Z and Staphylococcal enterotoxin A (SEA).I3 Studies in the mouse have indicated that the y8 TCR is expressed before the aP TCR during thymic ontogeny. Populations of y8 TCR' cells are intermittently generated that express characteristic TCRS.'~,'' These waves appear to home specifically to certain epithelia in the skin and in the gut.I6 It has already been shown that rearrangements involving Cy1 precede those involving Cy2, and recent results indicate that, during early fetal ontogeny, rearrangements involving the Cy1 constant region occur before those involving the Cy2 constant region gene ~egment.'~"~ Also, early in human fetal development a wave of TCRy8 cells exists that preferentially uses the V82 gene segment." The kinetics of engraftment after bone marrow transplantation (BMT) may serve as a model system to study the TCRy8 repopulation. After BMT, relative and absolute increased numbers of TCRyG cells have been reported, suggesting differences in repopulation rates between TCRaP and TCRy8 cells after

Upon disulfide bond reduction, the alpha 2-subunit of the dihydropyridine-sensitive Ca2+ channel undergoes a characteristic mobility shift on sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis with the concurrent appearance of the three delta peptides delta 1 (25,000 Da), delta 2 (22,000 Da), and delta 3 (17,000 Da). Densitometric scanning of Coomassie Blue-stained gels shows a stoichiometric ration of 1.0:0.31.47:0.08 for the alpha 2-subunit and the delta peptides 1, 2, and 3, respectively. Characterization of the delta peptides using antibodies, photoincorporation of a hydrophobic probe, and lectin staining shows tham to be antigenically similar hydrophobic glycoproteins. Amino-terminal sequence analysis of the delta peptides reveals three identical sequences that match the predicted amino acid sequence of the alpha 2-subunit starting at Ala935. Enzymatic deglycosylation of the reduced alpha 2.delta complex produces individual core peptides of 105,000 and 17,000 Da, respectively. Treatment of skeletal muscle membranes with high pH in the presence of reducing agents is able to extract the larger amino-terminal peptide but not the smaller carboxyl (delta) peptide, consistent with a single transmembrane domain in the carboxyl (delta) region. The data support a model of the alpha 2-subunit in which the propeptide is processed into two chains that remain attached through disulfide linkages.

A high frequency (greater than 80%) of acute lymphoblastic leukemias (ALL) exhibit a recombination of the T-cell receptor (TCR) delta chain locus. Interestingly, distinct TCR delta elements are preferentially used in immunologic subtypes. In a recent series of 201 children with common ALL (cALL) we observed a TCR delta rearrangement in 162 patients, 57% of the latter showing a hybridization pattern in Southern blots suggestive of a V delta 2 to D delta 3 recombination. To verify this interpretation and to elucidate in more detail the diversity of this common type of TCR delta recombination we amplified and sequenced the junctional region of nine cALL patients and cell line REH-6 by polymerase chain reaction (PCR). A V delta 2 D delta 3 recombination was confirmed in all cases; convincing evidence for the participation of D delta 1 or D delta 2 elements was not obtained. Eight of nine patients and REH-6 showed complete 5′ D delta 3 boundaries within V delta 2 D delta 3 segments, a limitation of junctional diversity also detected in 50% of peripheral blood cell clones derived from two healthy probands. Notably, sequence identity at the V delta 2 D delta 3 junction was demonstrated for a cALL and one of the control clones. Another group of 35 of 162 cALL patients was characterized by V delta 2 rearrangements and biallelic deletion of J delta and C delta sequences. Using a J alpha consensus primer, PCR-directed sequence analysis demonstrated V delta 2 D delta 3 J alpha recombinations in all four cases analyzed by this approach. The J alpha segments of these patients differed, but were identical or homologous to published J alpha elements. Our data suggest a recombination pathway of the TCR delta/alpha locus leading to chimeric TCR alpha molecules, containing V delta and, remarkably, also D delta sequences.

The low-frequency FeCN vibrations of cyanoferric myeloperoxidase (MPO) and horseradish peroxidase (HRP) have been measured by resonance Raman spectroscopy. The ordering of the frequencies of the predominantly FeC stretching and FeCN bending normal vibrational modes in the two peroxidases differs. These normal mode vibrations are identified by their wavenumber shifts upon isotopic substitution of the cyanide ligand. For MPO, the stretching mode nu 1 (361 cm-1) occurs at a lower frequency than the bending mode delta 2 (454 cm-1). For HRP, the order is reversed as nu 1 (456 cm-1) is at a higher frequency than delta 2 (404 cm-1). Normal coordinate analyses and model complexes have been used to address the origin of this behavior. The nu 1 stretching frequencies in cyanide complexes of iron porphyrin and iron chlorin model compounds are similar to one another and to that of HRP. Thus, the inverted order and altered frequencies of the nu 1 and delta 2 vibrations in MPO, relative to those in HRP and the model compounds, are not inherent to the proposed iron chlorin prosthetic group in MPO but, rather, are attributed to distinct distal environmental effects in the MPO active site. The normal coordinate analyses for MPO and HRP showed that the nu 1 and delta 2 vibrational frequencies are not pure; the potential energy distributions for these modes respond not only to the geometry but also to the force constants of the nu(FeC) and delta(FeCN) internal coordinates.(ABSTRACT TRUNCATED AT 250 WORDS)

We identified two types of gamma/delta T-cell clones: (1) BB3+ clones, which express the disulphide-linked form of the gamma/delta heterodimer (type 1 receptor) and carry rearrangements of the C gamma 1 gene; and (2) delta TCS1+ clones, which express the nondisulphide-linked form of the gamma/delta heterodimer (type 2 receptor) and carry rearrangements of the C gamma 2 gene. To determine the joining (J) and variable (V) region usage of the two receptor types, T gamma and T delta gene rearrangements and expression were analyzed in each gamma/delta T-cell clone. The results showed that the J and V regions used by the two receptor types are distinct: clones that express the type 1 receptor carry potentially functional rearrangements in J gamma P-V gamma 9 for gamma and in J delta 1-V delta 2 for delta; and clones that express the type 2 receptor had potentially functional rearrangements in J gamma 2- V gamma 2/V gamma 4 for gamma and in J delta 1-V delta 1 for delta. Therefore, two clearly different gamma/delta receptors (V gamma 9-J gamma P-C gamma 1/V delta 2-J delta 1-C delta and V gamma 2/V gamma 4-J gamma 2-C gamma 2/V delta 1-J delta 1-C delta) predominate in peripheral blood.

X-ray diffraction analysis (at 2.1-A resolution) of an acid alpha-amylase from Aspergillus niger allowed a detailed description of the stereochemistry of the calcium-binding sites. The primary site (which is essential in maintaining proper folding around the active site) contains a tightly bound Ca2+ with an unusually high number of eight ligands (O delta 1 and O delta 2 of Asp175, O delta of Asn121, main-chain carbonyl oxygens of Glu162 and Glu210, and three water molecules). A secondary binding site was identified at the bottom of the substrate binding cleft; it involves the residues presumed to play a catalytic role (Asp206 and Glu230). This explains the inhibitory effect of calcium observed at higher concentrations. Neutral Aspergillus oryzae (TAKA) alpha-amylase was also refined in a new crystal at 2.1-A resolution. The structure of this homologous (over 80%) enzyme and additional kinetic studies support all the structural conclusions regarding both calcium-binding sites.

Rearrangement of the immunoglobulin (Ig) and T-cell receptor (TcR) genes generally has been considered a useful marker of B- and T-cell lineage in lymphoproliferative disorders. However, concomitant rearrangements of Ig and TcR genes have been commonly reported in the most immature lymphoid malignancies, mainly in B-cell precursor acute lymphoblastic leukemia (ALL). To better characterize the nature of this lineage promiscuity, we have analyzed the configuration of the TcR delta locus in 75 B-precursor ALL. The large majority of cases (87%) displayed a rearrangement or deletion at the delta locus. Among the 57 nondeletional rearrangements, two patterns were predominant and both appeared to derive from partial joining: a V delta-(D)-D delta 3 (32/57) and a D delta 1/2-D delta 3 (11/57) type. A single V delta gene (V delta 2) appeared to be involved in the first type of rearrangement. These findings are at variance with T-ALL, where rearrangements 5′ to V delta 2 are usually found. It remains to be elucidated whether this incomplete attempt of V delta 2 gene assembly is related to the neoplastic event or represents a physiologic predisposition occurring during early stages of the normal lymphocyte differentiation.

The C[bond]N coupling constants centered at the C(epsilon 1) and C(delta 2) carbons in histidine residues depend on the protonation state and tautomeric form of the imidazole ring, making them excellent indicators of pH or pK(a), and the ratio of the tautomeric states. In this paper, we demonstrate that the intensity ratios for the C(epsilon 1)-H and C(delta 2)-H cross-peaks measured with a constant time HSQC experiment without and with J(C[bond]N) amplitude modulation are determined by the ratios of the protonated and deprotonated forms and tautomeric states. This allows one to investigate the tautomeric state of histidines as well as their pK(a) in situations where changing the pH value by titration is difficult, for example, for in-cell NMR experiments. We apply this technique to the investigation of the bacterial protein NmerA and determine that the intracellular pH in the Escherichia coli cytoplasm is 7.1 +/- 0.1.

The capsid of filamentous virus Ff is assembled from approximately 2750 copies of a 50-residue alpha-helical subunit, the two tyrosines of which (Tyr 21 and Tyr 24) are located within a hydrophobic sequence that constitutes the subunit interface. We have determined the side chain orientations of Tyr 21 and Tyr 24 by polarized Raman microspectroscopy of oriented Ff fibers, utilizing a novel experimental approach that combines site-specific mutation and residue-specific deuteration of capsid subunits. The polarized Raman signature of Tyr 21 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 21 in an Ff mutant in which Tyr 24 is replaced with methionine. Similarly, the polarized Raman signature of Tyr 24 was obtained by incorporating C(delta 1),C(delta 2),C(epsilon 1),C(epsilon 2)-tetradeuteriotyrosine at position 24 in the analogous Tyr 21 --> Met mutant. Polarizations of the corresponding C-D stretching bands in the 2200-2400 cm(-1) interval of the Raman spectrum were measured and interpreted using tensors transferred from a polarized Raman analysis of L-tyrosine-2,3,5,6-d(4) single crystals. Polarized Raman analysis was extended to the bands of Ff near 642 and 855 cm(-1), which originate from vibrational modes of the tyrosine phenolic ring. The results indicate the following: (i) For both Tyr 21 and Tyr 24, the phenolic 2-fold axis (C(1)-C(4) line) is inclined at 41 +/- 5 degrees from the virion axis and the normal to the plane of the phenolic ring is inclined at 71 +/- 5 degrees from the virion axis; (ii) the mutation of Tyr 24, but not the mutation of Tyr 21, perturbs Raman markers of the subunit tryptophan (Trp 26), suggesting interdependence of Tyr 24 and Trp 26 orientations in native Ff; and (iii) polarization anisotropies observed for Raman markers of Ff DNA bases are unperturbed by mutation of either Tyr 21 or Tyr 24, indicating that nonrandom base orientations of packaged Ff DNA are independent of the mutation of either Tyr 21 or Tyr 24. A molecular model consistent with these findings is proposed.

A series of delta 2-isoxazoline derivatives structurally related to Broxaterol 1 and Falintolol 3 has been prepared and evaluated for their binding affinity to beta 1- and beta 2-adrenergic receptors. Among the tested compounds only the 3-isopropenyl anti derivative 4d is as active as the reference compounds. An electron-releasing group, probably operating through a pi-pi interaction, in the 3-position of the isoxazoline nucleus greatly enhances the affinity of the compounds. Conversely, the closest analogs of Broxaterol (3-bromo delta 2-isoxazolines 4a and 5a) are at least one order of magnitude less active than the model compound 1. Throughout the series of derivatives the anti stereoisomers are invariably more active than their syn counterparts.

Calbindin D28K binds 3 mol of terbium per mol of protein. To determine which of six EF-hand structures in the protein are responsible for terbium binding, we constructed three mutant forms of this protein, one lacking EF-hand 2 (RCaBP delta 2), the other lacking EF-hands 2 and 6 (RCaBP delta 2,6), and the third containing only EF-hands 3 and 4 (RCaBP delta 1,2,5,6), and examined their binding properties by fluorescence techniques. Full-length calbindin D28K and RCaBP delta 2 and RCaBP delta 2,6 bound 3 mol of terbium per mol of protein with high affinity. Thus, EF-hand domains 2 and 6 are not essential for calcium binding to the proteins, and an absence of EF-hands 2 and/or 6 does not alter the pattern of terbium binding to the protein. Using resonance energy transfer from tryptophan residues, one of the high affinity terbium-binding sites (site A) had a greater affinity than the other two sites (sites B and C) of each protein. Site A was filled before the other two sites. Calcium competition experiments showed that a greater amount of calcium was required to displace terbium from site A than from sites B or C. Energy transfer experiments from terbium to holmium showed that two of the terbium-binding sites are in close proximity while the third site is distant from the other two sites. To determine whether EF-hand 3 or 4 was responsible for binding of terbium, we examined the terbium binding properties of a delta 1,2,5,6 RCaBP construct. The truncated protein RCaBP delta 1,2,5,6 contained a single terbium-binding site. Analysis of the terbium binding to RCaBP delta 1,2,5,6 construct showed that site 4 bound terbium, whereas site 3 did not. Analysis of the terbium binding characteristics of the proteins suggests that EF-hands 1, 4, and 5 of rat brain calbindin D28K are responsible for terbium binding.

The temperature dependence of the efficiency of oxidative refolding was examined for hen lysozyme three-disulfide derivatives produced in Escherichia coli. Each derivative was designed to lack one of the four disulfide bridges in authentic lysozyme: delta 1 (Cys6-->Ser, Cys127-->Ser), delta 2 (Cys30-->Ser, Cys115-->Ser), delta 3 (Cys64-->Ser, Cys80-->Ser), delta 4 (Cys76-->Ser, Cys94-->Ser), delta 2Ala (Cys30-->Ala, Cys115-->Ala), and delta 4Ala (Cys76-->Ala, Cys94-->Ala). The optimal refolding temperature was lowest for delta 1 (19 degrees C) and highest for delta 4Ala (30 degrees C). The chromatographically purified, completely refolded three-disulfide species were not stable above the optimal refolding temperature in the presence of glutathione. The stability of each of them was determined from the far-UV CD thermal denaturation measurement at pH 3.9 in the absence of glutathione, where the denaturation was reversible. The transition temperature was lowest for delta 1 and highest for delta 4Ala. Precise values of difference in the transition temperature among the three-disulfide derivatives were found to correlate with those in the optimal refolding temperature. Next, the effect of glycerol, which has been shown to increase the refolding efficiency [Sawano et al. (1992) FEBS Lett. 303, 11-14], was examined for delta 1 in detail. The optimal temperature for refolding increased by 3-4 degrees C with the increase in glycerol concentration by 10%. The amount of increase in the optimal refolding temperature was nearly equal to the amount of the increase in thermal stability in the presence of glycerol of refolded and purified delta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Duck lens delta-crystallins were the translational products of two tandem arranged genes, delta 1 and delta 2. Two fractions of the delta-crystallin (delta a and delta b) were resolved when the duck eye ball lens extract was passed through a Pharmacia-LKB Q-Sepharose column. These fractions were further separated by chromatofocusing into multiple forms with pI value ranging from 5.05 to 5.45. The charge heterogeneity of delta-crystallins was also demonstrated on the polyacrylamide gel when electrophoresis was performed under nonreducing conditions. All of those multiple forms possessed endogenous argininosuccinate lyase activity with activation energy approximately 12.5 +/- 1.6 kcal/mol. delta b-Crystallins showed higher enzyme activity than delta a-crystallins. Slightly different kinetic parameters were observed for the multiple forms of delta b-crystallins. On the other hand, delta a-crystallins could be further divided into two subgroups according to their kinetic parameters. There is 12-fold difference in kcat value between these two subgroups. delta a-Crystallins also have lower Km values than the delta b-crystallins. When examined by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate, the multiple forms were shown to be composed of subunits with similar M(r) of 55,000. All of these forms showed same antigenicity toward the rabbit anti-duck delta-crystallin antiserum; however, different carbonyl contents were observed for these forms, indicating that the origin of these multiple forms was due to post-translational oxidative modification of the protein molecules. Two delta-crystallin forms were demonstrated to be the N-truncated delta 2-crystallin, lacking the first eight amino acid residues from N-terminus. Modification and/or truncation of the proteins resulted in changing of the intrinsic tryptophan and tyrosine fluorescence. Those forms with higher pI values were shown to be much more thermostable than those with lower pI values. Our system may represent the first in vivo information that oxidation does not always lead to inactivation of an enzyme.

Topographic design of peptide ligands using specialized topographically constrained amino acids can provide new insights into the stereochemical requirements for delta opioid receptors. A highly constrained tyrosine derivative, (2S,3S)-beta-methyl-2',6'-dimethyltyrosine [(2S,3S)-TMT], was prepared by asymmetric synthesis and incorporated in [D-Pen2,D-Pen5] enkephalin (delta 1) and Deltorphin I (delta 2). The results of binding assays and bioassays showed that the two analogues (3 and 4) acted very differently at delta opioid receptors. Further pharmacological evaluations suggested that they actually interact primarily with the delta 1 and delta 2 receptor subtypes, respectively. These results, and conformational studies using NMR and computer-assisted modeling, provided insights into the different stereochemical requirements for these two delta opioid ligands to recognize the delta opioid receptor and its subtypes.

In amphibian limb regeneration memory for position in the proximal-distal axis can be respecified by retinoic acid. The favoured candidates to mediate this effect are the retinoic acid receptors (RARs) and of the RARs identified in the regeneration blastema, the delta receptor is the most abundant. The presence in blastemal mesenchyme of at least two delta receptor isoforms, delta 1 and delta 2, alternatively spliced at the A-B junction, was demonstrated in expression studies and by PCR cloning. The delta 1 receptor is abundant in regenerative structures such as the limb and tail, whereas the delta 2 and alpha receptors show a more uniform pattern of expression across adult newt tissues. Full-length cloning of the delta 1 receptor established the presence of an unusually long open reading frame and N-terminal sequence that appears unique among vertebrate retinoic acid receptors. Transient transfection of expression constructs into COS cells followed by Western blotting confirmed the existence of at least three potential initiation sites for delta 1 translation. The possibility that delta 1 RAR expression may specify positional memory directly was tested in RNase protection experiments. delta 1 receptor message is increased on amputation, but does not exhibit a pronounced differential distribution along the proximal-distal axis in normal and regenerating limbs, nor does it show a persistent alteration in expression levels following a dose of retinoic acid sufficient to respecify position. The possibility that the morphogenetic effects of RA may be mediated through receptor interactions is raised by the finding that single mesenchymal blastemal cells in culture can express multiple RAR subtypes (delta 1 and alpha) and isoforms (delta 1 and delta 2).

The extent of diversity of the gamma delta T-cell receptor (TCR) in normal human skin and Oriental Cutaneous Leishmaniasis (OCL) was examined by molecular analysis of the variable (V) delta gene segment, junctional (J) delta gene segment and junctional regions. To examine the expression of TCR delta genes, segments of gamma delta T lymphocytes, DNA isolated from normal human skin and from OCL were subjected to enzymatic gene amplification by the polymerase chain reaction (PCR) method using TCR V delta- and J delta-specific oligonucleotides as primers. PCR amplification using these primers indicated that the V delta 2 gene segment was predominantly used by gamma delta T lymphocytes in both normal human skin and OCL. To determine the extent of junctional diversity in the delta gene of gamma delta T cells in normal human skin and OCL, we sequenced the nucleic acid sequences corresponding to the V delta 2/J delta 1 junctional regions. Sequence analysis of junctional regions demonstrated broad junctional diversity in normal skin but only limited diversity in OCL. Our findings support the hypothesis that skin gamma delta T lymphocytes may derive from a fetal subset of gamma delta T lymphocytes that leaves the thymus early and colonizes the periphery. The limited junctional diversity demonstrated in OCL lesions indicates that gamma delta T cells can undergo oligoclonal expansion following recognition of a specific ligand and supports the idea that junctional regions are important in the recognition of antigenic determinant.

The chlorophyll a-binding protein CP47 serves as core antenna to photosystem II (PS II). The predicted topology of CP47 exhibits six membrane-spanning regions and a large hydrophilic loop (loop E) which roughly includes 200 residues (255-455) and is presumably exposed to the lumenal side of the thylakoid membrane. Several lines of experimental evidence suggest that loop E might be involved in binding or stabilizing functional manganese in the catalytic site of water oxidation or in interacting with the extrinsic PS II-O protein (the 33-kDa manganese-stabilizing protein). To scan loop E for functionally important domains, oligonucleotide-directed mutagenesis has been used to introduce deletions of 3-8 residues in conserved and charged regions of loop E. In addition, one single-site mutation of the only histidine present in loop E was created (H343L). Domains deleted in delta 1 (I265-F268), delta 2 (T271-K277), delta 4 (T304-L309), delta 5 (F311-N317), and delta 12 (D440-P447) are required for stable assembly of functional PS II complexes. Deletion of domains delta 3 (K277-E283) and delta 11 (R422-E428) significantly reduces the level of assembled PS II and impairs photoautotrophic growth and oxygen evolution. Deletion of domain delta 8 (A373-D380) enhances the susceptibility to photoinhibition. In contrast, deletion of domains delta 6 (G333-I336), delta 7 (K347-R352), delta 9 (V392-Q394), and delta 10 (D416-F420) and mutation of H343 to leucine do not seem to severly interrupt PS II structure and function, although all mutants exhibit a slightly decreased stability of PS II as compared to the wild type. Thus, selected domains of the large hydrophilic loop of CP47 are important for PS II structure and function. With respect to possible sites of interaction between loop E of CP47 and the extrinsic PS II-O protein, our results indicate that none of the deletions in the region from residue 330 to 420 (delta 6, delta 7, delta 8, delta 9, delta 10) completely interrupts a functional association of the manganese-stabilizing protein to PS II, although the binding characteristics might be changed in some cases.

The pressure-induced reversible unfolding of lysozyme was investigated by high-resolution proton magnetic resonance spectroscopy by following the proton spectra of the following residues: His-15 epsilon 1, Trp-28 epsilon 3, Leu-17 delta 2, Cys-64 alpha, and Trp-108 epsilon 3. The experiments were performed at pH 3.9 and 68.5 degrees C in the pressure range from 1 bar to 5 kbar both in the absence and presence of tri-N-acetylglucosamine (tri-NAG). From the pressure-induced changes of the equilibrium between the native and denatured forms of lysozyme, the reaction volumes (delta V) were calculated for each residue. Small but statistically significant differences in delta V were found for residues located in different regions of the protein. For example, delta V for the disulfide bonded Cys-64 alpha is smaller than the delta V's found for the other residues. In particular, the effect of tri-NAG binding to lysozyme was a change of delta V from -10.3 +/- 0.6 cm3/mol to -18.1 +/- 1.7 cm3/mol for the Trp-108 epsilon 3 residue which is located close to the active site. It is important to note that the Cys-64 alpha residue also senses the binding of the substrate analog. The ability to detect statistically significant differences for delta V of individual residues located in different regions of lysozyme represents the main result of these experiments.

On determine les conditions optimales de transformation d'isoxazolines-2 en β-hydroxycetones en utilisant un catalyseur Ni de Raney, l'acide borique, un melange 5/1 methanol/eau et l'hydrogene gazeux

The chicken delta-crystallin locus consists of 2 nonallelic, tandemly arranged genes (5'-delta 1-delta 2-3'). Only the delta 1 gene is known to be expressed. The nucleotide sequence for the delta 1 gene has been reported recently (Nickerson, J.M., Wawrousek, E.F., Hawkins, J.W., Wakil, A.S., Wistow, G.J., Thomas, G., Norman, B.L., and Piatigorsky, J. (1985) J. Biol. Chem. 260, 9100-9105; Ohno, M., Sakamoto, H., Yasuda, K., Okada, T.S., and Shimura, Y. (1985) Nucleic Acids Res. 13, 1593-1606). We now report the sequence for the delta 2 gene and the 4-kilobase intergenic spacer between the two delta-crystallin genes. The delta 2 gene, like the delta 1 gene, has 17 exons and 16 introns. The homologous exons are remarkably similar: exons 3-17 are identical in size between delta 1 and delta 2, and the sequence homology ranges from 70% (exon 2) to 100% (exons 7, 12, and 15), with the remaining exons having 89-98% identity between the delta 1 and delta 2 genes. Consequently, the encoded delta 2 polypeptide is 91% identical to the delta 1 polypeptide. Considerable similarity also exists between homologous introns of delta 1 and delta 2, with most of the differences accounted for by insertions and/or deletions. The presence of a TATA box, consensus splice junctions (almost identical to the delta 1 gene), lariat branch sequences, and a polyadenylation signal strengthen the possibility that delta 2 is a functional gene.

Dietary methyl-2-hexadecynoate appeared to inhibit fatty acid elongation in intact animals (Wood, R., Lee, T., and Gershon, H. (1980) Lipids 15, 141-150). Data from the present in vitro studies indicate that the microsomal elongation system is inhibited preferentially to the mitochondrial system. A series of metabolic acyl-CoA thioester intermediates has been isolated, characterized, and identified from microsomal and mitochondrial incubations with the 2-hexadecynoic acid (16 identical to 1 delta 2). The data support the following conclusions: 1) 16 identical to 1 delta 2 is activated to the CoA ester; 2) 16 identical to 1 delta 2 is acted on by an isomerase to produce a 2,3-allene; 3) either 16 identical to 1 delta 2 or the allene, or both, are hydrated to yield a beta-keto-CoA thioester after rearrangement; 4) the beta-keto ester is reduced to the beta-hydroxyacyl-CoA; 5) dehydration of the beta-hydroxy ester gives rise to trans-delta 2-hexadecenoate which accumulates; and 6) accumulation of the latter results from the inhibition of enoyl-CoA reductase by the 2,3-allene. The occurrence of cis and trans delta 3-hexadecenoates indicates the allene is reduced, after which the delta 3 monoene isomer may be isomerized to the delta 2 monoene by the acetylene isomerase or a different enzyme. Indirect evidence suggests that the fatty acid elongation systems may also be inhibited at another site.

Dermal absorption of the insecticide lindane (1 delta, 2 delta, 3 beta, 4 delta, 5 delta, 6 beta-hexachlorocyclohexane) was determined in rats and rhesus monkeys. Lindane is in widespread use as a 1% cream or lotion scabicide formulation and as a 1% miticide shampoo for body lice control in humans. Results obtained following our in vivo dermal absorption procedure demonstrated that 18 +/- 4.1%, 34 +/- 5.2%, and 54 +/- 26.3% of the applied dose was absorbed following topical applications at a rate of 1.5 micrograms/cm2 (6.2 micrograms/100 microliters of acetone) of the 14C-labeled pesticide to 4.2-cm2 regions of the forearm (n = 8), forehead (n = 7), and palm (n = 4) of rhesus monkeys, respectively. Dose sites were washed with soapy water 24 h posttreatment. Comparative studies in rats (n = 5) dosed middorsally demonstrated 31 +/- 9.5% absorption. Statistical analysis of the 14C excretion kinetics demonstrated slower clearance of lindane from rats than monkey forearm, forehead, or palm. Intramuscular (im) injections of 14C-lindane gave 52 +/- 7.1% recovery in monkey (n = 8) and 64 +/- 5.9% in rats (n = 5), suggesting body storage of this lipophilic chemical.

A minor abnormal hemoglobin was found in two members of an Iraqi family. Structural studies showed this hemoglobin to be a δ chain variant in which glutamic acid is substituted for valine at position 20 (B2).

The present study examines the effect of the acetylenic thioester dec-2-ynoyl-CoA (delta 2 10 identical to 1-CoA) on the microsomal fatty acid chain elongation pathway in rat liver. When the individual reactions of the elongation system were measured in the presence of delta 2 10 identical to 1-CoA, the trans-2-enoyl-CoA reductase activity was markedly inhibited (Ki = 2.5 microM), whereas the activities of the condensing enzyme, the beta-ketoacyl-CoA reductase, and the beta-hydroxyacyl-CoA dehydrase were not affected. The absence of inhibition of total microsomal fatty acid elongation was attributed to the significant accumulation of the intermediates, beta-hydroxyacyl-CoA and trans-2-enoyl-CoA, without formation of the saturated elongated product, indicating that the trans-2-enoyl-CoA reductase-catalyzed reaction was the only site affected by the inhibitor. The nature of the inhibition was noncompetitive. In contrast to the delta 2 10 identical to 1-CoA, delta 3 10 identical to 1-CoA did not inhibit trans-2-enoyl-CoA reductase activity, suggesting that the mode of inhibition was not via formation of the 2,3-allene derivative. Based on the observation (a) that p-chloromercuribenzoate markedly inhibits reductase activity, (b) that dithiothreitol protects the enzyme against inactivation by delta 2 10 identical to 1-CoA, (c) of the spectral manifestation of the interaction between thiol reagents and delta 2 10 identical to 1-CoA depicting an absorbance peak similar to that of the beta-ketoacyl thioester-Mg2+ enolate complex, (d) of a similar absorbance spectrum formed by the interaction between delta 2 10 identical to 1-CoA and liver microsomes, and (e) of the absence of formation of a similar spectrum by delta 3 10 identical to 1-CoA, trans-2-10:1-CoA, or delta 2 10 identical to 1 free acid with liver microsomes, we propose that delta 2 10 identical to 1-CoA inactivates trans-2-enoyl-CoA reductase by covalently binding to a critical sulfhydryl group at or in close proximity to the active site of the enzyme.

Cytochrome c1 is an amphiphilic protein which binds to the mitochondrial inner membrane, presumably through a hydrophobic region near the carboxyl (C)-terminus. In the preceding study (Hase, T., et al. (1987) J. Biochem. 102, 401-410), two cytochrome c1 mutations were constructed: delta 1 and delta 2 cytochromes c1, in which the C-terminal segments of 17 and 71 residues were replaced by foreign sequences of 20 and 15 residues, respectively. delta 2 cytochrome c1 had lost the putative membrane anchor. The two cytochrome c1 mutants were localized in mitochondria, but succinate-cytochrome c1 reductase activity was detected only in the mitochondria containing delta 1 cytochrome c1. The membrane association of the two mutant molecules as well as that of authentic cytochrome c1 was investigated. These three molecules were firmly attached to mitochondrial membranes and not solubilized on either sonication or sodium carbonate (pH 11) treatment. However, when the membranes were solubilized with Triton X-100, both the delta 1 and authentic cytochromes c1 were extracted from the membranes more easily than delta 2 cytochrome c1. By fractionating cholate extracts of mitochondrial membranes with ammonium sulfate, delta 1 cytochrome c1 was cofractionated with the enzymatic activity of complex III, but delta 2 cytochrome c1 was clearly separated from the complex III fraction. Trypsin treatment of mitochondria and mitoplasts showed that delta 2 cytochrome c1 was exposed to the intermembrane space, with such a topology that its trypsin susceptibility became much higher than that of the authentic molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Lithiation/alkylation en α- de la chaine laterale en 3 d'isoxazolines-2 trisubstituees-3,4,5