Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of the binding site for fibrinogen recognition peptide gamma 383-395 within the alpha(M)I-domain of integrin alpha(M)beta2

The leukocyte integrin alpha(M)beta(2) (Mac-1, CD11b/CD18) is a cell surface adhesion receptor for fibrinogen. The interaction between fibrinogen and alpha(M)beta(2) mediates a range of adhesive reactions during the immune-inflammatory response. The sequence gamma(383)TMKIIPFNRLTIG(395), P2-C, within the gamma-module of the D-domain of fibrinogen, is a recognition site for alpha(M)beta(2) and alpha(X)beta(2). We have now identified the complementary sequences within the alpha(M)I-domain of the receptor responsible for recognition of P2-C. The strategy to localize the binding site for P2-C was based on distinct P2-C binding properties of the three structurally similar I-domains of alpha(M)beta(2), alpha(X)beta(2), and alpha(L)beta(2), i.e. the alpha(M)I- and alpha(X)I-domains bind P2-C, and the alpha(L)I-domain did not bind this ligand. The Lys(245)-Arg(261) sequence, which forms a loop betaD-alpha5 and an adjacent helix alpha5 in the three-dimensional structure of the alpha(M)I-domain, was identified as the binding site for P2-C. This conclusion is supported by the following data: 1) mutant cell lines in which the alpha(M)I-domain segments (245)KFG and Glu(253)-Arg(261) were switched to the homologous alpha(L)I-domain segments failed to support adhesion to P2-C; 2) synthetic peptides duplicating the Lys(245)-Tyr(252) and Glu(253)-Arg(261) sequences directly bound the D fragment and P2-C derivative, gamma384-402, and this interaction was blocked efficiently by the P2-C peptide; 3) mutation of three amino acid residues within the Lys(245)-Arg(261) segment, Phe(246), Asp(254), and Pro(257), resulted in the loss of the binding function of the recombinant alpha(M)I-domains; and 4) grafting the alpha(M)(Lys(245)-Arg(261)) segment into the alpha(L)I-domain converted it to a P2-C-binding protein. These results demonstrate that the alpha(M)(Lys(245)-Arg(261)) segment, a site of the major sequence and structure difference among alpha(M)I-, alpha(X)I-, and alpha(L)I-domains, is responsible for recognition of a small segment of fibrinogen, gammaThr(383)-Gly(395), by serving as ligand binding site.     

Identification of sequence segments forming the alpha-bungarotoxin binding sites on two nicotinic acetylcholine receptor alpha subunits from the avian brain

The relationship between neuronal alpha-bungarotoxin binding proteins (alpha BGTBPs) and nicotinic acetylcholine receptor function in the brain of higher vertebrates has remained controversial for over a decade. Recently, the cDNAs for two homologous putative ligand binding subunits, designated alpha BGTBP alpha 1 and alpha BGTBP alpha 2, have been isolated on the basis of their homology to the N terminus of an alpha BGTBP purified from chick brain. In the present study, a panel of overlapping synthetic peptides corresponding to the complete chick brain alpha BGTBP alpha 1 subunit and residues 166-215 of the alpha BGTBP alpha 2 subunits were tested for their ability to bind 125I-alpha BGT. The sequence segments corresponding to alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were found to consistently and specifically bind 125I-alpha BGT. The ability of these peptides to bind alpha BGT was significantly decreased by reduction and alkylation of the Cys residues at positions 190/191, whereas oxidation had little effect on alpha BGT binding activity. The relative affinities for alpha BGT of the peptide sequences alpha BGTBP alpha 1-(181-200) and alpha BGTBP alpha 2-(181-200) were compared with those of peptides corresponding to the sequence segments Torpedo alpha 1-(181-200) and chick muscle alpha 1-(179-198). In competition assays, the IC50 for alpha BGTBP alpha 1-(181-200) was 20-fold higher than that obtained for the other peptides (approximately 2 versus 40 microM). These results indicate that alpha BGTBP alpha 1 and alpha BGTBP alpha 2 are ligand binding subunits able to bind alpha BGT at sites homologous with nAChR alpha subunits and that these subunits may confer differential ligand binding properties on the two alpha BGTBP subtypes of which they are components.     

Electrophysiological evidence for the coexistence of alpha1 and alpha6 subunits in a single functional GABA(A) receptor

The subunit combinations alpha1beta2gamma2, alpha6beta2gamma2, and alpha1alpha6beta2gamma2 of the GABA(A) receptor were functionally expressed in Xenopus oocytes. The properties of the resulting ion currents were characterized by using electrophysiological techniques. The concentration-response curve of the channel agonist GABA for alpha1alpha6beta2gamma2 showed a single apparent component characterized by an EC(50) of 107 +/- 26 microM (n = 4). It was different from the one for alpha1beta2gamma2, which had an EC(50) of 41 +/- 9 microM (n = 4), that for alpha6beta2gamma2, with an EC(50) of 6.7 +/- 1.9 microM (n = 5), and those for alpha1beta2 and alpha1alpha6beta2. There was no appreciable functional expression of alpha6beta2. Allosteric responses of alpha1alpha6beta2gamma2 to diazepam were intermediate to those of alpha1beta2gamma2 and alpha6beta2gamma2, and allosteric responses to flumazenil were comparable to the ones for alpha1beta2gamma2. The inhibition by furosemide of the currents elicited by GABA in alpha1alpha6beta2gamma2 [IC(50) = 298 +/- 116 microM (n = 7), assuming only one component] was not identical with inhibition of alpha6beta2gamma2 (IC(50) = 38 +/- 2 microM, n = 4), alpha1beta2gamma2 (IC(50) = 5,610 +/- 910 microM, n = 5), or a mixture of these components (assuming two components). These findings indicate unambiguously the formation of functional GABA(A) receptors containing two different alpha subunits, alpha1 and alpha6, with properties different from those of alpha1beta2gamma2 and alpha6beta2gamma2. Furthermore, we provide evidence for the facts that in the Xenopus oocyte (a) the formation of the different receptor types depends on the relative abundance of cRNAs coding for the different receptor subunits and (b) that functional dual subunit combinations alphabeta do not form in the presence of cRNA coding for the gamma subunit.     

Identification of the Goodpasture antigen as the alpha 3(IV) chain of collagen IV

The organizational relationship between the recently identified alpha 3 chain of basement membrane collagen (Butkowski, R.J., Langeveld, J.P.M., Wieslander, J., Hamilton, J., and Hudson, B.G. (1987) J. Biol. Chem. 262, 7874-7877) and collagen IV was determined. This was accomplished by the identification of subunits in hexamers of the NC1 domain of collagen IV that were immunoprecipitated with antibodies prepared against subunits M1, corresponding to alpha 1(IV)NC1 and alpha 2(IV)NC1, and M2, corresponding to alpha 3NC1, and by amino acid sequence analysis. The presence of at least two distinct types of hexamers was revealed, one enriched in M1 and the other enriched in M2, but in both types, M1 and M2 coexist. Evidence was also obtained for the existence of heterodimers comprised of M1 and M2. These results indicate that M2 is an integral component of the NC1 hexamer of collagen IV. The amino acid sequence of the NH2-terminal region of M2 was found to be highly related to the collagenous-NC1 junctional region of the alpha 1 chain of collagen IV. Therefore, M2 is designated alpha 3(IV)NC1 and its parent chain alpha 3(IV). These findings lead to a new concept about the structure of collagen IV: namely, 1) collagen IV is comprised of a third chain (alpha 3) together with the two classical ones (alpha 1 and alpha 2); the alpha 3(IV) chain exists within the same triple-helical molecule together with the alpha 1(IV) and alpha 2(IV) chains and/or within a separate triple-helical molecule, exclusive of alpha 1(IV) and alpha 2(IV) chains, but connected through the NC1 domains to the classical triple-helical molecule comprised of alpha 1(IV) and alpha 2(IV) chains. Additionally, a portion of those triple-helical molecules exclusive of alpha 1(IV) and alpha 2(IV) chains may be connected to each other through their NC1 domains; and 3) the epitope to which the major reactivity of autoantibodies are targeted in glomerular basement membrane in patients with Goodpasture syndrome is localized to the NC1 domain of the alpha 3(IV) chain.     

Synthesis and processing of the dimorphic forms of rat alpha 2u-globulin

alpha 2u-Globulin the androgen-dependent male rat urinary protein, can be resolved into two distinct molecular forms by SDS-polyacrylamide slab gel electrophoresis. These two forms designated as alpha 2u-A (M, 18,800) and alpha 2u (Mr 18,100) are found both in urine and in the liver cells. Translation of rat liver mRNA in the rabbit reticulocyte lysate produced two preprotein forms of alpha 2u-globulin, designated as alpha 2uA' (Mr 20,300) and alpha 2uB' (Mr 19,600). Cell-free translation of rat liver mRNA in the presence of dog pancreas microsomal membrane or in Xenopus oocytes produced the two processed forms of alpha 2u-globulin (alpha 2uA and alpha 2uB). Quantitation of alpha 2uA and alpha 2uB within the in vitro translation products of the hepatic mRNA from albino rats of Yale, Sprague-Dawley and Fischer strains showed genetic differences in the proportion of translatable mRNA for alpha 2uA and alpha 2uB. The ratio of alpha 2uA: alpha 2uB in the translation products of liver mRNA from Yale rats was found to be 1:2.5 while in the case of both Sprague-Dawley and Fischer rats, the ratio was 1:4. A small portion of the alpha 2uA and alpha 2uB synthesized in the cultured hepatocytes, in the Xenopus oocytes or in the membrane-supplemented cell-free system appeared as two additional forms, designated as alpha 2uA" (Mr 21,200) and alpha 2uB" (Mr 20,600). Unlike alpha 2uA and alpha 2uB both alpha 2uA" and alpha 2uB" were found to bind to Con A-Sepharose, suggesting their glycoprotein nature.     

Phenoxybenzamine binding reveals the helical orientation of the third transmembrane domain of adrenergic receptors

Phenoxybenzamine (PB), a classical alpha-adrenergic antagonist, binds irreversibly to the alpha-adrenergic receptors (ARs). Amino acid sequence alignments and the predicted helical arrangement of the seven transmembrane (TM) domains suggested an accessible cysteine residue in transmembrane 3 of the alpha(2)-ARs, in position C(3.36) (in subtypes A, B, and C corresponding to amino acid residue numbers 117/96/135, respectively), as a possible site for the PB interaction. Irreversible binding of PB to recombinant human alpha(2)-ARs (90 nm, 30 min) reduced the ligand binding capacity of alpha(2A)-, alpha(2B)-, and alpha(2C)-AR by 81, 96, and 77%. When the TM3 cysteine, Cys(117), of alpha(2A)-AR was mutated to valine (alpha(2A)-C117V), the receptor became resistant to PB (inactivation, 10%). The beta(2)-AR contains a valine in this position (V(3.36); position number 117) and a cysteine in the preceding position (Cys(116)) and was not inactivated by PB (10 microm, 30 min) (inactivation 26%). The helical orientation of TM3 was tested by exchanging the amino acids at positions 116 and 117 of the alpha(2A)-AR and beta(2)-AR. The alpha(2A)-F116C/C117V mutant was resistant to PB (inactivation, 7%), whereas beta(2)-V117C was irreversibly inactivated (inactivation, 93%), confirming that position 3.36 is exposed to receptor ligands, and position 3.35 is not exposed in the binding pocket.     

Absence of uterokinetic effects of prostaglandin F 2 alpha on oxytocin-reactive uterus in the mare

In most cyclic females, prostaglandin F(2alpha) (PGF(2alpha)) triggers a uterine motility response resembling that of oxytocin (OT). To determine if PGF(2alpha) is a uterokinetic substance in the cycling mare, uterine motility was measured by intrauterine balloon technique in 12 conscious, normally cyclic mares. After 60 min of saline infusion, continuous intravenous (i.v.) infusion with OT (1 i.u./min) was followed by PGF(2alpha) (200 mug/min) for 60 min each. The experiment was repeated 3 wk later except with PGF(2alpha) preceeding OT. A second group of mares was administered OT (60 i.u.) either i.v., intramuscularly (i.m.), or intrauterinely (i.u.). Plasma samples were studied for progesterone concentration. Control uterine motility for the first group of mares was (mean +/- SEM) 545.83 +/- 45.10 mm(2). Significant (P<0.05) elevation in uterine motility was recorded for OT (1118.60 +/- 70.56 mm(2)) regardless if PGF(2alpha) preceded OT infusion or vice-versa. No significant difference (P>0.05) was seen in motility after PGF(2alpha) (423.33 +/- 31.12 mm(2)) infusion. The uterokinetic effect of OT was greatest when OT was administered i.v. (1696.50 +/- 195.46 mm(2)) followed by i.m. (819.82 +/- 39.96 mm(2)), and it was least effective when administered i.u. (607.83 +/- 21.56 mm(2)) as compared to control uterine motility (279.78 +/- 22.33 mm(2)). Skin electrical resistance values rose from 0 to 2000 ohms with PGF(2alpha) infusion (but not with OT), indicating that PGF(2alpha) was bioactive. It was concluded that PGF(2alpha) was not a uterokinetic substance in the cyclic mare.     

Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

The presence of an N-acetylglucosaminyltransferase (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in alpha 1,6 linkage to the beta-linked mannose. GlcNAc beta 1,2Man alpha 1,3(Man alpha 1,6)Man beta 1,4GlcNAc was an excellent acceptor while Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, Man alpha 1,6(Man alpha 1,3)Man alpha 1,6(Man alpha 1,3)Man beta 1,4GlcNAc, and Man alpha 1,6(Man apha 1,3)Man alpha 1,6[GlcNAcMan alpha 1,3]Man beta 1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in beta 1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 microM and that for GlcNAcMan3GlcNAc about 16 microM.     

Expression of the alpha(2)delta subunit interferes with prepulse facilitation in cardiac L-type calcium channels

We investigated the role of the accessory alpha(2)delta subunit on the voltage-dependent facilitation of cardiac L-type Ca(2+) channels (alpha(1C)). alpha(1C) Channels were coexpressed in Xenopus oocytes with beta(3) and alpha(2)delta calcium channel subunits. In alpha(1C) + beta(3), the amplitude of the ionic current (measured during pulses to 10 mV) was in average approximately 1.9-fold larger after the application of a 200-ms prepulse to +80 mV. This phenomenon, commonly referred to as voltage-dependent facilitation, was not observed when alpha(2)delta was coexpressed with alpha(1C) + beta(3). In alpha(1C) + beta(3), the prepulse produced a left shift ( approximately 40 mV) of the activation curve. Instead, the activation curve for alpha(1C) + beta(3) + alpha(2)delta was minimally affected by the prepulse and had a voltage dependence very similar to the G-V curve of the alpha(1C) + beta(3) channel facilitated by the prepulse. Coexpression of alpha(2)delta with alpha(1C) + beta(3) seems to mimic the prepulse effect by shifting the activation curve toward more negative potentials, leaving little room for facilitation. The facilitation of alpha(1C) + beta(3) was associated with an increase of the charge movement. In the presence of alpha(2)delta, the charge remained unaffected after the prepulse. Coexpression of alpha(2)delta seems to set all the channels in a conformational state from where the open state can be easily reached, even without prepulse.     

Prevalence between different alpha subunits performing the benzodiazepine binding sites in native heterologous GABA(A) receptors containing the alpha2 subunit

The presence of two heterologous alpha subunits and a single benzodiazepine binding site in the GABA(A) receptor implicates the existence of pharmacologically active and inactive alpha subunits. This fact raises the question of whether a particular alpha subtype could predominate performing the benzodiazepine binding site. The hippocampal formation expresses high levels of alpha subunits with different benzodiazepine binding properties (alpha1, alpha2 and alpha5). Thus, we first demonstrated the existence of alpha2-alpha1 (36.3 +/- 5.2% of the alpha2 population) and alpha2-alpha5 (20.2 +/- 2.1%) heterologous receptors. A similar alpha2-alpha1 association was observed in cortex. This association allows the direct comparison of the pharmacological properties of heterologous native GABA(A) receptors containing a common (alpha2) and a different (alpha1 or alpha5) alpha subunit. The alpha2 subunit pharmacologically prevailed over the alpha1 subunit in both cortex and hippocampus (there was an absence of high-affinity binding sites for Cl218,872, zolpidem and [3H]zolpidem). This prevalence was directly probed by zolpidem displacement experiments in alpha2-alpha1 double immunopurified receptors (K(i) = 295 +/- 56 nM and 200 +/- 8 nM in hippocampus and cortex, respectively). On the contrary, the alpha5 subunit pharmacologically prevailed over the alpha2 subunit (low- and high-affinity binding sites for zolpidem and [3H]L-655,708, respectively). This prevalence was probed in alpha2-alpha5 double immunopurified receptors. Zolpidem displayed a single low-affinity binding site (K(i) = 1.73 +/- 0.54 microM). These results demonstrated the existence of a differential dominance between the different alpha subunits performing the benzodiazepine binding sites in the native GABA(A) receptors.     

Interaction of fully liganded valency hybrid hemoglobin with inositol hexaphosphate. Implication of the IHP-induced T state of human adult methemoglobin in the low-spin state

To gain further insight into the quaternary structures of methemoglobin derivatives in the low-spin state, the interaction of fully liganded valency hybrid human hemoglobins with IHP was studied by proton NMR spectroscopy. Upon addition of IHP to (alpha CO beta + N3-)2, the same resonances as the previously reported IHP-induced NMR peaks for azidomethemoglobin (alpha + N3-beta +N3-)2 appeared, whereas the binding of IHP did not significantly affect the NMR spectra for (alpha + N3-beta CO)2. The binding of IHP also brought about more pronounced spectral changes for (alpha CO beta + Im)2 and (alpha CO beta + H2O)2 than for (alpha + Im beta CO)2 and (alpha + H2O beta CO)2. Therefore, the IHP-induced NMR peaks for azidomethemoglobin are attributed to the beta heme methyl group. Such IHP-induced beta heme methyl resonances were also observed for (alpha NO beta + N3-)2, which undergoes quaternary structural change, analogously to the R-T transition by the binding of IHP. From the above results, it was suggested that the IHP-induced heme methyl resonances for azidomethemoglobin and (alpha CO beta +N3-)2 may also be associated with the quaternary structure of these Hbs, implying the presence of the IHP-induced "T-like" state in low-spin metHb A.     

Point mutation in the first transmembrane region of the beta 2 subunit of the gamma--aminobutyric acid type A receptor alters desensitization kinetics of gamma--aminobutyric acid- and anesthetic-induced channel gating

A conserved glycine residue in the first transmembrane (TM1) domain of the beta2 subunit has been identified to be involved with desensitization induced by gamma-aminobutyric acid (GABA) and anesthetics. Recombinant GABA(A) receptors expressed in Sf9 cells were recorded using semi-fast agonist application. Upon direct activation by GABA or anesthetics, the main effect of the TM1 point mutation on the beta2 subunit (G219F) was to slow the time constant (tau) of desensitization. At GABA concentrations eliciting maximum currents, the corresponding median tau values were 0.87 s (25-75% interval (0.76; 1.04 s)), 0.93 s (0.76; 1.23 s), and 1.36 s (1.17; 1.57 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than alpha1beta2gamma2 (p < 0.01) and alpha1(G223F)beta2gamma2 (p < 0.05). For pentobarbital-induced currents (500 microm), the corresponding median tau values were 1.36 s (0.81; 1.41 s), 1.47 s (1.31; 2.38 s), and 2.82 s (2.21; 5.56 s) for alpha1beta2gamma2, alpha1(G223F)beta2gamma2, and alpha1beta2(G219F)gamma2, respectively. The tau value for the beta2-mutant receptor was significantly longer than that for alpha1beta2gamma2 (p < 0.01). The present findings suggest that this TM1 glycine residue is critical for the rate at which desensitization occurs and that both GABA and intravenous anesthetics implement an analogous pathway for generating desensitization.     

Biochemical characterization of various catalytic complexes of the brain platelet-activating factor acetylhydrolase.

Brain intracellular platelet-activating factor acetylhydrolase (PAF-AH) isoform I is a member of a family of complex enzymes composed of mutually homologous alpha(1) and alpha(2) subunits, both of which account for catalytic activity, and the beta subunit. We previously demonstrated that the expression of one catalytic subunit, alpha(1), is developmentally regulated, resulting in a switching of the catalytic complex from alpha(1)/alpha(2) to alpha(2)/alpha(2) during brain development (Manya, H., Aoki, J., Watanabe, M., Adachi, T., Asou, H., Inoue, Y., Arai, H., and Inoue, K. (1998) J. Biol. Chem. 273, 18567-18572). In this study, we explored the biochemical differences in three possible catalytic dimers, alpha(1)/alpha(1), alpha(1)/alpha(2), and alpha(2)/alpha(2). The alpha(2)/alpha(2) homodimer exhibited different substrate specificity from the alpha(1)/alpha(1) homodimer and the alpha(1)/alpha(2) heterodimer, both of which showed similar substrate specificity. The alpha(2)/alpha(2) homodimer hydrolyzed PAF and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphorylethanolamine (AAGPE) most efficiently among 1-O-alkyl-2-acetyl-phospholipids. In contrast, both alpha(1)/alpha(1) and alpha(1)/alpha(2) hydrolyzed 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoric acid more efficiently than PAF. AAGPE was the poorest substrate for these enzymes. The beta subunit bound to all three catalytic dimers but modulated the enzyme activity in a catalytic dimer composition-dependent manner. The beta subunit strongly accelerated the enzyme activity of the alpha(2)/alpha(2) homodimer but rather suppressed the activity of the alpha(1)/alpha(1) homodimer and had little effect on that of the alpha(1)/alpha(2) heterodimer. The (His(149) to Arg) mutant beta, which has been recently identified in isolated lissencephaly sequence patients, lost the ability to either associate with the catalytic complexes or modulate their enzyme activity. The enzyme activity of PAF-AH isoform I may be regulated in multiple ways by switching the composition of the catalytic subunit and by manipulating the beta subunit.     

Role of calcium ion in the generation of factor XIII activity

The involvement of calcium ion in the activation of both plasma factor XIII (alpha 2 beta 2) and platelet factor XIII (alpha 2) was investigated. The second-order dependence of the rate constant for exposure of the active-site thiol group of alpha-thrombin-cleaved plasma factor XIII (alpha 2'beta 2) on the concentration of calcium ion suggested that the binding of two calcium ions is required for transformation of the alpha 2'beta 2 tetramer to enzymatically active factor XIIIa. Fibrinogen, previously reported to lower the calcium ion concentration required for efficient activation of alpha 2'beta 2 [Credo, R. B., Curtis, C. G., & Lorand, L. (1978) Proc. Natl. Acad. Sci. U.S.A. 75, 4234-4237], was found in the present study to increase the rate of exposure of the active-site thiol group. Whereas calcium ion is required for exposure of the active-site thiol group in cleaved plasma factor XIII (alpha 2'beta 2), exposure of an active-site thiol group in cleaved platelet factor XIII (alpha 2') occurs in the absence of calcium ion. The rate constant (2.2 x 10(5) M-1 s-1) for alpha-thrombin-catalyed exposure of the active-site thiol group of platelet factor XIII zymogen (alpha 2) in the presence of calcium ion was greater than the rate constant (0.7 x 10(5) M-1 s-1) determined in the absence of calcium ion.(ABSTRACT TRUNCATED AT 250 WORDS)     

The triad targeting signal of the skeletal muscle calcium channel is localized in the COOH terminus of the alpha(1S) subunit

The specific localization of L-type Ca(2+) channels in skeletal muscle triads is critical for their normal function in excitation-contraction (EC) coupling. Reconstitution of dysgenic myotubes with the skeletal muscle Ca(2+) channel alpha(1S) subunit restores Ca(2+) currents, EC coupling, and the normal localization of alpha(1S) in the triads. In contrast, expression of the neuronal alpha(1A) subunit gives rise to robust Ca(2+) currents but not to triad localization. To identify regions in the primary structure of alpha(1S) involved in the targeting of the Ca(2+) channel into the triads, chimeras of alpha(1S) and alpha(1A) were constructed, expressed in dysgenic myotubes, and their subcellular distribution was analyzed with double immunofluorescence labeling of the alpha(1S)/alpha(1A) chimeras and the ryanodine receptor. Whereas chimeras containing the COOH terminus of alpha(1A) were not incorporated into triads, chimeras containing the COOH terminus of alpha(1S) were correctly targeted. Mapping of the COOH terminus revealed a triad-targeting signal contained in the 55 amino-acid sequence (1607-1661) proximal to the putative clipping site of alpha(1S). Transferring this triad targeting signal to alpha(1A) was sufficient for targeting and clustering the neuronal isoform into skeletal muscle triads and caused a marked restoration of Ca(2+)-dependent EC coupling.     

Participation of the alpha 2(I) chain of bovine skin collagen in the formation of mature crosslinks

It is shown that regions of unreduced, insoluble cow hide collagen, represented by the peptides alpha 1(I)-CB6, alpha 2(I)-CB4 and the alpha 2(I)-CB3,5, are involved in the formation of unreducible acid-stable and mature-type crosslinks. The characteristic ratio of the CNBr peptides in soluble type I collagen was found to be changed in the insoluble collagen of cow hides. The intensity of the bands of alpha 1(I)-CB6, alpha 2(I)-CB4 and alpha 2(I)-CB3,5, shown by dodecyl sulfate polyacrylamide gel electrophoresis, is significantly reduced in such samples, which indicates an involvement of these peptides in crosslink formation. The purified highly polymeric CNBr peptide fraction was also investigated to confirm the participation of the alpha 2 chain of type I collagen in mature crosslink formation. Chymotryptic digests of such material contain peptides which originate from alpha 2(I)-CB4, alpha 2(I)-CB3,5, and alpha 1(I)-CB6. Finally, acid hydrolysates of crosslinked material were screened carefully for crosslinks down to concentrations of 1 in 1000 amino acids. Only two compounds were detected, one identified as "hydroxyaldol-histidine" and the other an as yet unknown compound. These results indicate that both the alpha 1(I) and the alpha 2(I) chains are involved in mature crosslink formation and that the polymeric CNBr peptide fraction contains components crosslinked by so far uncharacterized, nonreducible crosslinks.     

Synthesis of a repeating unit and the dimer of the repeating unit of the major chain of Shigella flexneri O-antigen polysaccharide

Methyl glycoside of the tetrasaccharide GlcNAc(beta 1-2)Rha(alpha 1-2)Rha(alpha 1-3)Rha, which represents a repeating unit of the basic chain of Shigella flexneri O-antigenic polysaccharides, was synthesized using acylated monosaccharide synthons. A dimer of the repeating unit, octasaccharide [GlcNAc(beta 1-2)Rha(alpha 1-2) Rha(alpha 1-3)Rha(alpha 1-3)]2-OMe was obtained by TrClO4-catalyzed condensation of two tetrasaccharide blocks.     

Intercellular adhesion molecule-3 binding of integrin alphaL beta2 requires both extension and opening of the integrin headpiece

The leukocyte-restricted integrin alpha(L)beta(2) is required in immune processes such as leukocyte adhesion, migration, and immune synapse formation. Activation of alpha(L)beta(2) by conformational changes promotes alpha(L)beta(2) binding to its ligands, ICAMs. It was reported that different affinity states of alpha(L)beta(2) are required for binding ICAM-1 and ICAM-3. Recently, the bent, extended with a closed headpiece, and extended with open headpiece conformations of alpha(L)beta(2), was reported. To address the overall conformational requirements of alpha(L)beta(2) that allow selective binding of these ICAMs, we examined the adhesion properties of these alpha(L)beta(2) conformers. alpha(L)beta(2) with different conformations were generated by mutations, and verified by using a panel of reporter mAbs that detect alpha(L)beta(2) extension, hybrid domain movement, or I-like domain activation. We report a marked difference between extended alpha(L)beta(2) with closed and open headpieces in their adhesive properties to ICAM-1 and ICAM-3. Our data show that the extension of alpha(L)beta(2) alone is sufficient to mediate ICAM-1 adhesion. By contrast, an extended alpha(L)beta(2) with an open headpiece is required for ICAM-3 adhesion.     

Two guanine nucleotide-binding proteins in rat brain serving as the specific substrate of islet-activating protein, pertussis toxin. Interaction of the alpha-subunits with beta gamma-subunits in development of their biological activities

Two proteins serving as substrates for ADP-ribosylation catalyzed by islet-activating protein (IAP), pertussis toxin, and binding guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinities were purified from the cholate extract of rat brain membranes. The purified proteins had the same heterotrimeric structure (alpha beta gamma) as the IAP substrates previously purified from rabbit liver and bovine brain and differed from each other in alpha only; the molecular weight of alpha was 41,000 (alpha 41 beta gamma) and 39,000 (alpha 39 beta gamma). Both were further resolved into alpha (alpha 41 or alpha 39) and beta gamma which were also purified to homogeneity to compare the activities of alpha-monomers with the original trimers. The maintenance of the rigid trimeric structure by combining alpha 41 or alpha 39 with beta gamma in the absence of Mg2+ was essential for the alpha-subunit to be ADP-ribosylated by IAP. The alpha-subunit was very stable but displayed the only partial GTP gamma S-binding activity under these conditions. Isolated alpha-monomers exhibited high GTPase activities when assayed in the presence of submicromolar Mg2+ but were very unstable at 30 degrees C and not ADP-ribosylated by IAP. The most favorable conditions for the GTP gamma S binding to alpha-subunits were achieved by combining alpha 41 or alpha 39 with beta gamma in the presence of millimolar Mg2+, probably due to the increase in stability and unmasking of the GTP-binding sites. There was no qualitative difference in these properties between alpha 41 beta gamma (alpha 41) and alpha 39 beta gamma (alpha 39). But alpha 39 beta gamma (or alpha 39) was usually more active than alpha 41 beta gamma (or alpha 41), at least partly due to its higher affinity for Mg2+ and lower affinity for beta gamma. Relation of these differences in activity between alpha 41 beta gamma and alpha 39 beta gamma to their physiological roles in signal transduction is discussed.