An Origin of Cooperative Oxygen Binding of Human Adult Hemoglobin: Different Roles of the α and ß Subunits in the α2ß2 Tetramer.

Human hemoglobin (Hb), which is an α2ß2 tetramer and binds four O2 molecules, changes its O2-affinity from low to high as an increase of bound O2, that is characterized by 'cooperativity'. This property is indispensable for its function of O2 transfer from a lung to tissues and is accounted for in terms of T/R quaternary structure change, assuming the presence of a strain on the Fe-histidine (His) bond in the T state caused by the formation of hydrogen bonds at the subunit interfaces. However, the difference between the α and ß subunits has been neglected. To investigate the different roles of the Fe-His(F8) bonds in the α and ß subunits, we investigated cavity mutant Hbs in which the Fe-His(F8) in either α or ß subunits was replaced by Fe-imidazole and F8-glycine. Thus, in cavity mutant Hbs, the movement of Fe upon O2-binding is detached from the movement of the F-helix, which is supposed to play a role of communication. Recombinant Hb (rHb)(αH87G), in which only the Fe-His in the α subunits is replaced by Fe-imidazole, showed a biphasic O2-binding with no cooperativity, indicating the coexistence of two independent hemes with different O2-affinities. In contrast, rHb(ßH92G), in which only the Fe-His in the ß subunits is replaced by Fe-imidazole, gave a simple high-affinity O2-binding curve with no cooperativity. Resonance Raman, 1H NMR, and near-UV circular dichroism measurements revealed that the quaternary structure change did not occur upon O2-binding to rHb(αH87G), but it did partially occur with O2-binding to rHb(ßH92G). The quaternary structure of rHb(αH87G) appears to be frozen in T while its tertiary structure is changeable. Thus, the absence of the Fe-His bond in the α subunit inhibits the T to R quaternary structure change upon O2-binding, but its absence in the ß subunit simply enhances the O2-affinity of α subunit.

Involvement of propionate side chains of the heme in circular dichroism of myoglobin: experimental and theoretical analyses.

Incorporation of the heme into globin induces a prominent circular dichroism (CD) band in the Soret region. The appearance of heme optical activity is widely believed to arise from the interaction between the heme and aromatic residues of the globin. However, hemoglobin (Hb) containing the reversed heme exhibits a CD spectrum obviously different from that of native Hb, indicating that the interactions of heme side chains with globin contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native myoglobin (Mb) and those of Mb reconstituted with synthetic hemes lacking vinyl and/or propionate. Replacement of 2,4-vinyl groups with methyl induced moderate changes. In contrast, replacement of 6,7-propionate groups with carboxylate resulted in complete disappearance of the positive Soret CD band. To get theoretical basis for the contributions of 6,7-side chains on the band, we investigated the CD spectra at a time-dependent density functional theory level. In the antiparallel conformation of the 6,7-side chains, the rotational strengths were calculated to be positive, on the other hand in the parallel conformation to be negative. We also found that the weak Soret CD band in 2,4-dimethyl-6,7-dicarboxyheme can be explained by canceling between different carboxyl conformers.

The structure of a deoxygenated 400 kDa haemoglobin reveals ternary- and quaternary-structural changes of giant haemoglobins.

The quaternary structures of invertebrate haemoglobins (Hbs) are quite different from those of vertebrate Hbs. The extracellular giant Hbs of molecular masses of about 400 and 3600 kDa are composed of a dome-shaped dodecameric subassembly which consists of four individual globin subunits. Several crystal structures of 400 kDa Hbs from annelids have been reported, including structures in oxygenated and partially unliganded states, but the structure of the fully deoxygenated state has not been reported. In the present study, crystal structures of V2Hb from the tube worm Lamellibrachia satsuma have been determined in both the fully oxygenated and deoxygenated states. A glycosylation site and novel metal-binding sites for divalent cations were clearly observed with no intersubunit interactions in V2Hb. A comparison of the oxygenated and the deoxygenated forms of V2Hb reveals that the ternary- and quaternary-structural changes occur in a manner that maintains the molecular D3 symmetry. These structures suggest that the mechanisms of quaternary-structural changes between the oxy and deoxy states for the giant Hbs are identical across species.

Circular dichroism of hemoglobin and myoglobin.

While heme alone does not exhibit circular dichroism (CD) spectrum, it exhibits a prominent positive CD band in the Soret region when incorporated into apoglobin of myoglobin (Mb) and hemoglobin (Hb). The appearance of this optical activity is widely accepted to arise from the interactions between the heme and aromatic residues of the globin. However, the reversed heme orientation in Hb was found to exhibit a CD spectrum obviously different from that of native Hb, indicating that the interactions of side chains of heme with globin also contribute to the appearance of heme optical activity. We examined this possibility by comparing CD spectra of native Mb and those of Mb reconstituted with unnatural heme lacking the vinyl and/or propionate. Replacement of vinyls at the 2,4-positions with methyls induced a 30% decrease in CD intensity of the positive Soret CD band without changes of spectral shape. In contrast, the replacement of the propionate at the 6,7-positions with carboxylic acid groups resulted in almost complete disappearance of the Soret CD band. These results seem to suggest that interactions of heme side chains, especially 2,4-vinyls and 6,7-propionates, with globin, as well as the electronic coupling of the heme bands with those of intrinsic protein chromophores, contribute to the appearance of the prominent positive Soret CD band of Mb and Hb.

Electronic control of discrimination between O2 and CO in myoglobin lacking the distal histidine residue.

We analyzed the oxygen (O2) and carbon monoxide (CO) binding properties of the H64L mutant of myoglobin reconstituted with chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities, in order to elucidate the effect of the removal of the distal His64 on the control of both the O2 affinity and discrimination between O2 and CO of the protein by the intrinsic heme Fe reactivity through the electron density of the heme Fe atom (ρFe). The study revealed that, as in the case of the native protein, the O2 affinity of the H64L mutant protein is regulated by the ρFe value in such a manner that the O2 affinity of the protein decreases, due to an increase in the O2 dissociation rate constant, with a decrease in the ρFe value, and that the O2 affinities of the mutant and native proteins are affected comparably by a given change in the ρFe value. On the other hand, the CO affinity of the H64L mutant protein was found to increase, due to a decrease in the CO dissociation rate constant, with a decrease in the ρFe value, whereas that of the native protein was essentially independent of a change in the ρFe value. As a result, the regulation of the O2/CO discrimination in the protein through the ρFe value is affected by the distal His64. Thus, the study revealed that the electronic tuning of the intrinsic heme Fe reactivity through the ρFe value plays a vital role in the regulation of the protein function, as the heme environment furnished by the distal His64 does.

Relationship between the electron density of the heme Fe atom and the vibrational frequencies of the Fe-bound carbon monoxide in myoglobin.

We analyzed the vibrational frequencies of the Fe-bound carbon monoxide (CO) of myoglobin reconstituted with a series of chemically modified heme cofactors possessing a heme Fe atom with a variety of electron densities. The study revealed that the stretching frequency of Fe-bound CO (ν(CO)) increases with decreasing electron density of the heme Fe atom (ρ(Fe)). This finding demonstrated that the ν(CO) value can be used as a sensitive measure of the ρ(Fe) value and that the π back-donation of the heme Fe atom to CO is affected by the heme π-system perturbation induced through peripheral side chain modifications.

Relationship between oxygen affinity and autoxidation of myoglobin.

Studies using myoglobins reconstituted with a variety of chemically modified heme cofactors revealed that the oxygen affinity and autoxidation reaction rate of the proteins are highly correlated to each other, both decreasing with decreasing the electron density of the heme iron atom. An Fe(3+)-O(2)(-)-like species has been expected for the Fe(2+)-O(2) bond in the protein, and the electron density of the heme iron atom influences the resonance process between the two forms. A shift of the resonance toward the Fe(2+)-O(2) form results in lowering of the O(2) affinity due to an increase in the O(2) dissociation rate. On the other hand, a shift of the resonance toward the Fe(3+)-O(2)(-)-like species results in acceleration of the autoxidation through increasing H(+) affinity of the bound ligand.

Structural and oxygen binding properties of dimeric horse myoglobin.

Myoglobin (Mb) stores dioxygen in muscles, and is a fundamental model protein widely used in molecular design. The presence of dimeric Mb has been known for more than forty years, but its structural and oxygen binding properties remain unknown. From an X-ray crystallographic analysis at 1.05 Å resolution, we found that dimeric metMb exhibits a domain-swapped structure with two extended α-helices. Each new long α-helix is formed by the E and F helices and the EF-loop of the original monomer, and as a result the proximal and distal histidines of the heme originate from different protomers. The heme orientation in the dimer was in the normal mode as in the monomer, but regulated faster from the reverse to normal orientation. The dimer possessed the oxygen binding property, although it exhibited a slightly higher oxygen binding affinity (∼1.4 fold) compared to the monomer and showed no cooperativity for oxygen binding. The oxygen binding rate constant (k(on)) of the dimer ((14.0 ± 0.7) × 10(6) M(-1) s(-1)) was similar to that of the monomer, whereas the oxygen dissociation rate constant (k(off)) of the dimer (8 ± 1 s(-1)) was smaller than that of the monomer (12 ± 1 s(-1)). We attribute the similar k(on) values to their active site structures being similar, whereas the faster regulation of the heme orientation and the smaller k(off) in the dimer are presumably due to the slight change in the active site structure and/or more rigid structure compared to the monomer. These results show that domain swapping may be a new tool for protein engineering.

Near-UV circular dichroism and UV resonance Raman spectra of individual tryptophan residues in human hemoglobin and their changes upon the quaternary structure transition.

The aromatic residues such as tryptophan (Trp) and tyrosine (Tyr) in human adult hemoglobin (Hb A) are known to contribute to near-UV circular dichroism (CD) and UV resonance Raman (RR) spectral changes upon the R → T quaternary structure transition. In Hb A, there are three Trp residues per αß dimer: at α14, ß15, and ß37. To evaluate their individual contributions to the R → T spectral changes, we produced three mutant hemoglobins in E. coli; rHb (α14Trp→Leu), rHb (ß15Trp→Leu), and rHb (ß37Trp→His). Near-UV CD and UVRR spectra of these mutant Hbs were compared with those of Hb A under solvent conditions where mutant rHbs exhibited significant cooperativity in oxygen binding. Near-UV CD and UVRR spectra for individual Trp residues were extracted by the difference calculations between Hb A and the mutants. α14 and ß15Trp exhibited negative CD bands in both oxy- and deoxy-Hb A, whereas ß37Trp showed positive CD bands in oxy-Hb A but decreased intensity in deoxy-form. These differences in CD spectra among the three Trp residues in Hb A were ascribed to surrounding hydrophobicity by examining the spectral changes of a model compound of Trp, N-acetyl-l-Trp ethyl ester, in various solvents. Intensity enhancement of Trp UVRR bands upon the R → T transition was ascribed mostly to the hydrogen-bond formation of ß37Trp in deoxy-Hb A because similar UVRR spectral changes were detected with N-acetyl-l-Trp ethyl ester upon addition of a hydrogen-bond acceptor.

Liposome-encapsulated hemoglobin alleviates hearing loss after transient cochlear ischemia and reperfusion in the gerbil.

To test liposome-encapsulated hemoglobin (LEH) in transient cochlear ischemia/reperfusion as a model of sudden deafness, Mongolian gerbils were randomly assigned to receive 2 mL/kg of either low-affinity LEH (l-LEH, P5002 = 40 mm Hg), high-affinity LEH (h-LEH, P5002 = 10 mm Hg), homologous red blood cells (RBCs), or saline (each group n = 6) 30 min before 15-min occlusion of the bilateral vertebral arteries and reperfusion. Sequential changes in hearing were assessed by auditory brain response 1, 4, and 7 days after ischemia/reperfusion, when the animals were sacrificed for pathological studies. h-LEH was significantly more protective than l-LEH in suppressing hearing loss, in contrast to RBC or saline treatment, at 8, 16, and 32 kHz, where hearing loss was most severe (P < 0.05 between any two groups) on the first day after cochlear ischemia/reperfusion. Thereafter, hearing loss improved gradually in all groups, with a significant difference among groups up to 7 days, when morphological studies revealed that the inner hair cells but not the outer hair cells, were significantly lost in the groups in the same order. The results suggest that pretreatment with h-LEH is significantly more protective than l-LEH in mitigating hearing loss and underlying pathological damage, in contrast to transfusion or saline infusion 7 days after transient cochlear ischemia/reperfusion.

Molecular insight into intrinsic heme distortion in ligand binding in hemoprotein.

A pair of myoglobins containing inherently distorted alpha-ethyl-2,4-dimethyldeuteroheme or undistorted 2,4-dimethyldeuteroheme were prepared, and the functional consequence of intrinsic heme deformation was investigated. The visible absorption peaks of the myoglobin bearing the distorted heme exhibited a bathochromic shift, indicating that the heme was deformed in the protein pocket. Ligand affinities for the ferric myoglobin with the distorted heme were found to be higher than those of the myoglobin bearing the undistorted heme. The observation suggested that the iron atom was more displaced toward the proximal histidine to weaken the coordination of the water molecule. In the paramagnetic proton NMR spectrum of ferrous deoxy protein, the deformed heme caused a 3.2 ppm lower-field shift of the proximal histidine signal, supporting an enhanced iron-histidine interaction. The deformed heme in ferrous myoglobin lowered the oxygen and carbon monoxide affinities by 25- and 480-fold, respectively, and caused the cleavage of the iron-histidine bond in a fractional population of the nitric oxide derivative. These results demonstrate a distinctive controlling mechanism for ligand binding by the deformed heme. Upon the heme distortion, the iron atom is more attracted by the proximal histidine to reduce the affinity of exogenous ligands for the ferrous heme.

Effect of heme modification on oxygen affinity of myoglobin and equilibrium of the acid-alkaline transition in metmyoglobin.

Functional regulation of myoglobin (Mb) is thought to be achieved through the heme environment furnished by nearby amino acid residues, and subtle tuning of the intrinsic heme Fe reactivity. We have performed substitution of strongly electron-withdrawing perfluoromethyl (CF(3)) group(s) as heme side chain(s) of Mb to obtain large alterations of the heme electronic structure in order to elucidate the relationship between the O(2) affinity of Mb and the electronic properties of heme peripheral side chains. We have utilized the equilibrium constant (pK(a)) of the "acid-alkaline transition" in metmyoglobin in order to quantitatively assess the effects of the CF(3) substitutions for the electron density of heme Fe atom (rho(Fe)) of the protein. The pK(a) value of the protein was found to decrease by approximately 1 pH unit upon the introduction of one CF(3) group, and the decrease in the pK(a) value with decreasing the rho(Fe) value was confirmed by density functional theory calculations on some model compounds. The O(2) affinity of Mb was found to correlate well with the pK(a) value in such a manner that the P(50) value, which is the partial pressure of O(2) required to achieve 50% oxygenation, increases by a factor of 2.7 with a decrease of 1 pK(a) unit. Kinetic studies on the proteins revealed that the decrease in O(2) affinity upon the introduction of an electron-withdrawing CF(3) group is due to an increase in the O(2) dissociation rate. Since the introduction of a CF(3) group substitution is thought to prevent further Fe(2+)-O(2) bond polarization and hence formation of a putative Fe(3+)-O(2)(-)-like species of the oxy form of the protein [Maxwell, J. C.; Volpe, J. A.; Barlow, C. H.; Caughey, W. S. Biochem. Biophys. Res. Commun. 1974, 58, 166-171], the O(2) dissociation is expected to be enhanced by the substitution of electron-withdrawing groups as heme side chains. We also found that, in sharp contrast to the case of the O(2) binding to the protein, the CO association and dissociation rates are essentially independent of the rho(Fe) value. As a result, the introduction of electron-withdrawing group(s) enhances the preferential binding of CO to the protein over that of O(2). These findings not only resolve the long-standing issue of the mechanism underlying the subtle tuning of the intrinsic heme Fe reactivity, but also provide new insights into the structure-function relationship of the protein.

Differences in coordination states of substituted tyrosine residues and quaternary structures among hemoglobin M probed by resonance Raman spectroscopy.

Among the four types of hemoglobin (Hb) M with a substitution of a tyrosine (Tyr) for either the proximal (F8) or distal (E7) histidine in the alpha or beta subunits, only Hb M Saskatoon (betaE7Tyr) assumes a hexacoordinate structure and its abnormal subunits can be reduced readily by methemoglobin (metHb) reductase. This is distinct from the other three M Hbs. To gain new insight into the cause of the difference, we examined the ionization states of E7 and F8 Tyrs by UV resonance Raman (RR) spectroscopy and Fe-O(Tyr) bonding by visible RR spectroscopy. Hb M Iwate (alphaF8Tyr), Hb M Boston (alphaE7Tyr), and Hb M Hyde Park (betaF8Tyr) exhibited two extra UV RR bands at 1,603 cm(-1) (Y8a') and 1,167 cm(-1) (Y9a') arising from deprotonated (ionized) Tyr, but Hb M Saskatoon displayed the UV RR bands of protonated (unionized) Tyr at 1,620 and 1,175 cm(-1) in addition to those of deprotonated Tyr. Evidence for the bonding of both ionization states of Tyr to the heme in Hb M Saskatoon was provided by visible RR spectroscopy. These results indicate that betaE7Tyr of Hb M Saskatoon is in equilibrium between protonated and deprotonated forms, which is responsible for facile reducibility. Comparison of the UV RR spectral features of metHb M with that of metHb A has revealed that metHb M Saskatoon and metHb M Hyde Park are in the R (relaxed) structure, similar to that of metHb A, whereas metHb M Iwate, metHb M Boston and metHb M Milwaukee are in the T (tense) quaternary structure.

Functional characterization of the Bombyx mori fatty acid transport protein (BmFATP) within the silkmoth pheromone gland.

Fatty acid transport protein (FATP) is an evolutionarily conserved membrane-bound protein that facilitates the uptake of extracellular long chain fatty acids. In humans and mice, six FATP isoforms have been identified and their tissue-specific distributions suggest that each plays a discrete role in lipid metabolism in association with fatty acid uptake. While the presence of FATP homologs in insects has been demonstrated, their functional role remains to be characterized. Pheromonogenesis is defined as the dynamic period in which all machinery required for sex pheromone biosynthesis is generated and organized within the pheromone gland (PG) cells. By exploiting this unique system in the PG of the silkmoth, Bombyx mori, we found that BmFATP is predominantly expressed in the PG and undergoes up-regulation 1 day prior to eclosion. Before eclosion, B. mori PG cells accumulate cytoplasmic lipid droplets (LDs), which play a role in storing the pheromone (bombykol) precursor fatty acid in the form of triacylglycerol. RNAi-mediated gene silencing of BmFATP in vivo significantly suppressed LD accumulation by preventing the synthesis of triacylglycerols and resulted in a significant reduction in bombykol production. These results, in conjunction with the findings that BmFATP stimulates the uptake of extracellular long-chain fatty acids and BmFATP knockdown reduces cellular long-chain acyl-CoA synthetase activity, suggest that BmFATP plays an essential role in bombykol biosynthesis by stimulating both LD accumulation and triacylglycerol synthesis via a process called vectorial acylation that couples the uptake of extracellular fatty acids with activation to CoA thioesters during pheromonogenesis.

Functional evaluation of iron oxypyriporphyrin in protein heme pocket.

The iron complex of oxypyriporphyrin, a porphyrinoid containing a keto-substituted pyridine, was coupled with apomyoglobin. The reconstituted ferric myoglobin was found to be five-coordinate without iron-bound water molecules. The anionic ligands such as CN (-) and N 3 (-) bound the myoglobin with high affinities, while neutral imidazole did not. The IR observation indicated that the azide complex was pure high-spin, although the corresponding native protein was in the spin-state equilibrium. The reduced myoglobin was five-coordinate but exhibited no measurable affinity for O 2. The affinity for CO was lowered down to 1/2400 as compared with native myoglobin. These anomalies were ascribed to the deformation in the iron coordination core after the replacement of one of the four pyrroles with a larger pyridine ring. The ligand binding analyses for the ferric and ferrous myoglobin suggest that the proximal histidine pulls the iron atom from the deformed core to reduce the interaction between the iron and exogenous ligands. Similarity of the reconstituted myoglobin with guanylate cyclase, a NO-responsive signaling hemoprotein, was pointed out.

Reduction in hemoglobin-oxygen affinity results in the improvement of exercise capacity in mice with chronic heart failure.

OBJECTIVES: This study examined whether a reduction in hemoglobin-oxygen affinity improves exercise capacity in mice with heart failure. BACKGROUND: Exercise intolerance is a major determinant of quality of life in patients with chronic heart failure. One of the major goals of the treatment for chronic heart failure is to improve quality of life. METHODS: Four weeks after left coronary ligation, we transplanted bone marrow cells isolated from the transgenic mice expressing a hemoglobin variant with low oxygen affinity, Presbyterian, into the lethally irradiated mice with heart failure or administered a synthetic allosteric modifier of hemoglobin. The mice were then exercised on a treadmill. RESULTS: Four weeks after the left coronary artery ligation, mice showed cardiac dysfunction and chamber dilation, which were characteristics of heart failure. The transplantation led to a reduction in hemoglobin-oxygen affinity and an increase in oxygen supply for skeletal muscle without changes in muscle properties. The transplanted mice showed improved running performance on a treadmill despite impaired cardiac contractility. Furthermore, administration of the synthetic allosteric modifier of hemoglobin showed a similar effect. CONCLUSIONS: Allosteric modification of hemoglobin represents a therapeutic option for improving exercise capacity in patients with chronic heart failure. One mechanism of improvement in exercise capacity is enhanced oxygen delivery in the skeletal muscle.

Effect of reversed heme orientation on circular dichroism and cooperative oxygen binding of human adult hemoglobin.

We found that recombinant human adult hemoglobin (rHb A) expressed in Escherichia coli showed heterogeneity of components with the intensity of a positive CD band at 260 nm and that it could be resolved into three components (SP-1, SP-2, and SP-3) by SP-Sepharose column chromatography. 1H NMR revealed that SP-1 is identical with native Hb A, while SP-2 and SP-3 largely contain the reversed heme isomer in both the alpha and beta subunits, with contents of approximately 50 and >80% in SP-2 and SP-3, respectively. Rotation of the heme 180 degrees about the 5,15-meso axis (reversed heme) causes an interexchange of the methyl groups at positions 2 and 7 with the vinyl groups at positions 8 and 3, respectively. To examine the effect of the modification of the heme-protein contact on the structure and function of Hb A, we compared the 1H NMR, CD, and oxygen binding properties of the three components with those of native Hb A. Native Hb A exhibits a distinct positive CD band in both the near-UV and Soret regions, but rHb A with reversed heme exhibits a very weak positive CD band at 260 nm and a prominent negative CD band in the Soret region. Cooperativity, as measured by Hill's n value, decreased from 3.18 (SP-1) to 2.94 (SP-2) to 2.63 (SP-3) with an increase in the reversed heme orientation. The effect of an allosteric effector, inositol hexaphosphate (IHP), on the oxygen binding properties was also reduced in rHb A with reversed heme. These results indicate that changes in the heme-globin contact exert a discernible influence on CD spectra and cooperative oxygen binding.

Sex pheromone production in the silkworm, Bombyx mori, is mediated by store-operated Ca2+ channels.

In most female moths, pheromone biosynthesis activating neuropeptide (PBAN) regulates sex pheromone production by stimulating an influx of extracellular Ca(2+). Little is known about the plasma membrane channel or how the PBAN stimulus is communicated to the channel. Fluorescent Ca(2+) imaging techniques confirmed PBAN-induced Ca(2+) influx in the silkworm, Bombyx mori, and showed that the PBAN response is reduced with repeated stimulation. Compounds known to impact Ca(2+) signaling were examined for their effects on sex pheromone production. These experiments demonstrated that the PBAN signal is likely mediated by a store-operated channel (SOC). SOC blockers, SKF-96365 and 2-aminoethoxydiphenyl borate, abolished sex pheromone production, as did flufenamic acid, a blocker of transient receptor potential (TRP) channels. Thapsigargin mimicked the pheromonotropic effects of PBAN. Similar results were seen when PBAN-induced lipase activity was assayed. Conversely, 1-oleoyl-2-acetyl-sn-glycerol and arachidonic acid, activators of diacylglycerol-dependent Ca(2+) channels, had no effect on bombykol production.

Oxygenation properties of extracellular giant hemoglobin from Oligobrachia mashikoi.

Oxygenation properties of hemoglobin (Hb) from Oligobrachia mashikoi were extensively investigated. Compared to human Hb, Oligobrachia Hb showed a high oxygen affinity (P(50)=1.4 mmHg), low cooperativity (n =1.4), and a small Bohr effect (deltaH(+)=-0.28) at pH 7.4 in the presence of minimum salts. Addition of NaCl caused no change in the oxygenation properties of Oligobrachia Hb, indicating that Na(+) and Cl(-) had no effect. Mg(2+) and Ca(2+) remarkably increased the oxygen affinity and cooperativity. The dependence of the oxygen affinity on Ca(2+) concentration indicated that ca. 0.6 Ca(2+) per heme is bound to the protein moiety upon oxygen binding. CO(2) and a polyanion, inositol hexaphosphate, showed a null effect on the oxygenation properties. Thus, unlike the vertebrate Hbs, but like the annelid extracellular Hbs, the oxygen binding properties of Oligobrachia Hb are regulated by divalent cations which preferentially bind to the oxy form.

The Bombyx mori sex pheromone biosynthetic pathway is not mediated by cAMP.

In most moths, sex pheromone production is regulated by pheromone biosynthesis-activating neuropeptide (PBAN). How the extracellular PBAN signal is turned into a biological response has been the focus of numerous studies. In the classical scheme of signal transduction, activated G proteins relay the extracellular signal to downstream effector molecules such as calcium channels and adenylyl cyclase. The role of calcium in PBAN signaling has been clearly demonstrated, but the possible involvement of cAMP is not as straightforward. While cAMP has been shown to be necessary for PBAN signaling in most heliothine species, there has been no definitive demonstration of its role in Bombyx mori. To address this question, we used degenerate RT-PCR to clone two Gs subunits, designated P50Gs1 and P50Gs2, from B. mori pheromone gland (PG) cDNAs. The two Gs proteins were expressed in all tissues examined and were not up-regulated in accordance with adult eclosion. Even though two bands corresponding to the approximate molecular weights of P50Gs1 and P50Gs2 were detected in PG homogenates, the Gs antagonist, NF449, had no effect on sex pheromone production. Furthermore, no changes in the intracellular cAMP levels were detected following PBAN stimulation.