Equilibrium and kinetic studies of oxygen binding to the haemocyanin from the freshwater snail Lymnaea stagnalis.

The binding of oxygen by the haemocyanin of the gastropod Lymnaea stagnalis was studied by equilibrium and kinetic methods. The studies were performed under conditions in which the haemocyanin molecule was in the native state. Over the pH range 6.8-7.6, in the presence of 10mM-CaCl2 the haemocyanin bound O2 cooperatively. Over this pH range the haemocyanin molecule displayed a normal Bohr effect whereby the O2 affinity of the molecule decreased with a fall in the pH of the solution. The maximum slope of the Hill plot (hmax.) was 3.5, obtained at pH 7.5. An increase in the CaCl2 concentration from 5 to 20 mM at pH 6.8 resulted in a slight increase in the oxygen affinity, with hmax. remaining virtually unchanged. At constant pH and CaCl2 concentration, an increase in NaCl concentration from 0 to 50 mM resulted in a small decrease in O2 affinity, but a significant increase in the value of hmax. from 3.5 to 8.6. Temperature-jump relaxation experiments over a range of O2 concentrations produced single relaxation times. The dependence of the relaxation time on the reactant concentrations indicated a simple bimolecular binding process. The calculated association and dissociation rate constants for this process at pH 7.5 are 29.5 X 10(6) M-1 X S-1 and 49 S-1 respectively. The association rate constant kon was found to be essentially independent of pH and CaCl2 concentration. The dissociation rate constant, koff, however, increased with a decrease in the pH, but was also independent of CaCl2 concentration. These results indicate that the stability of the haemocyanin-O2 complex is determined by the dissociation rate constant.     

Kinetic and thermodynamic parameters for oxygen binding to the allosteric states of Panulirus interruptus hemocyanin

The temperature dependence of the oxygen binding equilibria and kinetics of Panulirus interruptus hemocyanin has been analyzed within the context of the two-state allosteric model. Oxygenation of the T-state is characterized by a more negative value of DeltaH than that of the R-state; therefore, cooperative effects in oxygen binding to P. interruptus hemocyanin are thermodynamically governed by favorable entropy changes. The allosteric transition in the unliganded derivative shows an enthalpy-entropy compensation effect. The activation enthalpies for oxygenation and deoxygenation of the T-state are larger than those for the R-state, while the activation entropies are favorable for the T-state and unfavorable for the R-state. Thus, the activation free energies for oxygen binding to the T- and R-states are similar, while for the deoxygenation reaction DeltaG++ is smaller for the T-state. The analysis reported confirms the applicability of the Monod-Wyman-Changeux two-state allosteric model to P. interruptus hemocyanin and yields a complete thermodynamic characterization of oxygen binding under both equilibrium and dynamic regimes.     

Primary structures of a second hyperglycemic peptide and of two truncated forms in the spiny lobster, Jasus lalandii

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.     

Characterization and sequence elucidation of a novel peptide with molt-inhibiting activity from the South African spiny lobster, Jasus lalandii

We have isolated a peptide from extracts of sinus glands from a South African spiny lobster species, Jasus lalandii, by high-performance liquid chromatography (HPLC) and identified it as a putative molt-inhibiting hormone (MIH) by (i) an in vitro assay with J. lalandii Y-organs to measure the inhibition of ecdysteroid synthesis and (ii) an immunoassay using antiserum raised against MIH of the edible crab. The MIH of J. lalandii has 74 amino acid residues, a molecular mass of 9006 Da, a free N-terminus and an amidated C-terminus. The full primary sequence has been obtained from sequencing various digest fragments (tryptic, endoproteinase Asp-N, cyanogen bromide) of the unreduced (native) peptide: RFTFDCPGMMGQRYLYEQVEQVCDDCYNLYREEKIAVNCRENCFLNSWFTVCLQATMREHETPRFDIWR SIILKA-NH(2). Structural comparisons with other peptides show that the J. lalandii MIH belongs to the peptide family which includes the crustacean hyperglycemic hormone, molt-inhibiting hormone and vitellogenesis-inhibiting hormone (cHH/MIH/VIH). This novel peptide has 36-43% sequence identity to putative MIHs from other decapod crustaceans and 32-34% identity to the two cHH peptides previously identified in this spiny lobster species. This is the first report of a peptide with MIH activity in the Palinuridae infraorder.     

Insulin's structural behavior and its relation to activity

This paper discusses the hypothesis that insulin undergoes a conformational change either before or during its binding to the receptor. The evidence for this is not conclusive but allows us to reconcile the following observations: (1) no chemical modification or deletion of invariant surface residues has abolished the hormone's activity—only reduced its potency. (2) Reduction in potency follows many modifications to different side chains, both variant and invariant. (3) There are insulins with perfectly preserved structure (by the criteria of aggregation, spectroscopy, and x-ray analysis) that have markedly reduced potency. (4) Insulins with disturbed structure still exhibit real, sometimes substantial activity.     

Nested allosteric interaction in tarantula hemocyanin revealed through the binding of oxygen and carbon monoxide

We have examined the competitive binding of oxygen and carbon monoxide to the multisubunit hemocyanin of the tarantula Eurypelma californicum. Employment of high-precision thin-layer methods has enabled detailed characterization of the pure oxygen and pure carbon monoxide binding curves, as well as binding curves performed under mixed-gas conditions. The pure oxygen binding curve and the displacement of oxygen by carbon monoxide at full ligand saturation are highly cooperative, but in the absence of oxygen, carbon monoxide binds noncooperatively. The results were analyzed globally within the framework of a nested allosteric model [Robert, C.H., Decker, H., Richey, B., Gill, S.J., & Wyman, J. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 1891-1895] which takes into account the hierarchy of subunit structure present in the macromolecule. The use of two ligands enables one to recognize two distinct levels of allosteric interaction functioning in the protein assembly. The binding characteristics of the allosteric states demonstrated for Eurypelma follow a similar pattern as those found earlier for Homarus americanus.     

Effects of polyvalent anion binding to hemoglobin on oxygen and oxidation-reduction equilibria and their relevance to allosteric transition.

The reaction of human hemoglobin and some of its derivatives with aliphatic and aromatic compounds carrying from two to six carboxylate groups has been studied. The effect of the polycarboxylates as well as of three co-ordinate anions (respectively tri-, tetra- and pentavalent) on the oxygenation and oxidation-reduction equilibria and optical spectra have been compared to those of 2,3-diphosphoglycerate and inositol hexaphosphate.All the polyvalent anions raise the P_0.5³ and the E_m values of human hemoglobin and are thus bound more strongly to deoxyhemoglobin than to oxy- or methemoglobin. Binding of benzene hexacarboxylate and benzene pentacarboxylate to oxyhemoglobin is demonstrated through a study of oxygenation curves, that of these reagents, and ferrocyanide, to methemoglobin through their effect on redox potential as well as on optical spectra. Methemoglobin and oxyhemoglobin are shown to bind more than one molecule of the carboxylates at high anion concentrations. Results bearing on the anion binding site for deoxy- as well as for methemoglobin are reported.Two appropriate human hemoglobin derivatives, namely Hb_BME and Hb_{NES desArg} have been examined in search of relations between the effect of anions on oxygen equilibria and that on quaternary structure: in both of these derivatives the chemical modifications inhibit quaternary conformational change that would result from oxygen binding, the deoxy structure being strongly destabilized. Several of the polyanions significantly raise the P_0.5 values of these derivatives but do not modify the quaternary structure, as judged from the absence of characteristic spectral changes. The results imply that anion binding by these proteins somehow inhibits the change in tertiary structure produced by oxygen binding; similar considerations may also apply in the case of the normal hemoglobin-diphosphoglycerate complex.     

The physiological ecology of haemocyanin in some selected crabs. II. The characteristics of haemocyanin in relation to terrestrialness

The haemocyanins of five crabs ranging in habit from aquatic to terrestrial have been investigated.

The mean P₅₀ values of the respiratory pigments were determined at 0 mm Hg CO₂ and 28 °C (the average environmental temperature of all the species). Comparison of these data adjusted to the individual mean physiological pH indicate an increase in P₅₀ with terrestrialization, perhaps related to the greater abundance of oxygen in the aerial than in some the aquatic habits, and the progressive elaboration of lung breathing with terrestrialization.

The Bohr shifts (Δ log P₅₀/ΔpH) were determined (using different P_CO2 values to vary pH) and were found to decrease with terrestrialization, perhaps in adaptation to an associated rise in internal P_CO2 (6–8-fold between the aquatic Callinectes sapidus Rathbun and the terrestrial Cardisoma guanhumi Latreille and probably resulting from progressive gill reduction.

The temperature shifts (ΔH cal/mol) of the haemoeyanins were found and it is suggested that they diminish with increasing evironmental temperature and temperature fluctuation accompanying terrestrialization.     

Analysis of oxygen binding to Panulirus japonicus hemocyanin. The effect of divalent cations on the allosteric transition

The effects of H+ and divalent cations on the O2 equilibrium of hexameric hemocyanin from a spiny lobster, Panulirus japonicus, were examined. The hemocyanin showed the normal Bohr effect. When divalent cations were removed by EDTA treatment, the protein showed a fivefold increase in the O2 affinity and a considerable decrease in the cooperativity. Several cooperativity models were tested for the conformity with the observed O2-binding isotherms by the least-square curve fitting. Among the models examined, the three-state concerted model was found to be most consistent with the results. It was postulated that in the absence of divalent cations deoxyhemocyanin is mainly in the intermediate-affinity state. The arthropod hemocyanins were found to be classifiable into two groups according to their functional responses to the divalent cations. It was suggested that the cations act differently on the allosteric transitions of the two groups of hemocyanins.     

Equilibrium and kinetic studies of oxygen binding to fragments of Lymnaea stagnalis (freshwater snail) haemocyanin obtained by proteolytic digestion.

Functional fragments of the haemocyanin from the gastropod mollusc Lymnaea stagnalis (freshwater snail) were obtained by partial digestion with trypsin and plasmin. The fragments were purified by ion-exchange chromatography and characterized by detergent/polyacrylamide-gel electrophoresis and crossed immunoelectrophoresis. Three types of single-functional unit fragment were isolated from the trypsin digest, and two immunologically distinct three-functional unit fragments and a single-functional unit fragment were isolated from the plasmin digest. The O2-binding behaviour of the fragments was investigated by equilibrium and kinetic methods. Over the pH range 7.0-8.2, in the presence of 10-20 mM-CaCl2, all of the single-functional unit fragments displayed non-co-operative O2 binding and showed no evidence of a Bohr or a salt effect. A Hill coefficient of less than 1.0 was obtained with one of the two three-functional unit fragments studied, whereas both of these fragments displayed a Bohr effect. Functional heterogeneity of the fragments was indicated by the variation in the O2 affinity, the P50 (partial pressure of O2 at half saturation) ranging between 0.26 and 0.77 kPa (approx. 2-6 mmHg). Stopped-flow data reflected the O2 equilibrium behaviour. Thus there was a fall in the value of the O2 dissociation rate constant from approx. 15 to 1s-1 in parallel with the increase in O2 affinity.     

Molecular cloning and localization of a cDNA encoding a crustacean hyperglycemic hormone from the South African spiny lobster,Jasus lalandii

A cDNA, encoding a crustacean hyperglycemic hormone (cHH) of the South African spiny lobster, Jasus lalandii has been cloned. The cDNA consists of 1773 bp with an open reading frame of 399 bp that encodes a preprohormone of 133 amino acid residues. The preprohormone consists of a 25 amino acid hydrophobic signal peptide, a 32 amino acid cHH precursor-related peptide (CPRP) and the cHH sequence of 72 amino acid residues. The cHH sequence is flanked N-terminally by a Lys-Arg cleavage site and C-terminally by Gly-Lys, where Gly serves as an amidation site. The deduced amino acid sequence of the CPRP is in complete agreement with a peptide previously elucidated from sinus glands of J. lalandii, code-named CPRP 2 and the sequence of the cHH peptide matches that of the minor cHH isoform of J. lalandii, i.e. crustacean hyperglycemic hormone-II (cHH-II), which was also previously obtained by peptide sequencing. In situ hybridization on eyestalks revealed strong cHH-II mRNA expression in a subset of neurosecretory cells of the X-organ.     

A note on a possible influence of traps on assessment of the diet of Jasus lalandii (H. Milne-Edwards)

Dietary analyses are important components of ecological studies. However, some methods of collecting organisms may expose them to exceptionally high densities of prey items, leading to inaccurate dietary assessments. These methods include the use of baited traps. We hypothesised that such a "trap effect" occurred during work on the diet of the rock lobster Jasus lalandii, because baited traps attracted isopods, which were then eaten opportunistically by trapped J. lalandii. To test this hypothesis, rock lobsters were collected at two sites using both baited-traps and Scuba diving. Results showed that large numbers of isopods were attracted to, and consumed, the trap-bait. Analyses of the stomach contents of trap-caught J. lalandii from both localities reflected a larger occurrence and significantly greater abundance of isopods in stomach samples from trap- rather than Scuba-caught rock lobsters. For probably similar reasons, small fish were significantly over-represented in the gut contents of trap-caught rock lobsters, although the evidence is less clear-cut and there may be other explanations for the high proportions of fish in the diet. Although isopods and fish may be naturally consumed at a low rate, predation on them is greatly inflated in traps, and the dietary analyses of trap-caught J. lalandii. All assessments of the diets of animals traditionally caught with traps should consider and account for such "trap effects", by calibrating the data for trap-caught animals against an alternative sampling method.     

Influence of allosteric effectors and temperature on oxygen binding properties and the Bohr effect of bovine hemoglobin

The O2 binding properties of bovine Hb were examined. The increase in Cl- and DPG concentration enhanced P50. A reduction in n(max) was observed at high Cl- concentration, while DPG had little effect on n(max). An increase in Cl- concentration enhanced the Bohr effect, the magnitude of which reached a maximum at 0.1 M Cl- and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [Cl-] plot, and also equal to the physiological Cl- concentration (0.1 M) of bovine blood. Furthermore, the influence of Cl- concentration on the Bohr effect is independent of temperature. On the other hand, in the absence of Cl-, bovine Hb is sensitive to DPG; an increase in DPG concentration enhanced the Bohr effect, which reached a maximum at 3 mM DPG and 20 degrees C. This concentration is nearly equal to that at the highest slope of the log P50 vs. log [DPG] plot. At low DPG concentrations, the DPG effect on the Bohr effect became small with increasing temperature, whereas at high DPG concentrations, the DPG effect was insensitive to temperature changes. At the physiological concentration of DPG (0.5 mM), increases in both Cl- concentration and temperature diminished the DPG effect. At the physiological concentrations of Cl- and DPG, the Bohr effect was -0.36 at 37 degrees C. The deltaH value at the physiological concentrations of Cl- and DPG was approximately -5.8 kcal/mol at pH 7.4. These results indicate that Cl- and temperature are important determinants of the O2 binding properties of bovine Hb.