The tetramer-dimer dissociation equilibria (K 4,2) of several fish hemoglobins have been examined by sedimentation velocity measurements with a scanner-computer system for the ultracentrifuge and by flash photolysis measurements using rapid kinetic methods. Samples studied in detail included hemoglobins from a marine teleost, Brevoortia tyrannus (common name, menhaden); a fresh water teleost, Cyprinus carpio, (common name, carp); and an elasmobranch Prionace glauca (common name, blue shark). For all three species in the CO form at pH 7, in 0.1 M phosphate buffer, sedimentation coefficients of 4.3 S (typical of tetrameric hemoglobin) are observed in the micromolar concentration range. In contrast, mammalian hemoglobins dissociate appreciably to dimers under these conditions. The inability to detect dissociation in three fish hemoglobins at the lowest concentrations examined indicates that K 4,2 must have a value of 10(-8) M or less. In flash photolysis experiments on very dilute solutions in long path length cells, two kinetic components were detected with their proportions varying as expected for an equilibrium between tetramers (the slower component) and dimers (the faster component); values of K 4,2 for the three fish hemoglobins in the range 10(-9) to 10(-8) M were calculated from these data. Thus, the values of K 4,2 for liganded forms of the fish hemoglobins appear to be midway between the value for liganded human hemoglobin (K 4,2 approximately 10(-6) M) and unliganded human hemoglobin (K 4,2 approximately 10(-12) M). This conclusion is supported by measurements on solutions containing guanidine hydrochloride to enhance the degree of dissociation. All three fish hemoglobins are appreciably dissociated at guanidine concentrations of about 0.8 M, which is roughly midway between the guanidine concentrations needed to cause comparable dissociation of liganded human hemoglobin (about 0.4 M) and unliganded human hemoglobin (about 1.6 M). Kinetic measurements on solutions containing guanidine hydrochloride indicated that there are changes in both the absolute rates and the proportions of the fast and slow components, which along with other factors complicated the analysis of the data in terms of dissociation constants. Measurements were also made in solutions containing urea to promote dissociation, but with this agent very high concentrations (about 6 M) were required to give measureable dissociation and the fish hemoglobins were unstable under these conditions, with appreciable loss of absorbance spectra in both the sedimentation and kinetic experiments. (+info)
Brain blood flow and blood pressure during hypoxia in the epaulette shark Hemiscyllium ocellatum, a hypoxia-tolerant elasmobranch.
The key to surviving hypoxia is to protect the brain from energy depletion. The epaulette shark (Hemiscyllium ocellatum) is an elasmobranch able to resist energy depletion and to survive hypoxia. Using epi-illumination microscopy in vivo to observe cerebral blood flow velocity on the brain surface, we show that cerebral blood flow in the epaulette shark is unaffected by 2 h of severe hypoxia (0.35 mg O2 l-1 in the respiratory water, 24 C). Thus, the epaulette shark differs from other hypoxia- and anoxia-tolerant species studied: there is no adenosine-mediated increase in cerebral blood flow such as that occurring in freshwater turtles and cyprinid fish. However, blood pressure showed a 50 % decrease in the epaulette shark during hypoxia, indicating that a compensatory cerebral vasodilatation occurs to maintain cerebral blood flow. We observed an increase in cerebral blood flow velocity when superfusing the normoxic brain with adenosine (making sharks the oldest vertebrate group in which this mechanism has been found). The adenosine-induced increase in cerebral blood flow velocity was reduced by the adenosine receptor antagonist aminophylline. Aminophylline had no effect upon the maintenance of cerebral blood flow during hypoxia, however, indicating that adenosine is not involved in maintaining cerebral blood flow in the epaulette shark during hypoxic hypotension. (+info)
Mechanics of ventilation in swellsharks, Cephaloscyllium ventriosum (Scyliorhinidae).
A simple two-pump model has served to describe the mechanics of ventilation in cartilaginous and bony fishes since the pioneering work of G. M. Hughes. A hallmark of this model is that water flow over the gills is continuous. Studies of feeding kinematics in the swellshark Cephaloscyllium ventriosum, however, suggested that a flow reversal occurred during prey capture and transport. Given that feeding is often considered to be simply an exaggeration of the kinematic events performed during respiration, I investigated whether flow reversals are potentially present during respiration. Pressure and impedance data were coupled with kinematic data from high-speed video footage and dye studies and used to infer patterns of water flow through the heads of respiring swellsharks. Swellsharks were implanted with pressure transducers to determine the pattern and magnitude of pressures generated within the buccal and parabranchial (gill) cavities during respiration. Pressure traces revealed extended periods of pressure reversal during the respiratory cycle. Further, impedance data suggested that pressures within the buccal and parabranchial cavities were not generated by the cyclic opening and closing of the jaws and gills in the manner previously suggested by Hughes. Thus, the classic model needs to be re-evaluated to determine its general applicability. Two alternative models for pressure patterns and their mechanism of generation during respiration are provided. The first depicts a double-reversal scenario common in the swellshark whereby pressures are reversed following both of the pump stages (the suction pump and the pressure pump) rather than after the pressure-pump stage only. The second model describes a scenario in which the suction pump is insufficient for generating a positive pressure differential across the gills; thus, a pressure reversal persists throughout this phase of respiration. Kinematic analysis based on high-speed video footage and dye studies, however, suggested that during respiration, as opposed to feeding, distinct flow reversals do not result from the pressure reversals. Thus, water is probably pooling around the gill filaments during the long periods of pressure reversal. (+info)
Electrical parameters of the isolated cornea of the dogfish, Squalus acanthias.
The electrical potential difference and electrical resistance of the nonswelling cornea of the dogfish, Squalus acanthias, were examined. It was found that routine procedures used in the procurement of fish invariably produce damage to the corneal epithelium which affects electrical measurements and possibly composition of the aqueous humor. We found no electrical evidence of ionic pumps in the corneal epithelium of this elasmobranch. The electrical resistance of corneas with apparently well-preserved epithelium was 300omega-cm.2 (compared to 30omega-cm.2 in corneas with damaged epithelium). (+info)
Substitution rates of organelle and nuclear genes in sharks: implicating metabolic rate (again).
Rates of nucleotide substitution for nuclear genes are thought to be governed primarily by the number of germ line replication events (the so-called "generation time" hypothesis). In contrast, rates of mitochondrial DNA evolution appear to be set primarily by DNA damage pathways of mutation mediated by mutagenic by-products of oxidative phosphorylation (the so-called "metabolic-rate" hypothesis). Comparison of synonymous substitution rates estimated for the mitochondrial cytochrome b gene and nuclear-encoded dlx, hsp70, and RAG-1 genes in mammals and sharks shows that rates of molecular evolution for sharks are approximately an order of magnitude slower than those for mammals for both nuclear and mitochondrial genes. In addition, there is significant positive covariation of substitution rate for mitochondrial and nuclear genes within sharks. These results, interpreted in light of the pervasiveness of DNA damage by mutagenic by-products of oxygen metabolism to both nuclear and mitochondrial genes and coupled with increasing evidence for cross-genome activity of DNA repair enzymes, suggest that molecular clocks for mitochondrial and nuclear genes may be set primarily by common mutational mechanisms. (+info)
Rate determination in phosphorylation of shark rectal Na,K-ATPase by ATP: temperature sensitivity and effects of ADP.
Phosphorylation of shark rectal Na,K-ATPase by ATP in the presence of Na(+) was characterized by chemical quench experiments and by stopped-flow RH421 fluorescence. The appearance of acid-stable phosphoenzyme was faster than the rate of fluorescence increase, suggesting that of the two acid-stable phosphoenzymes formed, RH421 exclusively detects formation of E(2)-P, which follows formation of E(1)-P. The stopped-flow RH421 fluorescence response to ATP phosphorylation was biphasic, with a major fast phase with k(obs) approximately 90 s(-1) and a minor slow phase with a k(obs) of approximately 9 s(-1) (20 degrees C, pH 7.4). The observed rate constants for both the slow and the fast phase could be fitted with identical second-degree functions of the ATP concentration with apparent binding constants of approximately 3.1 x 10(7) M(-1) and 1. 8 x 10(5) M(-1), respectively. Increasing [ADP] decreased k(obs) for the rate of the RH421 fluorescence response to ATP phosphorylation. This could be accounted for by the reaction of ADP with the initially formed E(1)-P followed by a conformational change to E(2)-P. The biphasic stopped-flow RH421 responses to ATP phosphorylation could be simulated, assuming that in the absence of K(+) the highly fluorescent E(2)-P is slowly transformed into the "K(+)-insensitive" E'(2)-P subconformation forming a side branch of the main cycle. (+info)
Retropositional parasitism of SINEs on LINEs: identification of SINEs and LINEs in elasmobranchs.
Some previously unidentified short interspersed repetitive elements (SINEs) and long interspersed repetitive element (LINEs) were isolated from various higher elasmobranchs (sharks, skates, and rays) and characterized. These SINEs, members of the HE1 SINE family, were tRNA-derived and were widespread in higher elasmobranches. The 3'-tail region of this SINE family was strongly conserved among elasmobranchs. The LINEs, members of the HER1 LINE family, encoded an amino acid sequence similar to that encoded by the chicken CR1 LINE family, and they contained a strongly conserved 3'-tail region in the 3' untranslated region. This tail region of the HER1 LINE family was almost identical to that of the HE1 SINE family. Thus, the HE1 SINE family and the HER1 LINE family provide a clear example of a pair of SINEs and LINEs that share the same tail region. Conservation of the secondary structures of the tail regions, as well as of the nucleotide sequences, between the HE1 SINE family and HER1 LINE family during evolution suggests that SINEs utilize the enzymatic machinery for retroposition of LINEs through the recognition of higher-order structures of the conserved 3'-tail region. A discussion is presented of the parasitism of SINEs on LINEs during the evolution of these retroposons. (+info)
The main fatty acid-binding protein in the liver of the shark (Halaetunus bivius) belongs to the liver basic type. Isolation, amino acid sequence determination and characterization.
Three fatty acid-binding proteins (FABPs) from the liver of the shark Halaetunus bivius were isolated and characterized: one of them belongs to the liver-type FABP family and the other two to the heart-type FABP family. The complete primary structure of the first FABP, and partial primary structures of the two others, were determined. The liver-type FABP constitutes 69% of the total FABPs, and its amino acid sequence presents the highest identity with chicken, catfish, iguana and elephant fish liver basic FABPs. The L-FABP protein has low affinity for palmitic and oleic acids and high affinity for linoleic and arachidonic acids and other hydrophobic ligands, all of them important for the metabolic functions of the liver. In contrast, both heart-type FABPs have the highest affinity for palmitic acid, the principal fatty acid mobilized from fat deposits for beta-oxidation. (+info)