Comparative Biochemistry and Physiology Part B: Comparative Biochemistry最新文献

Arginase activity (3.1 ± 0.5 units/g (wet wt) of tissue) was found associated to the cytosolic fraction of the gill cells of the bivalve Semele solida. The enzyme, with a molecular weight of 120,000 ± 3000, was partially purified, and some of the enzymic properties were were examined. The activation of the enzyme by Mn²⁺ followed hyperbolic kinetics with a K_Mn value of 0.10 ± 0.02 μM. In addition to Mn²⁺, the metal ion requirement of the enzyme was satisfied by Ni²⁺, Cd²⁺ and Co²⁺; Zn²⁺ was inhibitory to ail the Values of K_m for arginine and K_i for lysine inhibition, were the same, regardless of the metal ion used to activate the enzyme; K_m values were 20 mM at pH 7.5 and 12 mM at the optimum pH of 9.5. Competitive inhibition was caused by ornithine, lysine and proline, whereas branched chain amino acids were non competitive inhibitors of the enzyme.

The hemoglobins of three snake species: Liophis miliaris, Bothrops alternatus and Boa constrictor present a single ATP binding site per tetramer. The ATP association constant values for the deoxyhemoglobins at pH 7.5 were about K_D ≅ 10⁶ M⁻¹ (10⁷ M⁻¹ for B. contrictor), three to four orders of magnitude higher than the respective values for oxyhemoglobin of about K_O ≅ 10² M⁻¹. The deoxyhemoglobin constant values markedly decrease as a function of pH, becoming, at pH 8.5, about K_D ≅ 10³ M⁻¹ whereas for the oxyhemoglobin the constants remain of about the same, K_O ≅ 10² M⁻¹, at the pH range studied. The high ATP binding affinity constants, compared to those of human hemoglobin A, were explained from a molecular structural standpoint, considering L. miliaris hemoglobin, whose complete primary sequence is known. Two distinct amino acid residue differences were found in the β-chain, one being Trp (NA3) (more hydrophobic) in the snake hemoglobin which substitutes the Leu (NA3) in human hemoglobin, and the second being Val 101 β (G3) instead of Glu 101 β (G3). The substitutions could provide an un-neutralized, positively charged, residue Lys-104β and, taking into account its high pK value, the pH dependence of ATP binding affinity for the snake hemoglobin would originate from pH-dependent ionization of phosphate groups of the allosteric effector. The physiological implications of the high ATP binding constant, as well as the possible protective role of the nucleotide binding against the effect of high environmental temperatures on the oxygen dissociation curves, are discussed.

The fatty acyl composition of sarcoplasmic reticulum was analyzed in skeletal muscle from hibernating and aroused Richardson's ground squirrels, Spermophilus richardsonii.

Total levels of monounsaturated fatty acyl groups increased in the phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) fractions of aroused animals. Docosahexaenoic acid (22:6n-3) was lower in all phospholipids while the ratio $\text{20:4}\text{22:6}$ was higher in aroused animals.

These changes in fatty acyl composition have the potential to alter SR Ca²⁺-ATPase activity and Ca²⁺ uptake efficiency during the energetically demanding arousal process.

Flavin-containing monooxygenase (FMO) activity as N,N-dimethylaniline (DMA) N-oxygenation was characterized in microsomes from the smooth dogfish shark (Squalus acathias). DMA N-oxygenase activity from the liver of the dogfish shark was linear with increasing protein content and over 60 min. The optimal temperature for catalysis was 25°C with a 76 percent reduction in activity when incubated at 15°C and 99 percent loss of activity at 45°C. Optimal pH was approximately 9.6. The maximum velocity for DMA N-oxygenase activity was calculated to be 1.3 nmol min⁻¹ mg⁻¹ with an apparent Michaelis constant of 44 μM. Methimazole oxidase activity was also observed in dogfish liver microsomes which was inhibited by trimethylamine (TMA). Inhibition of DMA N-oxygenase activity by TMA and thiobenzamide was competitive, while inhibition by methimazole was not competitive. Western blot analysis indicated a single liver protein from both Squalus and Carcharhinus of approximately 50 kDa that bound to antibodies raised against FMO 2. An attempt was made to purify FMO as methimazole oxidase from the liver of the silky shark. A single peak of about 10-fold purity was observed following passage through two chromatographic media (CM-Sepharose and HA-Agarose). However, no activity was recoverable after the FMO-containing fractions were applied to a 2′5′ ADP-Sepharose column.

Angiotensin I-converting enzyme (ACE) was purified from lungs of pig, rat, monkey and human for comparison of its physicochemical, enzymatic and immunological properties. The protocol involved three chromatographic steps after detergent extraction, i.e. DEAE–Sphérodex ion exchange, lisinopril–Sepharose affinity and Superose 12 HPLC, plus Mono-Q HPLC for monkey ACE. Purified ACE's presented numerous homologies: in particular, closely similar specific activities, catalytic efficiencies, K_m's, optimal pH and chloride activations; the molecular weights were about 170 kDa by SDS-PAGE and 320 kDa by gel-filtration on Superose 12; the isoelectric points were about 4.5–4.7. Specific polyclonal antibodies recognized the antigen (porcine ACE) as well as rat, monkey and human ACEs. In contrast, three monoclonal antibodies (F02.4.1, F01.1.3 and F03) produced against porcine ACE showed some differences: they only reacted with pig enzyme and only one (F02.4.1) was anticatalytic. Moreover, the cross-reactivity judged on ELISA with porcine ACE characterized different epitopes specific for the porcine enzyme. In particular, the binding of F02.4.1 was not diminished by previous treatment with saturating concentrations of synthetic competitive ACE inhibitors. Thus, the extrapolation to human of data obtained on animal models should be possible at least for pharmacological and medical trials.

Malate dehydrogenase banding patterns were analyzed by polyacrylamide gel electrophoresis in Sarcophaga ruficornis, S. argyrostoma, S. dux, S. invaria and S. peregrina. Two distinct zones of MDH activity, attributed to be the product of two gene loci, namely, MDH-1 and MDH-2 were observed. The MDH-1 locus revealed polymorphism for three electrophoretic phenotypes which are governed by two electromorphs MDH-1^a and MDH-1^b. The MDH banding pattern does not reveal any notable interspecific difference.

The subunit locations of the component enzymes of the pig heart trifunctional mitochondrial β-oxidation complex are suggested by analyzing the primary structure of the large subunit of this membrane-bound multienzyme complex [Yang S.-Y.et al. (1994) Biochem. biophys. Res. Commun. 198, 431–437] with those of the subunits of the E. coli fatty acid oxidation complex and the corresponding mitochondrial matrix β-oxidation enzymes. Long-chain enoyl-CoA hydratase and long-chain 3-hydroxyacyl-CoA dehydrogenase are located in the amino-terminal and the central regions of the 79 kDa polypeptide, respectively, whereas the long-chain 3-ketoacyl-CoA thiolase is associated with the 46 kDa subunit of this complex. The pig heart mitochondrial bifunctional β-oxidation enzyme is more homologous to the large subunit of the prokaryotic fatty acid oxidation complex than to the peroxisomal trifunctional β-oxidation enzyme. The evolutionary trees of 3-hydroxyacyl-CoA dehydrogenases and enoyl-CoA hydratases suggest that the mitochondrial inner membrane-bound bifunctional β-oxidation enzyme and the corresponding matrix monofunctional β-oxidation enzymes are more remotely related to each other than to their corresponding prokaryotic enzymes, and that the genes of E. coli multifunctional fatty acid oxidation protein and pig heart mitochondrial bifunctional β-oxidation enzyme diverged after the appearance of eukaryotic cells.

Gangliosides were investigated in adult brains and in 5-vesicle stage embryos of representatives belonging to the four vertebrate classes: Chondrichthyes, Amphibia, Aves and Mammalia.

Considerable variability in brain ganglioside composition and concentration was observed among the adult vertebrates.

The ganglioside patterns of the developmentally matched vertebrate embryos were similar in that each comprised GD3 as the predominant ganglioside.

The phylogenetic conservation of abundant GD3 expression during early vertebrate ontogeny is interpreted as biochemical evidence consistent with von Baer's theory of increasing differentiation and suggests that GD3 is of critical importance for normal vertebrate development.