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

The complete amino acid sequence of a basic (pI 9.0) fatty acid-binding protein purified from liver of Gallus domesticus was determined by automated Edman degradation of tryptic, CNBr/HFBA and Staphylococcus aureus protease peptides. The protein contains 125 amino acid residues which correspond to a molecular mass of 14094. The identification of the blocked N-terminus Ac-Ala required digestion of a SV-8 peptide with the acylamino acid-releasing enzyme prior to sequence analysis. Sequence comparison shows that chicken liver basic-FABP has a significant similarity to other proteins belonging to the superfamily of intracellular lipid molecule binding proteins. Moreover, these sequence data confirm that basic-FABP probably binds its substrate in a slightly different way when compared with other FABPs. Basic-FABP was submitted to the EMBL Data Library with an accession number of P80226

Highly purified peroxisomes were obtained from the liver of untreated rats, and rates of peroxisomal beta-oxidation were measured using fatty acyl-CoAs differing in chain length and degree of unsaturation. A 20–24-fold purification of peroxisomes, indicated by the specific activities of the marker enzymes catalase and urate oxidase, respectively, was obtained from crude liver homogenate using differential centrifugation techniques followed by a 30% Nycodenz gradient separation. The use of a 30% Nycodenz gradient in the final step of purification was extremely effective (e.g. 5.5-fold reduction) in removing lysosomal contamination. The rate of peroxisomal beta-oxidation with lauroyl-CoA (C12:0) as substrate was the highest of all fatty acyl-CoAs tested. Butyryl-CoA (C4:0) was not oxidized by purified peroxisomes. In general, as chain length of the fatty acyl-CoAs increased above 12 carbons, the rates of beta-oxidation decreased.

PK from Artemia embryos displayed sigmoidal kinetics with respect to phosphoenolpyruvate while saturation curves for ADP followed regular hyperbolas. The enzyme was strongly affected by ATP, alanine and fructose 1,6-bisphosphate, the latter increasing the affinity for PEP up to 10-fold. In order to assess whether Artemia PK is regulated by covalent modification, kinetic parameters were determined at various stages of development as well as after exposure of Artemia embryos to anoxic conditions. Incubation under anoxic conditions had no strong effect on the kinetic parameters of the enzyme. In addition, only a small effect of pH changing on the PK kinetic constants was observed. The results of the present study provide no evidence to suggest that Artemia PK is covalently modified in response to anoxia. Therefore, it appears that alternative mechanisms are involved in the depression of metabolic rate observed during the transition of Artemia embryos into anaerobic dormancy.

The monomer molecular mass of glucose dehydrogenase (GDH, EC 1.1.1.47) from rainbow trout liver and beef liver were estimated to be 90 kDa for both enzymes, by electrophoresis in the presence of Na-dodecyl-SO₄ (SDS). The 90-kDa proteins were partially degraded to about 60 kDa when purified with a delayed procedure without protease inhibitors. Tryptic cleavage of the 90-kDa proteins gave fragments of about 60 kDa and 30 kDa, being similar for trout and beef GDH. Isoelectric points, kinetic and thermodynamic properties of the two enzymes are markedly different. Triton X-100 stimulated and stabilized the reactions catalysed by the purified enzymes.

Lactate dehydrogenase (LDH) is highly active in filariae and could be a valuable tool for phyllogeny studies. Unfortunately, the isoenzymatic diagnosis of filariae is often difficult for LDH because of a poor mobility of the enzymes in starch gels which are the most commonly used in such studies. We propose here a method to separate filarial LDH isoenzymes using disc electrophoresis. The experiments were carried out on male and female Molinema dessetae in order to compare their respective isoenzymes. The study of several parameters such as buffer systems, percentage of bisacrylamide and progression time led to optimize the enzyme separation. LDH from male and female filariae were compared to mammal LDH-H4 and LDH-M4. Five and four LDH isoenzymes were found, respectively, in male and female worms. Relative concentration of each isoenzyme diverged between male and female worms. Mammal muscle LDH-M4 type moved between LDH2 and LDH3 from female worms, and between LDH1 and LDH2 from male worms. Mammal heart H4 type enzyme was very different in electrophoretic mobility. The ratio of each isoenzyme was determined by densitometry. The major isoenzymes from female worms will be studied as a biochemical target for chemotherapeutic attack.

The fatty acid composition of the sponge Hymeniacidon sanguinea from the Black Sea has been determined by methods involving silver ion HPLC and GC-MS. More than a hundred different fatty acids were identified, of which many were similar to those in sponges from tropical seas. By contrast, some of the fatty acids identified, including trans-6-hexadecenoic acid and 5,15-tetracosadienoic acid, may not have been found previously in sponges and other marine sources, and perhaps are new to science.

Liver nucleotides (ATP, ADP, AMP, IMP), the adenylate energy charge (AEC), total adenylate concentration (TA), and IMP-load were used as measures of stress in rainbow trout (Oncorhynchus mykiss) acclimated to normoxic (10.0 mg/l), hypoxic (6.5 mg/l), and supersaturated (13.0 mg/l) dissolved oxygen concentrations and subjected to a challenge by confinement. Liver ATP (783.0 nmol/g) was significantly different in the normoxic fish compared to either hyperoxic (447.7 nmol/g) or hypoxic (402.0 nmol/g) fish at the end of the confinement. Within 6.0 hr in the confinement, liver AEC in the normoxic fish increased significantly (0.58) compared to hypoxic (0.42) and hyperoxic fish (0.42). Similarly, the IMP-load in normoxic fish (0.16) decreased to near prestress levels by 6.0 hr in confinement compared to either the hypoxic (0.31) or hyperoxic (0.30) fish. Nucleotides in liver were significantly affected by the dissolved oxygen treatments and the confinement stress in contrast to the muscle nucleotides which were not.

In the deep-lake invertebrate, Acanthogammarus grewingkii a number of unusual fatty acids such as cis-11,12-methylene-5-eicosenoate, cis,cis-11,12–14,15-bis-methylene-5-eicosenoate, and their homologues, were identified. The structures were determined from spectra (¹HNMR, ¹³CNMR, mass, IR) after their isolation and quantification by means of Ag⁺-TLC, semipreparative RP-HPLC and capillary GC-MS. The possibility of biosynthesis and function of these unusually fatty acids is discussed, e.g. they may represent a specific feature of animals dwelling in a deep lake or may also be metabolites that are formed in cellular membranes or be of dietary origin.

The blood of the domestic pigeon, Columba livia, was examined for its haematologieal characteristics and chemical constituents with a view to finding if there existed sexually oriented differences. The values obtained, such as red blood cell (RBC) count, white blood cell (WBC) count, cholesterol, uric acid and protein, were within the range of those reported for other avian species. However, mean corpuscular volume (MCV), haemoglobin content (Hb), blood volume, plasma glucose and length/width (L/W) ratio of red blood cells were found to be higher in pigeon blood. Values for haematocrit (Hct), RBC count, Hb, MCH, cytosome and nuclear length, nuclear volume, and cytosome and nuclear L/W ratio of RBC were significantly higher in males than females. Similarly, plasma levels of sodium, potassium, protein, albumin and uric acid were also higher in males than females. In contrast, MCV was significantly higher in females than males.

The fatty acid composition of various phospholipid classes, e.g. phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines and sphingomyelins, of the fish endoparasite Paratenuisentis ambiguus and of intestinal tissue of its host, eel (Anguilla anguilla), were investigated. Phospholipids from parasite and infected host intestine show considerable differences in the fatty acid compositions. High proportions of saturated long-chain and very long-chain acyl moieties were found in phosphatidylcholines, ethanolamines and serines of the parasite. Membrane phospholipids of P. ambiguus contain remarkably high proportions of polyunsaturated acyl moieties, in particular eicosapentaenoyl moieties (20:5 n-3), which undoubtedly originate from aquatic feed or host intestine. It is suggested that the parasite's membranes are stabilized by incorporation of high proportions of saturated long-chain and very long-chain fatty acids to counterbalance the destabilizing effects of the polyunsaturated fatty acids.