Comparative aspects of plasma membrane-bound enzymes in frog and rat liver

• 1.1. Plasma membranes have been isolated from frog (Rana esculenta) liver.
• 2.2. The average yield was 0.194 mg protein/g wet liver.
• 3.3. The activities of plasma membrane-bound enzymes (Na⁺-K⁺-ATPase and 5'-nucleotidase as well as of (Mg²⁺)-ATPase have been determined in the liver homogenate and in isolated plasma membranes.
• 4.4. (Na⁺-K⁺-ATPase, 5'-nucleotidase and (Mg²⁺)-ATPase activities of frog liver are compared with the same enzymatic activities observed in rat liver.

     

Chemical structures of mono-, di-, tri- and tetraglycosyl glycerides in rice bran

• 1.1. Monoglycosyl monoglyceride, mono-, di-, tri- and tetraglycosyl diglycerides were isolated from rice bran and characterized for their chemical structures.
• 2.2. Monoglycosyl monoglycerides were characterized as Gal(β1' → 3)-1- or 2-monoacyl-sn-glycerol and Glc(β1' → 3)-1- or 2-monoacyl-sn-glycerol.
• 3.3. The structures ofmonoglycosyl diglyceride were Gal(β1' → 3)-1,2-diacyl-sn-glycerol and Glc(β1' → 3)-1,2-diacyl-sn-glycerol. Epimeric separation of the galactosyl and glucosyl glycerides was for the first time achieved by thin-layer chromatography.
• 4.4. The main diglycosyl diglyceride was shown to be Gal(α1' → 6')-Gal(β1' → 3)-1,2-diacyl-sn-glycerol.
• 5.5. The major structure of triglycosyl diglyceride was characterized as Gal(α1″' → 6″)-Gal(α1″ → 6')-Gal(β1' → 3)-1,2-diacyl-sn-glycerol.
• 6.6. The representative structure of tetraglycosyl diglyceride was for the first time established as Gal(α1″ → 6″')-Gal(α1″' α 6″)-Gal(α1″ → 6')-Gal(βl' → 3)-1, 2-diacyl-sn-glycerol.

     

Why,when and how does the poly(A) tail shorten during mRNA translation?

• 1.1. The length of the poly(A) tail at the 3'-end of mRNA may control protein synthesis by bringing the 3'-end in close proximity to the 5'-end of the noncoding region as well as increasing the duration of mRNA translation by its binding to the poly(A) binding protein.
• 2.2. The rate-limiting step in the decay of the body of the message is the shortening of a long poly(A) tail during mRNA translation. The shortening of the poly(A) tail occurs during pre-elongation in the protein synthesis cycle.
• 3.3. The shortening of the poly(A) tail during mRNA translation may not involve RNase activity, however poly(A) binding protein seems to play a role, at least in part, in shortening of the poly(A) tail.

     

Nucleoside monophosphoramidate hydrolase from rat liver: Purification and characterization

• 1.1. Adenosine 5'-phosphoramidate hydrolase of 29 kDa was isolated from rat liver cytosol.
• 2.2. It consisted of two subunits of 14 kDa.
• 3.3. It hydrolyzed nucleoside 5'-monophosphoramidates into nucleoside 5'-monophosphates and ammonia, while it did not hydrolyze adenylyl phosphoramidate, adenylyl imidodiphosphate and N-phosphorylated compounds like phosphocreatine, N^ω-phosphoarginine, 6-phospholysine and 3-phosphohistidine.
• 4.4. Divalent cations and cyclic AMP had no effect on the hydrolytic activity.

     

Troponin C of the Antarctic icefish (Champsocephalus gunnari) white muscle

• 1.1. The troponin C (TN-C) from the Antarctic icefish (_{Champsocephalus gunnari}) has been isolated to homogeneity by a procedure involving extraction from acetone powder, DEAE-Sepharose 4B and AcA 54 column chromatography.
• 2.2. The calcium-induced conformational changes of apo TN-C have been studied by absorption difference spectroscopy, circular dichroism and intrinsic fluorescence.
• 3.3. The results indicate that the overall characteristics of icefish TN-C (such as amino acid composition, modifications of helix content and of the microenvironment of aromatic residues as a function of calcium binding) are quite similar to those of rabbit TN-C.
• 4.4. The intrinsic fluorescence properties are close to those reported for pike and carp TN-C.

     

Thermoreceptor distribution in different skin layers and its significance for thermoregulation

• 1.1.|The activity pattern of 50 cold receptors of the rabbit nose back skin was investigated.
• 2.2.|The latency of the response of individual cold receptors to identical cold stimuli varied between 0.8 ± 0.3 to 29.4 ± 4.5 s; maximal firing rates are attended after 5.5 ± 0.5 to 72.2 ± 6.2 s. Characteristic phasic responses are only demonstrated by short latency receptors.
• 3.3.|The results suggest that cold receptors are distributed throughout the skin of the rabbit's nose.
• 4.4.|Changes of temperature gradients between different skin layers were measured at different ambient temperatures.
• 5.5.|It is suggested that cold receptors might indicate heat flow through the skin.

     

Characterization of two major non-histone protein fractions from chromatin of sea urchin embryos

• 1.1. Chromatin of sea urchin embryos was chromatographed on a hydroxyapatite column, and the two largest fractions of proteins desorbed with 0.05 M and 0.2 M Na-phosphate, pH 6.8, were comparatively characterized.
• 2.2. The mol. wt ranges of these fractions were about 18,000 and 14,000 daltons, respectively, at both molarities of the phosphate buffer.
• 3.3. Both fractions at each elution molarity were found to be acidic, and to differ somewhat in their respective amino acid compositions. No important differences related to the stage of development could be detected in this respect.
• 4.4. Upon re-electrophoresis both large fractions resolved in one major and three minor bands. The amino acid composition of the major band was different for the blastula and gastrula chromatin.
• 5.5. It is likely that these fractions contain glycoprotein subunits.

     

Properties of sweat,burro (Equus asinus) and man

• 1.1. Sweat of two burros (Equus asinus) was collected after two desert walks, one at 38°C, one at 41°C.
• 2.2. Sweat from one burro was about twice as concentrated as from the other. In that respect and in respect to concentrations of chloride, sodium and potassium their sweat was like that of man.
• 3.3. Low concentrations of bicarbonate were present in burros' sweat contrasted with little if any in man's.
• 4.4. Urea nitrogen plus ammonia nitrogen were found in higher concentrations in burros' sweat than in man's sweat.

     

Distribution of beta-adrenergic binding in fractionated porcine adipocytes

• 1.1. The subcellular distribution of the porcine adipocyte beta-adrenergic receptor was studied in fractionated adipocytes.
• 2.2. The 30,000 g pellet obtained from hypotonically lysed cells contained membrane vesicles and mitochondria; it yielded approx 200–300 fmol dihydroalprenolol-bound receptors/mg protein.
• 3.3. Activity was increased to about 1000 fmol/mg protein after isolation of a plasma membrane fraction on a Percoll gradient.
• 4.4. The 5'-nucleotidase, succinate dehydrogenase and lactate dehydrogenase activities were usually enriched in compartments different from the ligand-binding activity.
• 5.5. Activity of porcine adipocyte 5'-nucleotidase, a purported plasma membrane marker enzyme, was not distributed in the same manner as the beta-adrenergic receptor.

     

The influence of environmental and incubation temperature on fatty acid synthetase from flounder (Platichthys flesus L.) liver

• 1.1. Fatty acid synthetase from liver of cold and warm adapted flounder and rabbit was purified to homogenity and compared.
• 2.2. The mol. wt of the cold and warm flounder enzyme was estimated to be about 457,000.
• 3.3. The kinetic properties were found to be similar for warm and cold adapted flounder liver enzyme and not different from the rabbit liver enzyme when measured at 5, 10, 15, 20 and 37°C.
• 4.4. Palmitic acid was the main product of both the flounder and rabbit enzyme, but significant amounts of butyric acid were also synthesized. The product composition did not change for any of the enzymes tested when the incubation temperature was changed.
• 5.5. It was concluded that fatty acid synthetase from flounder liver is similar to mammalian fatty acid synthetase with regard to molecular weight and kinetic properties.

     

Metabolic rate and body temperature of adult and juvenile hyrax (Procavia capensis)

• 1.1. Resting metabolic rates (RMR) below thermoneutrality in adult hyrax acclimated to 26, 15 and 10°C remained unchanged, i.e. thermal conductance (K) remained constant.
• 2.2. Conductance in juveniles decreased with acclimation to lower ambient temperatures (T_a).
• 3.3. Body temperature (T_b) dropped by 3.8°C in adults exposed to T_a of 30 – 5°C. The decrease was constant.
• 4.4. Body temperature fell by 1.5°C in juveniles exposed to T_a of 30 – 20°C but stabilized between 20 and 5°C.
• 5.5. The labile T_b, associated with behavioural strategies and lower than predicted RMR, can be seen as an energy-conserving mechanism of particular importance during winter conditions.

     

Lysosomal acid deoxyribonuclease from vervet monkey livers—II: Enzymic properties

• 1.1. Primate liver lysosomal acid DNase is an endonucleolytic enzyme.
• 2.2. The enzyme has both 3'- and 5'-nucleotidohydrolase activities.
• 3.3. The oligonucleotides produced by DNase are polymers mainly about 30 mononucleotides long.
• 4.4. The Arrhenius plot shows a discontinuity with a transition temperature at 47°C, with an activation energy of 107 kJ/mol below and 67 kJ/mol above this temperature.
• 5.5. The activation enthalpy is 104kJ/mol and the entropy −0.498 kJ/mol/K.
• 6.6. The enzyme is subject to substrate inhibition and the K_m value is 159 × 10⁻³mM DNA-P.

     

Cloning and characterization of rabbit pancreatic colipase

• 1.1. Among the digestive enzymes synthesized by pancreas, lipase is the principle lipolytic enzyme which hydrolyses dietary glycerides.
• 2.2. For its action it requires a coenzyme, colipase.
• 3.3. The molecular mechanisms of the interaction of these two are not fully understood.
• 4.4. Further, molecular events that regulate and influence lipid absorption are ill denned.
• 5.5. The rabbit is the conventional animal model for the study of lipid absorption. We have undertaken the molecular cloning, and characterization of rabbit pancreatic colipase, the coenzyme for pancreatic lipase.
• 6.6. Colipase has been cloned from a gt 11 library of an adult rabbit pancreatic cDNA by probing with an oligonucleotide derived from human colipase sequence.
• 7.7. The total reading frame consists of 321 nucleotides coding for 90 amino acids of the functional protein and 17 nucleotides of the leader peptide.
• 8.8. Northern blot analysis revealed a distinct band around 0.5kb. Comparison with other species revealed an over all homology of 75% at the nucleotide level.
• 9.9. At the amino acid level highest conservation is observed at the lipase-binding region (AA 53–73).
• 10.10. Rabbit enzyme also retained the N-terminal pentapeptide of it preform.
• 11.11. The regions of homology and conservation may aid to define the sites of interaction of colipase with lipase.

     

Study of yeast D-glyceraldehyde-3-phosphate3 dehydrogenase with the use of a fluorescent probe, 1-anilino-8-naphthalene sulfonate

• 1.1. The effect of NAD⁺ and its fragments, adenosine and 5'-AMP on the interaction of yeast d-glyceraldehyde-3-phosphate dehydrogenase with l-anilino-8-naphthalene sulfonate has been studied.
• 2.2. A competitive relationship between NAD⁺ and the dye has been demonstrated using fluorimetric technique; in the case of adenosine and 5'-AMP a direct method of analytical ultracentrifugation was employed.
• 3.3. The results obtained suggest the dye binding at the adenine subsite of the dehydrogenase.
• 4.4. The fluorescence of 1-anilino-8-naphthalene sulfonate bound to d-glyceraldehyde-3-phosphate dehydrogenase increases and the emission maximum shifts to shorter wavelengths on addition of nicotinamide mononucleotide.
• 5.5. This suggests some conformational changes to occur in the micro-environment of the bound dye in response to the interaction with the ligand in the nicotinamide subsite.
• 6.6. A quantitative relationship between the nicotinamide mononucleotide concentration on one hand, and the fluorescence change on the other, was used to determine the dissociation constant of the ligand, which was found to be 13.0 mM (0.1 M glycine buffer pH 8.5 at 20°C).

     

Isolation and characterization of glycosaminoglycans from atria of the human heart

• 1.1. Five glycosaminoglycans were isolated from tryptic digest of atria of the human heart and were assayed by determining the carbohydrate content of materials.
• 2.2. Separation of these 5 polymers was achieved by Dowex 1 × 2 column chromatography.
• 3.3. They were identified as hyaluronic acid, heparan sulfate, chondroitin-4-sulfate, dermatan sulfate and chondroitin-6-sulfate, respectively.

     

Amino acid substitutions within the analogous nucleotide binding loop (P-loop) of aminoglycoside 3'-phosphotransferase-II

• 1.1. Oligonucleotide-directed mutagenesis of APH(3')-II was used to investigate the functions of key amino acids in the P-loop analogous motif of the enzyme.
• 2.2. The mutations of Gly205 → Glu, Gly210 → Ala and Arg211 → Pro considerably reduced the resistance of the resulting strains to KM and to related drugs, e.g. G418.
• 3.3. Similarly, enzyme activity in the crude extracts of these mutants was substantially reduced as well as the enzyme's affinity for Mg²⁺ ATP.
• 4.4. Alternatively substitutions at a highly conserved basic residue (Arg211 → Lys and Arg211 → His) were not sufficient for the enzyme to sustain the activity at a level comparable to that of the wildtype.
• 5.5. Moreover, an Arg211 → His mutation drastically reduced affinity of the enzyme for Mg²⁺ ATP.
• 6.6. This argues the importance of Arg211 residue in contributing to the formation of the P-loop structure in addition to its involvement in phosphoryl transfer reaction.
• 7.7. Computer analysis of the secondary structure predicted that the APH(3')-II loop connects a β -strand to an α-helix and that the above mutations caused varying degrees of structural distortions at the corresponding regions of the protein.

     

Purification of the flavin-containing monooxygenase from mouse and pig liver microsomes

• 1.1. The microsomal flavin-containing monooxygenase has been purified from mouse and pig liver utilizing Cibacron-Blue Sepharose, Procion-Red agarose, and 2'5'-ADP Sepharose.
• 2.2. The enzymes had a final specific activity of 1200 and 954 nmol/min/mg protein from mouse and pig liver respectively.
• 3.3. The enzyme from both mouse and pig liver displayed typical flavoprotein spectra and appeared homogeneous by denaturing polyacrylamide gel electrophoresis.

     

Metabolism of testosterone in human,rat and rabbit fetal lungs and in rabbit neonatal lung in vitro

• 1.1. Metabolism of 4-¹⁴C-testosterone was investigated in human, rat and rabbit fetal lung subcellular fractions and also in rabbit neonatal lungs. Androst-4-ene-3,17-dione, 17β-hydroxy-5α-androstan-3-one and both 5α- and 5β-androstane-3α,17β-diols were identified as metabolites of testosterone.
• 2.2. The microsomal fraction produced mainly 5α-reduced epimers while the cytosol incubations resulted in 5β-reduced metabolites.
• 3.3. No conjugation was found.
• 4.4. The amounts of polar metabolites in the microsomal incubations and the amounts of dihydroxy-lated metabolites in the soluble fraction incubations were statistically significantly greater in the neonatal rabbit lung incubations compared with those of fetal lungs.

     

Serum lipoproteins of sea bass (Dicentrarchus Labrax L.). Purification and partial characterization by density gradient ultracentrifugation and agarose column chromatography

• 1.1. Five classes of sea bass serum lipoproteins were purified by single vertical spin ultracentrifugation and agarose column chromatography
• 2.2. VLDL, beta migrating, are the larger and less dense lipoproteins.
• 3.3. LDL are the more heterogeneous in size, ranging from 11 × 10⁶ to 1 × 10⁶.
• 4.4. HDL represent the predominant class which, on the basis of density and electrophoresis migration, is differentiated in three subclasses.
• 5.5. VHDL float at a density > 1.22 mg/ml, which corresponds to the density of the other serum lipoproteins. This subclass, with an apparent molecular weight of 1.5 × 10⁵, resembles the albumin-like fatty acids binding proteins, shown in mammals and teleosts and absent in elasmobranchs.

     

Ultrastructure of maturing fish (Oreochromis niloticus) and snake (Waglerophis merremii) erythroid cells with regard to hemoglobin biosynthesis

• 1.1. Fish and snake immature erythrocytes were submitted to a comparative ultrastructural study, analysing changes in organelles involved in hemoglobin (Hb) biosynthesis.
• 2.2. Iron uptake occurs probably via transferrin, and ferruginous compounds accumulate as siderosomes, taken as iron sources for heme biosynthesis, later on caught by a double lamella.
• 3.3. Mitochondrial membrane of the inner camera differentiates to lamellated bodies that, sucessively, give rise to expansions for ferruginous material and globin chains captation, constituting prehemosomal vesicles, which become condensed vesicles, followed by prohemosomes.
• 4.4. Through an internal membrane rearrangement, prohemosomes change to hemosomes wherein, hypothetically, heme and the globin chains assembly may occur.
• 5.5. In both fish and snake erythroid cells, all stages for hemosomegenesis are similar to the stages found in erythroid cells of other vertebrate species, including humans, except that fish cells often present single organelles of still unknown function, void of internal membrane.
• 6.6. Through electrophoresis of the respective supernatants obtained after osmotical lysis of the organellar fractions, it was shown that fish hemosomes contain three Hb patterns, while snake hemosomes present two patterns.