Main serum protein of rainbow trout (Salmo gairdnerii): its biological properties and significance

• 1.1. Main serum α₁-protein (α₁P) of rainbow trout was purified and its biochemical and physico-pathological properties were studied.
• 2.2. α₁P was suggested to be a primitive protein having both properties of albumin and AFP in serum proteins of mammals according to the following results.
• 3.3. Molecular weight (75,000), two kinds of molecules (pI 4.55 and 5.05) and amino acid composition.
• 4.4. Dye- or ConA binding activity.
• 5.5. Estrogen binding activity and inhibitory effect on lymphoblastoid-forming activity.
• 6.6. Possible osmotic regulator.
• 7.7. Significant elevation of blood α₁P level in the course of hepatoma induction.

     

Kinetic properties of cat liver microsomal glucose-6-phosphatase

• 1.1. Cat liver microsomes contain the multifunctional enzyme glucose-6-phosphatase.
• 2.2. High specificity was shown for the phosphohydrolase as well as for the transferase activity.
• 3.3. Both activities have high V_max values determined in optimized conditions.
• 4.4. The phosphate transfer with carbamyl-phosphate as a phosphoryl donor and d-glucose as acceptor is consistent with a random mechanism in which the binding of one substrate decreases the enzyme's affinity for the second substrate.

     

A novel inhibitor of glycogen phosphorylase from crayfish hepatopancreas

• 1.1. A novel glycogen phosphorylase inhibitor was partially purified from crayfish hepatopancreas.
• 2.2. The inhibitor was found only in two species of crayfish examined, and not in lobster, fresh and salt water clams, mussels or cockroaches.
• 3.3. The inhibitor is a small protein (M_r = 23,000) which did not show proteolytic activity.
• 4.4. Preliminary kinetic analysis of the inhibitory mechanism indicated that it bound to both glycogen and the glycogen phosphorylase protein.
• 5.5. Inhibitor binding to glycogen resulted in a competitive inhibition pattern with respect to glycogen phosphorylase (inhibition constant of ca 10 μg/ml).
• 6.6. The inhibitor also bound glycogen phosphorylase directly with a binding coefficient of 100 μg/ml resulting in a partially non-competitive inhibition pattern with respect to phosphate.

     

The binding and degradation of 125I-labelled insulin by rat kidney brush-border membranes

• 1.1. The interaction of insulin with purified brush-border membranes from rat kidney was studied with the use of [¹²⁵I]insulin.
• 2.2. The specific binding of insulin by brush-borders could be demonstrated, and was time- and temperature-dependent.
• 3.3. [¹²⁵I]insulin was displaced by unlabelled insulin. A₁-B₂₉ dodecoyl insulin and insulin A- and B-chains in proportion to their relative bioactivity.
• 4.4. Brush-border membranes showed high insulin-degrading activity with an apparent K_m of 2.2 μM.
• 5.5. A number of proteinase inhibitors were effective in inhibiting insulin degradation but the greatest degree of inhibition was achieved by the use of thiol-blocking reagents.
• 6.6. No evidence was obtained for the involvement of the enzyme glutathione-insulin transhydrogenase.

     

Characterization of lactoferrin binding to the MAC-T bovine mammary epithelial cell line using a biotin-avidin technique

• 1.1. A non-radioisotopic method utilizing a biotin-avidin approach was used to characterize lactoferrin binding to the clonal MAC-T bovine mammary epithelial cell line.
• 2.2. Binding of lactoferrin to MAC-T cells and isolated membranes was specific and saturable.
• 3.3. Unlabeled lactoferrin competed for and displaced biotin-labeled lactoferrin from binding sites on mammary epithelial cells. In contrast, unlabeled transferrin did not compete.
• 4.4. Scatchard analysis of lactoferrin binding to MAC-T cell crude membranes was nonlinear, revealing two classes of binding sites with association constants (K_a) of 2.36 × 10⁷ and 3.36 × 10⁶M⁻¹.
• 5.5. Binding of lactoferrin to MAC-T cells may be associated with the initial events which result in decreased MAC-T cell proliferation.

     

Lactoferrin binding to human platelets

• 1.1. Platelets bind specifically lactoferrin.
• 2.2. The lactoferrin binding to the platelets depends on the concentration of labelled lactoferrin, the number of platelets, the time of incubation and pH.
• 3.3. The binding was characterized by two types of binding site: one with high affinity and low capacity, and another with low affinity and high capacity (respectively k_{aff 1} = 13.6 × 10⁹1/mol and about 40 binding sites, and K_{aff 2} = 1.23 × 10⁹1/mol and about 135 binding sites per platelet).
• 4.4. Both human transferrin and bovine lactoferrin compete with human lactoferrin for the receptors.
• 5.5. The presence of lactoferrin receptors on the platelet membrane surface is connected most probably with the effect(s) on the cell function(s) of these cells.

     

Essential tyrosyl residues of human placental alkaline phosphatase

• 1.1. Human placental alkaline phosphatase was inactivated with tetranitromethane in a biphasic process.
• 2.2. Spectral and amino acid analysis demonstrated that the inactivation was due to the conversion of tyrosine residues to 3-nitrotyrosine.
• 3.3. The inactivation process showed saturation kinetics.
• 4.4. Protection of the enzyme against tetranitromethane inactivation was afforded by inorganic phosphate.
• 5.5. The binding affinity between the modified enzyme and inorganic phosphate was decreased.
• 6.6. Our results suggest the involvement of tyrosyl residues in the locus of phosphoryl site of the phosphorylated enzyme forms.

     

Characteristics of Crassostrea virginica crystalline style chitin digestion

• 1.1. Fundamental chitin digestion characteristics of Crassostrea virginica crystalline style were investigated.
• 2.2. Optimum temperature and pH were 34°C and 4.8. respectively.
• 3.3. The colloidal regenerated chitin (0.56mol/0.5 ml: GlcNAc equivalents) was saturating under all enzyme levels encountered.
• 4.4. There was no evidence of end product inhibition, even after 100 hr incubation.
• 5.5. Calculated K_m for the chitinase complex was 1.19mM when determined using a 30 min assay, but was only 0.70 mM when determined using a 4.6 hr assay.
• 6.6. Both K_m values are lower than reported for similar assays in other molluscs and for most bacteria.
• 7.7. Effect of substrate preparation on the kinetics are discussed.
• 8.8. Eight peaks of chitinase activity were resolved by DEAE-Fractogel ion exchange chromatography.

     

Hen's egg yolk alkaline phosphatase: General characterization and kinetic stuy with inhibitors

• 1.1. The non-specific hen's egg yolk alkaline phosphatase is a metalloprotein (Zn²⁺?) composed of two identical inactive subunits.
• 2.2. A second metal site preferably binds Mg²⁺ (15-fold activation). Me(II))H₂O)H⁺, a charged arginine, and tyrosine in the active site are involved in positioning and binding of the substrate and metal ion.
• 3.3. Substrate inhibition differs with pH. This may be related to the presence of two active sites in the enzyme, one in each subunit.
• 4.4. Uncompetitive inhibition with L-phenylalanine and analogues suggests a phosphorylated intermediate.
• 5.5. Inhibition is weakly competitive with P_i strong non-competitive with PPi as compared to Mg²⁺-free PP_i, and partially competitive with arsenate.
• 6.6. The purified enzyme is stabilized and activated by amines and proteins.

     

Sex-specific androgen binding in spotted hyaena (Crocuta crocuta) plasma

• 1.1. A charcoal adsorption assay demonstrated a large variance in androgen binding ability in female spotted hyaenas.
• 2.2. A positive correlation between plasma androgen binding ability and ovarian steroid concentrations was demonstrated in adult females.
• 3.3. The strong plasma binding affinity for testosterone and dihydrotestosterone (DHT) (nM) together with the lack of cortisol and weaker oestradiol-17β binding suggests that a specific androgen binding substance, possibly a protein, is present in adult females of this species.
• 4.4. The lack of high affinity binding in male spotted hyaenas is unusual and deserves further investigation.
• 5.5. Some androgen binding in all, including males and immature animals suggests that albumin may bind some plasma androgens in this species.

     

Synaptic interaction between the giant interneuron and the giant motorneuron in the hermit crab,Pagurus pollicarus

• 1.1. An anatomical and physiological investigation was made of several components in the withdrawal escape response of the hermit crab, Pagurus pollicarus.
• 2.2. In the fused last thoracic-first abdominal ganglion, the giant axon (GA) synapses with the giant motor (GM) neuron which innervates the abdominal flexor muscles.
• 3.3. The synapse is unidirectional, conducting impulses only from the GA to the GM.
• 4.4. The synaptic delay time is less than 0.4 msec.
• 5.5. Concentrations of MgCl₂ and MnCl₂ which normally block chemical synapses were ineffective in interfering with GA-GM transmission.
• 6.6. The GA-GM synapse was reversibly blocked by 0.5 mM DNP.
• 7.7. It is concluded that GA-GM connection is a rectifying electrical synapse.

     

Isohematoporphyrin-diesters and diethers

• 1.1. A series of diesters of isohematoporphyrin (isoHp), from dimethyl to dioctyl were prepared according to Rimington et al. (1989b). Their optical absorption, fluorescence spectra and high performance liquid chromatography (HPLC) retention times were recorded.
• 2.2. A plot of HPLC retention time against number of C atoms in the alcohol used for esterification was approximately linear at first then rising steeply from diamyl to diocyi ester, whether a gradient elution was used or only methanol: water, 95/5, at pH 7.5.
• 3.3. Preparation of the diethers of isoHp was much more difficult than that of the corresponding derivatives of hematoporphyrin (Hp). Several different methods were investigated, varying both times and temperatures.
• 4.4. These methods included reaction of isoHp or its demethyl ester with
  • 4.1.(i) a bromoalkane in presence of anhydrous K₂CO₃;
  • 4.2.(ii) reaction with bromoalkane and Ag₂O;
  • 4.3.(iii) reaction of brominated-isoHp, prepared by using thionylbromide, with the selected alcohol, or corresponding sodium alcoholate;
  • 4.4.(iv) heating of isoHp alone with an alcohol containing 20% (w/v) H₂SCO₄ (temp. range from 45° to 118°C),
  • 4.5.(v) refluxing as in (iv) at the b.p. of the alcohol; and
  • 4.6.(vi) carrying out this reaction in refluxing ethyleneglycoldimethyl ether (b.p. 85°C) or diethyleneglycoldimethyl ether (b.p. 155°C).
• 5.5. Some diether formation was observable by all these methods but yields were small, a considerable quantity of unreacted isoHp and other products remaining.
• 6.6. Examined by HPLC, the diethers consistently afforded a forked peak which on thin layer chromatography was only resolved into two very closely associated bands by a solvent mixture carefully selected for development.
• 7.7. On elution these materials had virtually identical optical absorption and fluoresence spectra.
• 8.8. The nature of the association is discussed, atropisomers (Gottwald and Ullman, 1969) and possible stacked monomer: dimers (Abraham et al., 1963) being considered as possibilities.

     

Electrical transport characteristics of Aplysia californica intestinal epithelium

• 1.1. Replacing chloride (Cl⁻) with sulfate (SO₄²⁻) in the bathing medium drastically reduced the mucosal membrane potential difference (ψ_m).
• 2.2. The voltage divider ratio was significantly greater than one.
• 3.3. Mucosal ^d-glucose decreased the input resistance of the intestinal epithelium.
• 4.4. Addition of furosemide to the mucosal bathing medium inhibited transepithelial potential difference and short-circuit current.
• 5.5. Addition of SITS to the mucosal bathing medium partially inhibited transepithelial potential difference and short-circuit current.
• 6.6. Diffusion potentials in the intestinal epithelium were symmetrical.

     

Domain structure of the 90-kDa stress protein: Heparin- and antibody-binding domain

• 1.1. To understand the physiological roles of the 90-kDa stress protein (HSP90), we investigated the heparin- and antibody-binding domains of the protein.
• 2.2. For heparin-binding sites, HSP90 was digested completely with trypsin, and the digests were applied to a heparin-Sepharose column and eluted with 1.0 M NaCl, followed by 8.0 M urea.
• 3.3. Each elutant was purified by a reverse-phase C₁₈ column.
• 4.4. Two peptides from the NaCl-eluted fraction and no peptide from the urea-eluted fraction were purified.
• 5.5. The purified peptides were sequenced by an automated peptide sequencer.
• 6.6. One of the heparin-binding sites was present between Leu-362 and Arg-365; another was present between Leu-645 and Lys-648.
• 7.7. These two peptides were basic and considerably hydrophilic.
• 8.8. For antibody-binding sites, HSP90 was mildly digested with trypsin, electrophoresed on SDS-polyacrylamide gels and transferred to PVDF membranes.
• 9.9. The four bound of the trypsin fragments could be sequenced with a peptide sequencer.
• 10.10. There was only one antibody-binding peptide, 38 kDa, starting from Pro-2. The others showed no cross-reactivity with the antibody and started from Leu-283.
• 11.11. Therefore, the epitopes of HSP90 are present between Pro-2 and Leu-282.
• 12.12. The heparin-binding sites are present from the middle region of the HSP90 molecule, and the antigen sites are at the N-terminal domain.

     

Oxidation of methionine residues in equine growth hormone by chloramine-T

• 1.1. Reactivity of methionine residues towards Chloramine-T was studied in the equine growth hormone.
• 2.2. With a 20.0-fold molar excess of reagent over methionine, full oxidation of the four residues of the protein is achieved.
• 3.3. Methionine 4 is the most reactive group, followed by methionines 72 and 178—methionine 123 being the less reactive residue.
• 4.4. As judged by circular dichroism spectra and binding assays, protein conformation and binding capacity to specific receptors remains unchanged even after full oxidation of all four methionine residues.
• 5.5. Results agree with data previously obtained with bovine growth hormone.

     

Effect of bovine milk antigens and egg lysozyme on the binding of 59Fe-lactoferrin to platelet plasma membranes

• 1.1. Platelets bind specifically to lactoferrin. A significant similarity between human lactoferrin and some bovine milk proteins has been established.
• 2.2. Because of the structural homology of lactoferrin and cows milk proteins they are able to influence lactoferrins regulatory function on the level of its binding to membrane receptors on platelets.
• 3.3. An inhibitory effect of bovine α-lactalbumin and of β-lactoglobulin on lactoferrin-receptor interaction was shown.
• 4.4. Bovine α-lactalbumin competes with lactoferrin for the binding sites.
• 5.5. Scatchard plot analysis of data shows one binding site for lactoferrin in the presence of α-lactalbumin with an affinity constant, Ka = 0.46 × 10⁹ mol/1 and 335 receptors/cell.
• 6.6. The inhibitory effect of β-lactoglobulin reaches 62% and is different for the common fraction ⨿-lactoglobulin and the genetic variants β-lactoglobulin A and B.
• 7.7. β-lactoglobulin does not compete with lactoferrin for the membrane receptors.
• 8.8. Bovine casein and egg lysozyme stimulate ⁵⁹Fe-lactoferrin binding to the receptors. The mechanism of these effects is still unknown.
• 9.9. Tested alimentary antigens are able to interact with lactoferrin and also with some platelet membrane structures.
• 10.10. Established changes in lactoferrin binding to the platelet membrane might be in relation to lactoferrins regulatory function and (or) eliminating mechanisms of these alimentary antigens.

     

The influences of individual variations in metabolic rate and tidal conditions on the response to hypoxia in Carcinus maenas (L.)

• 1.1. The oxygen consumption of crabs in normoxic and hypoxic (50% O₂) seawater was measured directly after collection.
• 2.2. The influences of size and lunar cycles were removed by scaling the data.
• 3.3. Strong negative correlations between low individual levels of O₂ consumption and the ability to compensate for hypoxia were apparent in Wicklow (subtidal) crabs.
• 4.4. Compensation for hypoxia was much greater on the flood tide than on the ebb.
• 5.5. Crabs from Roscoff (intertidal) had lower levels of compensation than those from Wicklow.
• 6.6. Size, sex and condition had no apparent effect upon these relationships.
• 7.7. Crabs acclimated to laboratory conditions have not shown this tidal variation in compensation for hypoxia.

     

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Respiration in the eastern water dragon,Physignathus lesueurii (agamidae)

• 1.1. Some aspects of the gas exchange system of a diving lizard, Physignathus lesuewii were studied.
• 2.2. Breathing patterns were analysed.
• 3.3. Breathing rate increases logarithmically with temperature and Q₁₀ = 1.8. LogBR = −0.237 + 0.0256 T.
• 4.4. Gas tensions in lung air and arterial and venous blood were measured. Arterial pH declines with increasing temperature.
• 5.5. Temperature has a marked effect on oxygen affinity of the blood (ΔH = −10.1 kcal mol⁻). A Bohr effect was also noted.
• 6.6. CO₂ equilibrium curves were drawn.
• 7.7. The results are considered with a view to anticipating the efficiency of the gas exchange system of this species under conditions of variable temperature and during diving.