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.

     

Solubilization,characterization and photoaffinity labeling of the mitochondrial dihydropyridine receptor from bovine adrenal medulla

• 1.1. The mitochondrial dihydropyridine receptor was solubilized with Chaps at a detergent/ protein ratio of 2.5, during 45 min at 4°C.
• 2.2. From the rate constants of association (8.10 ± 0.25 × 10⁴ M⁻¹ min⁻¹) and dissociation (0.022 ± 0.001 min⁻¹ a K_d of 275 nM was calculated, while from saturation experiments a K_d of 270 ± 30 nM and a density of receptors of 106 ± 9 pmol/mg protein was obtained.
• 3.4. The solubilized receptors are heat-resistant, sensitive to the trypsin and to the reduction of disulfide bonds.
• 4.5. In native membranes, a polypeptide of 50 kDa was specifically photolabelled with [³H]Azidopine.

     

Characterization of hemoglobin from Phoronis architecta (phoronida)

• 1.1. Phoronis architecta hemoglobin is composed of four distinct hemoglobin subunits with minimum MW's of 16–17,000 or 17–19,000 daltons. All four hemoglobins are monomeric when oxygenated. Two of the monomers combine to form dimers when bound with carbon monoxide.
• 2.2. In cellulo, Phoronis architecta hemoglobin has a half-saturation (P₅₀) value of 1.3 ± 0.1 mm Hg, shows cooperative oxygen binding (Hill coefficient = 2.7 ± 0.3), and no Bohr effect from pH 6.6 to 7.9. In vitro, the hemoglobin has a P₅₀ of 0.76 ± 0.21 mm Hg but shows no cooperativity (0.90 ± 0.15 (SD)).
• 3.3. The oxygen dissociation constant (K_off) from hemoglobin is 2.7 ± 0.2 sec⁻¹, and the computed oxygen association constant (K_on) is 2.5 × 10⁶ M⁻¹ · sec⁻¹ (1.9–3.6 × 10⁶ M⁻¹ · sec⁻¹).

     

The pH dependence of the affinity,kinetics and cooperativity of ligand binding to the root effect haemoglobin of the goldfish Carassius auratus

• 1.1. The binding of O₂ to goldfish haemoglobin showed a strong pH dependence P₅₀=5.5 mmHg; n = 2.4 at pH 8.0 and P₅₀ = 170 mmHg; n = 1.0 at pH 5.5 such that the protein is only 50% saturated in a solution of air equilibrated buffer at pH 5.5.
• 2.2. The binding of CO is cooperative at high pH (n = 2.8; L = 1000; K_R = 0.1 μM; K_T = 4 μM) and non-cooperative (n = 1) at pH 5.5.
• 3.3. The rate of O₂ dissociation is extremely fast and pH dependent; being 30 sec⁻¹ at pH 8.0 and 400 sec⁻¹ at pH 6.0 at 1°C. At 23°C the rate of this process is too fast to obtain accurate data using stopped-flow techniques.
• 4.4. Partial photolysis of the oxyhaemoglobin species leads to homogeneous recombination kinetics at pH 8.0 with an associated rate constant of 4.7 × 10⁷ M⁻¹ sec⁻¹. At pH < 7.5 the recombination process occurs in two steps. One rate is equal to that observed at pH 8.0. The slower process is favoured at low pH.
• 5.5. Photolysis of the CO haemoglobin complex indicates that, at high pH, combination of CO with deoxyhaemoglobin is cooperative, whilst recombination with Hb(CO)₃ is non-cooperative and occurs at a rate of 1.2 × 10⁶ M⁻¹ sec⁻¹.
• 6.6. At neutral pH recombination of CO with partially linganded haemoglobin occurs in a two-step process. The proportion contributed by each of these two steps in pH dependent.
• 7.7. The functioning of this Root effect haemoglobin is discussed in terms of the two state-model of cooperativity in which the αβ chain heterogeneity is minimal

     

Purification and some properties of a Mg2+-activated acid phosphatase from rat testis

• 1.1. The acid phosphatase (AcPase, EC 3.1.3.2) IV from rat testicular tissue was purified to apparent homogeneity.
• 2.2. The enzyme displays a native molecular weight of 70 kDa determined on gel permeation chromatography on a Sephadex G-100 column and 68 kDa using linear 5–20% sucrose density gradient centrifugation. The subunit molecular weight on SDS-PAGE analysis is 67 kDa, suggesting that the enzyme is a monomeric protein.
• 3.3. The enzyme does not bind to Concanavaline A-Sepharose 4B column, indicating that it is not a glycoprotein.
• 4.4. The rat testis AcPase IV is a metal activated enzyme in which Mg²⁺ is the metal activating agent with a K_a, = 0.88 × 10⁻³ M. The Michaelis constant for p-nitrophenylphosphate, in the presence of saturating concentrations of Mg²⁺ ions, is 0.23 × 10⁻³ M.
• 5.5. The enzyme preferentially hydrolizes p-nitrophenylphosphate, phenylphosphate and ATP.

     

Demonstration of highly specific and sensitive antibodies to a naturally occurring tetrahydroisoquinoline alkaloid,salsolidine

• 1.1. We report for the first time on the production and characterization of antibodies against a naturally occurring tetrahydroisoquinoline, namely salsolidine (6,7-dimethoxy-1-methyl-1,2,3,4-tetrahydroisoquinoline).
• 2.2. Immunogen synthesis was carried out by coupling the hapten salsolidine to bovine serum albumin (BSA) as carrier protein on the basis of reductive amination.
• 3.3. By immunization of rabbits with salsolidine-BSA conjugate antisalsolidine antibodies were produced.
• 4.4. At a final dilution of 1:1700 the highest-litre antiserum bound 35% of 0.21 pmol [³H] salsolidine. This antiserum was used to develop a radioimmunoassay for salsolidine.
• 5.5. Cross-reactivity studies revealed a high specificity of the antiserum to the hapten.
• 6.6. The antibodies had a high affinity to salsolidine (K_a = 1.5 × 10⁹ M⁻¹).
• 7.7. Standard curves covered a measuring range of 0.5–70 pmol/tube and the detection limit was found to be 0.27 pmol/tube.

     

Comparative nuclear magnetic resonance studies of diffusional water permeability of red blood cells from different species. V—Rabbit (Oryctolagus Cuniculus)

• 1.1. The diffusional water permeability (P_d) of rabbit red blood cell (RBC) membrane has been monitored by a doping nuclear magnetic resonance (NMR) technique on control cells and following inhibition with p-chloromercuribenzene sulfonate (PCMBS).
• 2.2. The values of P_d were around 6.3 × 10⁻³ cm/sec at 15°C, 7.0 × 10⁻³cm/sec at 20°C, 8.0 × 10⁻³ cm/sec at 25°C, 9.1 × 10⁻³ cm/sec at 30°C and10.7 × 10⁻³ cm/sec at 37°C.
• 3.3. Systematic studies on the effects of PCMBS on water diffusion indicated that the maximal inhibition was reached in 15 min at 37°C with 0.5 mM PCMBS.
• 4.4. The values of maximal inhibition were around 71–74% at all temperatures.
• 5.5. The basal permeability to water was estimated as 1.6 × 10⁻³cm/sec at 15°C, 2.0 × 10⁻³cm/sec at 20°C, 2.4 × 10⁻³cm/sec at 25°C, 2.6 × 10⁻³cm/sec at 30°C, and 3.1× 10^{−3 cm/secat 37°C}.
• 6.6. The activation energy of water diffusion was around 18 kJ/mol and increased to 27 kcal/mol after incubation with PCMBS in conditions of maximal inhibition of water diffusion.
• 7.7. The membrane polypeptide electrophoretic pattern of rabbit RBCs has been compared with its human counterpart.
• 8.8. The rabbit membrane contained a higher amount of spectrin (bands 1 and 2), while the band 6 (glyceraldehyde-3-phosphate dehydrogenase) was markedly less intense.
• 9.9. Considerable differences in the electrophoretic patterns of the two sources of RBC membranes appeared in the bands migrating in the band 4.5 region and in front of band 7, where some polypeptides were apparent in higher amounts in the rabbit RBC membrane.

     

Energy metabolism and body temperature in 13 sunbird species (Nectariniidae)

• 1.1. Diurnal cycles of body temperature, T_b, and energy metabolism, M, at different ambient temperatures (T_a: +5 −+ 32°C) were tested in 13 sunbird species from various habitats and of different body masses (5.2–14.2 g) including one of the smallest passerines, Aethopyga christinae.
• 2.2. Resting M-level (night) reaches T_a-dependent mean values of 54% (+5°C) and 49% (+25°C) of activity M-levels (day). Expected level is ca 75%.
• 3.3. Resting metabolic rate of sunbirds lies within the range of theoretically expected values for birds.
• 4.4. Mean linear metabolism-weight regression of the night values follows: M = 0.102 × W^0.712 (M = energy metabolism in kJ/hr and W = body mass in g).
• 5.5. Thermal conductances, T_c, are lower (−24%) than the predicted values. This is caused by a decrease of T_b at low T_a. Mean nocturnal T_c is 3.2 J/g × hr × °C, mean day-time value is 4.3 J/g × hr × °C.
• 6.6. The zone of thermoneutrality is, in most species, within a T_a-range of 24–28°C.
• 7.7. Normal day and night levels of T_b are in the same range as reported for other birds of the same weight class. T_b decreases slightly with falling T_a (partial heterothermia). Lowest recorded T_b was 34.2°C.
• 8.8. No species tested showed any sign of torpor at night, independent of T_a, body mass or habitat origin.

     

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.

     

Purification,characterization and biological activities of phospholipase a from russell's viper (Vipera russelli) venom

• 1.1. The major phospholipase A has been purified to electrophoretic homogeneity from the venom of Vipera russelli (Russell's viper).
• 2.2. The molecular weight of the purified enzyme was estimated to be 31,000 by Sephadex G-75 gel filtration chromatography and 29,000 by SDS-polyacrylamide gel electrophoresis. The enzyme exhibited an apparent K_m value of 2.3 × 10⁻² M.
• 3.3. The phospholipase A showed edema forming, indirect hemolytic and myonecrotic activities but not hemorrhagic activity.

     

Purification and properties of a β-1,4-xylanase from a cellulolytic extreme thermophile expressed in Escherichia coli

• 1.1. An endoxylanase (EC 3.2.1.8) was purified from an Escherichia coli strain carrying a xylanase gene from the extreme thermophile “Caldocellum saccharolyticum strain Tp8T6.3.3.1. It was found to have an M_r of 42,000 and an isoelectric point of approx. 5.0.
• 2.2. The enzyme showed optimum activity at pH 5.0–7.7 and had an activation energy of 44 kJ mol⁻¹. It was stable at room temperature at pH 4.5–11.5 in the presence of 0.5 mg ml⁻¹ bovine serum albumin. The half-life of the enzyme at 75°C was 20 min at pH 6.0 in the presence of 0.5 mg ml⁻¹ bovine serum albumin.
• 3.3. The xylanase had highest activity on oat spelts xylan, releasing xylobiose and some xylotriose. The K_m for oat spelts xylan was 0.021% (w/v) at pH6.0.
• 4.4. The enzyme had high activity on sugar cane bagasse hemicelluloses A and B, lower activity on larchwood xylan and also hydrolysed carboxymethylcellulose, 4-methylumbelliferyl β-D-cellobioside and p-nitrophenyl β-D-cellobioside, but could not hydrolyse xylobiose.
• 5.5. It showed transferase activity on p-nitrophenyl β-D-xylopyranoside. Xylose did not inhibit the enzyme.

     

Aluminium and calcium affect the perivitelline fluid in fish eggs

• 1.1. Microelectrodes have been used to measure K⁺ activities and electrical potential differences between the perivitelline fluid (pvf) of the eggs of pike (Esox lucius) and surrounding water in a range of pH, calcium and aluminium concentrations.
• 2.2. Potential differences between pvf and water are decreased by Ca²⁺ (10⁻³ M) while Al³⁺ (18 × 10⁻⁶ M) reverses the polarity of the potential difference.
• 3.3. K⁺ activities in the pvf of eggs in 10⁻⁴M KCl + 10⁻⁵M NaCl are decreased by Ca²⁺(10⁻³ M).
• 4.4. The results are discussed with reference to ion-exchange theory and chorion permeability.

     

A fluorescence study of substrate and inhibitor binding to bovine liver dihydrofolate reductase

• 1.1. Covalent coupling of fluorescein to methotrexate (MTX) by a 5-carbon spacer yields a dihydrofolate reductase (DHFR) inhibitor (FMTX) with K_i = 11 nM.
• 2.2. FMTX shows a fluorescence quenching with respect to fluorescein which is relieved by binding to the enzyme.
• 3.3. The dissociation constants (K_d) of MTX, FMTX, NADPH and 7,8-dihydrofolate (DHF) from bovine liver DHFR have been determined by fluorometric titrations.
• 4.4. The K_d values for NADPH, MTX and FMTX from the complementary binary complexes (MTX·DHFR, FMTX·DHFR and NADPH·DHFR) were also obtained; these show a 2- to 4-fold decrease with respect to those obtained by titration of the free enzyme.
• 5.5. A competitive assay for MTX has been developed by exploiting the fluorescence enhancement of DHFR-bound FMTX. This assay may be useful for the routine determination of MTX in the concentration range from 10⁻⁹ to 10⁻⁷ M.

     

Ouabain sensitive Na+/K+-transporting ATPase from the brain of Poekilocerus bufonius

• 1.1. The properties of Na⁺/K⁺-transporting ATPase in microsomal fractions from the nervous tissue of the grasshopper, Poekilocerus bufonius were investigated.
• 2.2. Two components of ATPase activity are present.
• 3.3. Inclusion of 1 mM ouabain in the incubation media reduced the activity of total and Na⁺/K⁺-ATPase by 57 and 79%, respectively.
• 4.4. The maximum velocity (V_max) was decreased by the addition of 1 mM ouabain, whereas the apparent K_m value was not affected indicating a non-competitive type of inhibition.
• 5.5. The calculated value of the pI₅₀ was 6.4 (I₅₀ = 3.98 × 10⁻⁷M) for ouabain inhibition of the enzyme showing great sensitivity to the cardiac glycoside ouabain.
• 6.6. The present results show that the physicochemical properties of Na⁺/K⁺-transporting ATPase from the brain of P. bufonius are essentially the same as for the enzyme prepared from the excretory system of the insect which has been previously investigated.
• 7.7. Dissimilarities were also observed between these tissues in the way that the enzyme from the brain was sensitive to ouabain inhibition with a non-competitive type rather than a ouabain-resistance and a competitive type of inhibition for the enzyme from the excretory system.
• 8.8. These dissimilarities are probably due to different isoenzyme patterns available in the same insect.

     

Inhibiton and gamma-aminobutyric acid-induced conductance on locust (Schistocerca gregaria) extensor tibiae muscle fibres

• 1.1. Increases in membrane conductance (g_m) were induced by GABA in distal bundles 32, 33 and 34 of extensor tibiae muscles of the locust (Schistocerca gregaria).
• 2.2. Bath application of GABA (10⁻⁵−5 × 10⁻³ M) induced reductions in muscle fibre space constant (λ).
• 3.3. GABA (5 × 10⁻³ M) induced additional membrane conductance of 2.21 ± 0.03 × 10⁻⁶ S/mm, 0.38 ± 0.03 × 10⁻⁶ S/mm and 0.29 ± 0.06 × 10⁻⁶ S/mm on muscle bundles 34, 33 and 32 respectively. The greater sensitivity of muscle fibres in bundle 34 to GABA is due at least in part to a larger number of GABA receptors on bundle 34 muscle fibres.
• 4.4. The decrement of electrotonic potentials in the presence of GABA were measured over distances of both half fibre length and whole fibre length. Good agreement was obtained between changes in space constant produced by GABA using half fibre length and whole fibre length data.
• 5.5. By taking into account changes in space constant induced by GABA it was possible to demonstrate that presynaptic GABA receptors were involved in the inhibition of slow excitatory postsynaptic potentials by GABA.
• 6.6. “Slow” excitatory postsynaptic potentials recorded under current clamp were inhibited in a dose-dependent manner by GABA. This inhibition was not dependent on muscle-fibre GABA sensitivity and could not be completely accounted for by GABA-induced changes in the cable properties of the muscle fibres.

     

δ-Aminolevulinic acid transport in Saccharomyces cerevisiae

• 1.1. This work represents the first approach to characterize the transport system of haem pathway precursors, such as δ-aminolevulinic acid (ALA), in two strains of Saccharomyces cerevisiae, a wild type, D27, and a HEM R⁺ mutant.
• 2.2. ALA transport occurs unidirectionally by a sole active system with an apparent K_M of 0.10 mM, at the optimum pH of 5.0. ALA uptake is influenced by both the carbon and nitrogen source; this suggests a rather complex regulation mechanism.
• 3.3. This transport is not mediated by the general amino acid permease (GAP).
• 4.4. ALA uptake is strongly inhibited by compounds harboring a methyl-amine terminus suggesting that this group is essential for ALA transport; however, the electric environment of the carboxylic group may be also important for the interaction between ALA and its transporter active site.
• 5.5. We have found differences in ALA transport which would indicate a different regulation mechanism for this system in both strain cells.

     

An alkaline phosphatase from Thermus sp strain Rt41A

• 1.1. A thermostable orthophosphoric monoester phosphohydrolase (EC 3.1.3.1) from Thermus sp strain Rt41A has been purified 400-fold to give a specific activity of 25 U/mg at 60°C in IM diethanolamine (pH 11.1).
• 2.2. The enzyme has a M_r of 160,000 and is trimeric.
• 3.3. The half-life of the enzyme is 5 min at 85°C.
• 4.4. The enzyme has a wide specificity for a number of phosphate monoesters.
• 5.5. The H_m of the enzyme is pH dependent, so the pH optimum of the enzyme is affected by the substrate concentration.
• 6.6. The enzyme is inhibited 50% by 20 mM Ca²⁺ or Mg²⁺.
• 7.7. The K_i for phosphate, EDTA-di sodium salt and arsenate (in 1 M diethanolamine, pH 11.1) is approx 1.2, 1.6 and 4mM respectively.
• 8.8. Urea (200 mM) is not inhibitory.

     

A peptide inhibitor for S-adenosyl-l-methionine-dependent transmethylation reactions: Purification and characterization

• 1.1. A proteinaceous inhibitor for S-adenosyl-l-methionine (AdoMet)-dependent transmethylation reactions has been purified to apparent homogeneity from rat liver cytosolic fraction.
• 2.2. The peptide was made up of 29 amino acid residues with a molecular weight of 2,584. Glycine accounted for 52% of the total amino acids.
• 3.3. Employing AdoMet: protein-carboxyl O-methyltransferase (Protein methylase II) and bovine serum γ-globulin as in vitro substrate, the mode of inhibition was found to be non-competitive with K_i value of 1.9 × 10⁻⁸ M.
• 4.4. When the inhibitor was present in the reaction mixture together with S-adenosyl-l-homocysteine (AdoHcy), which is a competitive inhibitor for AdoMet, the extent of inhibition exceeded that exerted by each individual inhibitor alone, suggesting that the sites of the inhibitors on the enzyme molecule are different.
• 5.5. Almost a stoichiometric relationship exists between the enzyme and the inhibitor molecule, the ratio being approx one.

     

l-lysinamidase from Cryptococcus laurenti 112. Purification and properties

• 1.1. The purified enzyme hydrolyzes the linear l-lysinamide and the cycle amide of l-lysine—l-α-amino-ϵ-caprolactam.
• 2.2. The apparent relative molecular mass is 180,000. The enzyme consists of four subunits and the molecular mass of a single subunit was found to be 47,000.
• 3.3. The coefficient of molecular sedimentation equals 8.3 S, the isoelectric point was determined to be pH 4.3
• 4.4. The enzyme is not a glycoprotein. p-Mercuribenzoate binds 10 SH-groups of the native enzyme molecule and 20 SH-groups in the presence of 0.7% SDS.
• 5.5. pH- optimum for the hydrolysis of l-lysine amides was observed to be 7.5–7.7. The enzyme is strictly dependent on Mn²⁺ and Mg²⁺.
• 6.6. The kinetic parameters for the hydrolysis of l-lysinamide where K_m = 3.8 mM and k_cat = 3000 sec⁻¹ For the hydrolysis of cyclic L-lysinamide K_m = 4.8 mM and k_cat = 2600 sec⁻.

     

Purification and properties of an ld-dipeptidase FROM Bacillus sphaericus

• 1.1. An ld-dipeptidase (EC 3.4.13.-) that hydrolyzes the unrelated dipeptides l-Ala-d-Glu (sp. act. 0.85 μmol·min⁻¹·mg⁻¹) and l-Lys-d-Ala (sp. act. 11 μmol · min⁻¹·mg⁻¹) has been purified 250-fold from the sporulation medium of Bacillus sphaericus with a 4% recovery of lytic activity.
• 2.2. Throughout the purification steps, followed with both substrates, the enzyme peaks of activities were congruent and the ratios of activities were constant. Both activities were activated 50-fold by cobalt. Polyacrylamide gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. The data are consistent with those activities being due to a single enzyme.
• 3.3. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the purified enzyme showed a single protein band (M_r 38,000).
• 4.4. This dipeptidase hydrolyzes some other ld-dipeptides with a free amino and carboxyl group. Although dipeptides having a di-amino acid as the amino terminus are the best of the substrates tested, the hydrolysis occurs also when neutral amino acids are N-terminal. The activity is higher with neutral C-terminal residues such as Gly or d-Ala than with a di-acid residue such as d-Glu.
• 5.5. This enzyme may have a function in peptidoglycan metabolism.