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.

     

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.

     

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.

     

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.

     

The dynamics of the permeation of an analogue of insect juvenile hormone into nematodes

• 1.1. ¹⁴C-dichlorofarnesoate permeated rapidly into Haemonchus contortus (infective juveniles) and Panagrellus redivivus (mixed cultures) and was strongly bound by hydrophobic association (K_s > 10⁻⁴M).
• 2.2. Uptake rose linearly with increases in temperature (5–38°C) and external concentration (C₀; 0.07–2.15 × 10⁻⁴ M). Within 1 hr the internal concentration, C₁ was >C C₀.
• 3.3. The pH of the medium (6–8) did not affect uptake.
• 4.4. Efflux of dichlorofarnesoate was low: the half-time of release was > 18 hr.
• 5.5. The uptake curve approximated to the expression C₁/C₀ = a(1 − e^−bt) with a and b as constants and t in hr.
• 6.6. These results clarify previous work on the inhibitory action of juvenile hormone on the development of nematodes.

     

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.

     

Histological and pharmacological investigations of the two symmetrically situated giant neurons,RPeNLN and LPeNLN,identified on the anterior surface of the pedal ganglia of an african giant snail (achatina fulica férussac)

• 1.1. Morphological and pharmacological investigations were made of two giant neurons, RPeNLN (right pedal nerve large neuron) and LPeNLN (left pedal nerve large neuron), situated symmetrically on the anterior surface of the pedal ganglia of an African giant snail (Achatina fulica Férussac).
• 2.]2. The two neurons (about 250–300 μm in diameter) were the largest ones identified in the ganglia of the snail species. The axonal pathways of the two neurons were symmetrical; of their four main axonal branches, the three main branches innervated the ipsilateral pedal nerves, whereas the last main branch projected to the contralateral pedal nerves.
• 3.]3. The pharmacological features of the two neurons were very similar. Both were inhibited markedly by dopamine [minimum effective concentrations (MECs): 3 × 10⁻⁶-10⁻⁵M], dl-octopamine (MECs: 2 × 10⁻⁶-2 × 10⁻⁵M), 5-hydroxytryptamine (MEC: 3 × 10⁻⁶M), GABA (MEC: 3 × 10⁻⁵ M), l-homocysteic acid (MECs: 3 × 10⁻⁵-10-10⁻⁴M) and erythro-β-hydroxy-l-ghitanuc acid (MEC: 3× 10⁻⁵M). Acetylcholine showed varied effects, either excitatory or inhibitory, on the two neurons examined. No substances were found to have any marked excitatory effects on the neurons.

     

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.

     

Charge interactions of cytochrome c with cytochrome c oxidase

• 1.1. The pyridoxal phosphate (PLP) modification of the lysine amino groups in cytochrome c causes decrease in the reaction rate with cytochrome c oxidase.
• 2.2. The rate constants for (PLP);-cyt. c, PLP(Lys 86)-cyt. c, PLP(Lys 79)-cyt. c and native cytochrome c (at pH 7.4, 1=0.02) are 3.6 × 10⁻³'sec-', 5.5 × 10⁻³, 5.2 × 10⁻³-'sec⁻¹ and 9.8 × 10⁻³sec⁻¹, respectively.
• 3.3. In spite of the same positive charge of singly PLP-cytochromes c the reaction between PLP(Lys 86)-cyt. c and cyt. c oxidase exhibits the ionic strength dependence that differs from those of the PLP(Lys 79)-cyt. c.
• 4.4. The rate constants at zero and infinite ionic strength for PLP(Lys 86)-cyt. c is 2-fold less than that for PLP(Lys 79)-cyt. c.
• 5.5. The positively charged cytochrome c lysines 86 and 79 form two from four or five predicted complementary charge interactions with carboxyl groups on cytochrome c oxidase.

     

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.

     

The chemical ecology of Biomphalaria glabrata (say): Sugars as attractants and arrestants

• 1.1. The behavioural responses of the freshwater snail Biomphalaria glabrata to chemical gradients of sugars were investigated by means of diffusion olfactometers.
• 2.2. The snails proved very discriminating in their responses. Thus, only nine (39.1%) of the 23 sugars tested proved to be statistically significant attractants or arrestants. None proved to be statistically significant repellents.
• 3.3. Of all the sugars tested maltose proved to be the most potent attractant or arrestant. The lower threshold of response to this sugar lies between 5 × 10⁻⁶ and 5 × 10⁻⁷M.
• 4.4. The results are compared with those obtained for amino and carboxylic acids and their ecological relevance is discussed.

     

Purification and properties of tetralysine endopeptidase from Escherichia coli AJ005

• 1.1. A new tetralysine endopeptidase from Escherichia coli AJ005 has been purified about 135-fold.
• 2.2. The peptidase seems to be specific to tetralysine among lysine homopolymers.
• 3.3. The optimal pH was about 7.5
• 4.4. The activity was inhibited by KCN but not inhibited by soybean trypsin inhibitor.
• 5.5. The apparent K_m value was 2.5 × 1O⁻³ M for tetralysine.

     

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.

     

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.

     

Characterization of NaK ATPase from the gills of the freshwater prawn Macrobrachium rosenbergii (de Man)

• 1.1. The specific activity of Na-K ATPase was determined from the microsomal preparation of gills dissected from adult Macrobrachium rosenbergii.
• 2.2. Maximal ATPase activity was achieved at a substrate concentration of 0.5 mM ATP.
• 3.3. Optimal enzyme activity was obtained at pH of 7.5.
• 4.4. The Arrhenius plot of Na-K ATPase activity revealed a marked discontinuity at 30°C. “Mg” ATPase activity did not exhibit a marked discontinuity.
• 5.5. The E_a for Na-K ATPase and “Mg” ATPase was 14.6 kCal/mole and 9.31 kCal/mole respectively. Q₁₀ values for Na-K ATPase was 2.34 and for “Mg” ATPase 1.65.
• 6.6. ATPase activity and gill homogenate protein concentration exhibited a linear relationship up to 130 μg protein/ml.
• 7.7. Na-K ATPase activity was inhibited by 10⁻³ M ouabain. It was equally inhibited by the removal of K⁺ from the reaction medium.

     

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.

     

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.

     

The effect of triiodothyronine on glucose utilization in adipocytes from obese rats

• 1.1. In the presence of insulin, 10⁻⁵ M 3,3',5-triiodothyronine (T₃) treatment for 1/2 hr decreased fatty acid synthesis 35% only in adipocytes from lean rats, whereas at 10⁻¹¹ M through 10⁻⁷M T₃ the obese adipocytes had nearly a 20% increase in fatty acid synthesis.
• 2.2. A 2 hr pretreatment of adipocytes with 10⁻⁹ and 10⁻⁷ M T₃ decreased insulin-stimulated fatty acid synthesis by nearly 20% in both lean and obese adipocytes.
• 3.3. In the absence of insulin, the 2 hr pretreatment with 10⁻⁹ M T₃ resulted in a 45% increase in lean adipocyte fatty acid synthesis, though the obese adipocytes required at least 10⁻⁷ M T₃ for 2 hr to increase the non-insulin-stimulated fatty acid synthesis by 50%.
• 4.4. At 10⁻⁹M T₃ concentrations non-insulin-stimulated fatty acid synthesis was increased by 200% in lean adipose tissue explants, but obese adipose expiants were not significantly affected under these conditions.
• 5.5. The addition of 10⁻⁹ M T₃ plus insulin to the explant media decreased fatty acid synthesis by 35% in both the lean and obese tissues.
• 6.6. The results also imply that the low T₃ status of the obese rat may be contributory to the elevated fatty acid synthesis observed in obese adipocytes.

     

Peritubular Na-K exchange ion pump in maleate-treated frog kidney proximal tubular cells

• 1.1. After perfusion of isolated frog kidneys for 1 hr with 10⁻³ or 10⁻² M maleate Ringer, the peritubular membrane potential gradually declined in a dose-dependent manner.
• 2.2. The ouabain-like effects of maleate on cell Na and K activities were dose-dependent and smaller than the effects of zero K or 10⁻⁴M ouabain. Intracellular pH was not altered in the presence of 10⁻²M maleate.
• 3.3. The driving force for Na entry into the cell was reduced, respectively, to 81.4 and 58.4% (of control) in the presence of 10⁻³ and 10⁻² M maleate.
• 4.4. There was no histochemically detectable inhibition of proximal tubule Na-K ATPase activity during 3 hr of perfusion with 10⁻² M maleate.

     

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.