Soluble cyclic amp-dependent protein kinases from chick liver: Effects of NaCl and heat-stable modulator

• 1.1. Two cyclic AMP-dependent protein kinases—Fraction I and II—have been isolated from chick liver soluble preparation on DEAE-cellulose.
• 2.2. Both fractions have an apparent K_m for ATP of 2 × 10⁻⁶M, are stimulated maximally by 5 × 10⁻⁸ M cyclic AMP and phosphorylate mainly basic proteins—histone and protamine.
• 3.3. They exhibit various pH values for optimal activity and show differences with respect to both sensitivity to NaCl and substrate specificity.
• 4.4. The heat-stable protein modulator inhibits the cyclic AMP-dependent protein kinase activity of both fractions, but with cyclic GMP one kinase is stimulated and the other inhibited.
• 5.5. Slight differences in histone triggered holoenzyme dissociation as well as the lack of difference between their ability for subunit reassociation do not allow to classify these isozymes as protein kinases of Type I and II, according to Corbin et al. (1975).

     

Identification and characterization of a third form (D3) of cyclic 3'5'-nucleotide phosphodiesterase from rhizobwm fredii

• 1.1. A third form (D3) of cyclic nucleotide phosphodiesterase from Rhizobiumfrediiv/as detected and characterized for the first time.
• 2.2. The enzyme could hydrolyse both cyclic AMP and cyclic GMP with apparent K_m for cyclic AMP of approx. 0.2 μM.
• 3.3. D3 cyclic nucleotide phosphodiesterase had a pH optimum of about 6.0 when hydrolysing cyclic AMP.
• 4.4. The enzyme lost almost all its activity when heated to 60°C for 20 min.
• 5.5. Gel filtration with Sephadex G-100 gave a mol. wt of approx. 42.5 kD for the native enzyme.

     

A halogenated amino acid-containing sperm activating peptide and its related peptides isolated from the egg jelly of sea urchins,Tripneustes gratilla,Pseudoboletia maculata,Strongylocentrotus nudus,Echinometra mathaei and Heterocentrotus mammillatus

• 1.1. Twenty-eight peptides were isolated from the egg jelly of sea urchins, Tripneustes gratilla, Pseudoboletia maculata, Strongylocentrotus nudus, Echinometra mathaei (type A and B) and Heterocentrotus mammillatus and their amino acid sequences were determined.
• 2.2. Two of the peptides obtained from T. gratilla egg jelly possessed a bromophenylalanine (Br-Phe) residue in their sequences (Gly-(Br-Phe)-Asn-Leu-Asn-Gly-Gly-Gly-Val-Gly and Gly-(Br-Phe)-Asp-Leu-Asn-Gly-Gly-Gly-Val-Gly).
• 3.3. All of the peptides elevated cyclic GMP concentrations in the spermatozoa of the respective sea urchin and caused a shift in the apparent mol. wt of a major sperm protein of the respective sea urchin.
• 4.4. They stimulated respiration rates of the spermatozoa of Hemicentrotus pulcherrimus as well as their own species.
• 5.5. One-half maximal concentrations of the peptides for respiratory stimulation of H. pulcherrimus spermatozoa were between 10⁻¹¹ M and 10⁻⁹ M except a methionine-containing peptide which was about 10⁻⁷ M.

     

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.

     

Intracellular localization and characterization of 3-hydroxykynureninase in human liver

• 1.1. 3-hydroxykynureninase in human liver was present in cytosol and mitoehondria.
• 2.2. The cytosolic enzyme and mitochondrial enzyme had the same physiological and enzymic properties.
• 3.3. The enzyme had a mol. wt of 130,000 by gel filtration and isoelectric point of pH 5.9.
• 4.4. The enzyme was active for 3-hydroxykynurenine and kynurenine, and its activity ratio was 15:1. The apparent K_m values of the enzyme were 7.7 × 10⁻⁵M for 3-hydroxykynurenine, 1.0×10⁻³M for kynurenine and 2.5 × 10⁻⁶M for pyridoxal 5'-phosphate with 3-hydroxykynurenine.
• 5.5. Some other properties of purified enzymes are described.

     

On the role of fumarate reductase in anaerobic carbohydrate catabolism of Mytilus edulis L

• 1.1. The role of the fumarate:NADH oxidoreduction in the anaerobic glycolysis of the sea mussel is examined and discussed.
• 2.2. Fumarate reductase activity is present in submitochondrial particles especially from adductor muscle, digestive gland and mantle.
• 3.3. The pH optimum of the enzyme complex is 7.9; the approx K_m's for NADH and fumarate are 4.0 × 10⁻⁵ M and 6.3 × 10⁻⁵ M, respectively.
• 4.4. The enzyme complex is inhibitied by amytal, antimycin, ethanol, malonate, phosphate, rotenone, and succinate, and stimulated by Mg²⁺.
• 5.5. It is concluded that part of the mitochondrial respiratory chain is involved in the reduction of fumarate by NADH, comprising site 1 of the oxidative phosphorylation.

     

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.

     

Effects of pentoxifylline on cell shape,atp content and deformability in rabbit erythrocytes under hyperosmolar conditions

• 1.1. Pentoxifylline (I) [3,7-dimethyl-1-(5-oxo-hexyl)-xanthine] (0–10⁻³M) inhibited the discocyte-echinocyte transformation caused by hyperosmolarity and improved the impaired filtrability of erythrocytes due to hyperosmolarity.
• 2.2. The ATP content of erythrocytes was increased at low concentrations (0–3 × 10⁻⁵M) of I. but decreased at high concentrations (3 × 10⁻⁵ M or above) of I.

     

Isolation and characterization of lipoamide dehydrogenase from mackerel dark muscle

• 1.1. Lipoamide dehydrogenase was purified 1500-fold from mackerel dark muscle.
• 2.2. The enzyme was homogeneous as judged by acrylamide gel electrophoresis in the presence and absence of SDS.
• 3.3. Molecular weights of 102,000 and 55,000 were estimated for the native and denatured enzyme, respectively.
• 4.4. Optimal activity for the enzyme was obtained at around pH 5.7 and enhanced with citri acid.
• 5.5. Loss of activity was less than 5% by incubating the enzyme at 70°C for 20 min.
• 6.6. An apparent K_m of 3.1 × 10⁻³ M was obtained for dl-lipoic acid and 1.5 × 10⁻⁵ M for NADH.
• 7.7. The properties of lipoamide dehydrogenase from mackerel dark muscle observed in this investigation were very similar to those reported for the enzyme from other sources.

     

Binding proteins for cyclic AMP in mammalian tissues

• 1.1. The distribution and physicochemical properties of proteins known to bind cyclic AMP in vitro and methodological aspects of their interaction with ligands is reviewed.
• 2.2. The interaction between such proteins and cyclic AMP is discussed, the allosteric binding of the nucleotide to cyclic AMP dependent protein kinase type I being considered in detail.
• 3.3. The use of naturally occurring binding proteins in assays for cyclic AMP is briefly reviewed.
• 4.4. Finally, some aspects of the control of cyclic AMP binding in the intact cell are considered.

     

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.

     

Fluxes of dissolved amino acids between sea water and Echinaster

• 1.1. Measurement of free amino acid (primary amine) influx and efflux into the starfish, Echinaster, were accomplished utilizing improved methods of sea water purification and analysis.
• 2.2. Specimens placed in amino acid depleted sea water (5 × 10⁻⁸ M) demonstrated net release as measured with the fluorescamine method. Similarly, specimens placed in the same water to which amino acid mixtures had been reintroduced to normal levels demonstrated net uptake.
• 3.3. A mathematical model indicated an equilibrium amino acid concentration (when influx equals efflux) of 5.26 × 10⁻⁷ M, or about one fourth the level of natural sea water.
• 4.4. Since at normal environmental levels (20.65 × 10⁻⁷ M) net flux is inward by a ratio of nearly 4-1, it is concluded that the previous suggestions of some workers that such would not be the case for marine invertebrates are no longer valid.
• 5.5. The net uptake of amino acid from environmental levels would account for 5.67% of the measured total respiration if all were being metabolized.
• 6.6. This figure appears to be in line with the previously developed hypothesis that the epidermis largely obtains its nutrition directly from the environment. However, the real benefit of the uptake mechanism may be to prevent loss of the body amino acid pools.

     

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.

     

Comparative aspects of xanthine dehydrogenase activity of Panstrongylus megistus

• 1.1. Organ extracts from Panstrongylus megistus were assayed for xanthine dehydrogenase (E.C. 1.2.1.37) and xanthine oxidase (E.C. 1.2.3.2) activities.
• 2.2. XDH was present in the fat body, Malpighian tubes, testis and ovary, higher levels being found in the fat body. XO activity was practically absent.
• 3.3. Specific enzyme activity was fairly constant regardless the feeding condition of the insects.
• 4.4. The enzyme was able to oxidize xanthine (K_m = 2.8 × 10⁻⁴ M) and hypoxanthine (K_m = 4.0 × 10⁻⁴ M) but unable to oxidize pterine under the experimental conditions.

     

The influence of phosphate,fluoride and potassium ions on the activity of amp-deaminase from human skeletal muscle

• 1.1. Kinetic properties of the inhibitory effect of inorganic phosphate and fluoride and of the activatory effect of potassium ion on human skeletal muscle AMP-deaminase (E.C. 3.5.4.6) have been investigated
• 2.2. It has been shown that phosphate is a competitive inhibitor (K₁, ≈0.8 × 10⁻³M) and fluoride a noncompetitive inhibitor (K₁≈3.2 × 10⁻³ M) of human muscle AMP-deaminase.
• 3.3. The changes of potassium ion concentration between 20 and 200 mM did not influence the Michaelis constant which was about 0.9 x 10⁻³ M at 30°C. Also the change of substrate concentration in the range 40–300 μM did not influence the activation constant for potassium (K_a≈0.4 × 10⁻¹ M).
• 4.4. Higher concentraion of potassium (200mM) was found to diminish the “temperature sensitivity” of the enzyme activity.
• 5.5. The energy of activation (E) in the presence of 150 mM KC1 calculated from Arrhenius plot was about 4600 cal/mole of substrate. The heat of the enzyme-substrate complex formation obtained from the plot of log K_m vs T⁻¹ was shown to have positive value (+2200 cal/mole) at the temperatures lower than 23°C and negative value (—4100 cal/mole) at the temperatures higher than 23°C.

     

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 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 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.

     

Mechanisms controlling pigment movements within swordtail (Xiphophoprus helleri) erythrophores in primary cell culture

• 1.1. Pigment organelle (pterinosomes and carotenoid vesicles) movements within isolated swordtail (Xiphophorus helleri) erythrophores cultured in vitro were studied.
• 2.2. Pterinosome and carotenoid vesicles migrate centripetally to a juxtanuclear position or centrifugally into the dendritic processes of erythrophores in response to catecholamines.
• 3.3. Cateeholamine stimulation of alpha adrenoceptors results in centripetal pigment organelle migration whereas beta adrenoceptors mediate centrifugal movements.
• 4.4. Melatonin also induces perinuclear aggregation of pigment organelles but by receptor mechanisms separate from adrenoceptors.
• 5.5. MSH and ACTH stimulate pigment organelle dispersion. Centrifugal dispersion of erythrophore pigments is also stimulated by cyclic AMP and dibutyryl cyclic AMP.

     

Effect of calcium ions on ecdysteroid secretion by testes of Heliothis virescens

• 1.1. Testes of Heliothis virescens synthesized ecdysteroid in media containing low titers of calcium; the optimum calcium titer for testis sheaths stimulated to synthesize ecdysteroid in vivo was ca 1 mM, while the optimum of testes stimulated in vitro with the peptide testis ecdysiotropin was ca 0.3 mM calcium.
• 2.2. Verapamil at concentrations lower than 10⁻³ M induced increases in ecdysteroid synthesis, indicating more efficient synthesis when calcium influx was inhibited.
• 3.3. Hemolymph of H. virescens was 7 mM in calcium, while whole testes were maintained at 1–2 μM calcium.