Studies on the active centre(s) of rat liver porphyrinogen carboxy-lyase. in vivo effect of hexachlorobenzene on decarboxylation site(s) of porphyrinogens

• 1.1. The role of histidine on the decarboxylation of porphyrinogens of 7-, 6-, and 5-COOH III brought about by porphyrinogen carboxy-lyase (PCL) was studied.
• 2.2. For this purpose hepatic PCL from normal and hexachlorobenzene (HCB) treated rats were modified with diethylpyrocarbonate.
• 3.3. The results indicated that the enzyme from both normal and porphyric animals had histidine at the binding sites of all the porphyrinogens assayed.
• 4.4. Comparative studies between the enzyme from normal and porphyric rats suggested that in vivo HCB treatment affected the active site for the decarboxylation of 7-, 6- and 5-COOH porphyrinogens III at histidine residues.
• 5.5. On the other hand arginine modification by 2,3-butanedione treatment altered 5-COOH porphyrinogen III decarboxylation for both enzymes. However this amino acid was not involved at the binding site of this substrate.

     

Metabolic capacity,fibre type area and capillarization of rat plantaris muscle. Effects of age,overload and training and relationship with fatigue resistance

• 1.1. The influences of age (5, 13 and 25-month-old rats), overload as obtained by denervation of synergists, and training on the metabolic capacity, relative muscle cross-sectional area occupied by each fibre type, capillarization and fatigue resistance of the rat m. plantaris were investigated.
• 2.2. Creatine kinase, phosphorylase and citrate synthase activities were lower in muscles of 25 than in those of 13-month-old rats (P < 0.001).
• 3.3. Overload resulted in an increased relative area of type I and II a fibres at all ages (P = 0.001).
• 4.4. Capillary density decreased with overload and increasing age (P < 0.001).
• 5.5. Fatigue resistance was higher in muscles of 13 than in those of 5-month-old rats (P < 0.05), and increased with overload (P < 0.05) at all ages.
• 6.6. Fatigue resistance of the whole muscle was not closely related to its oxidative capacity in contrast to what is generally found for single fibres or motor units.

     

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.

     

Myofibril and sarcoplasmic reticulum changes during muscle development: Activity vs inactivity

• 1.1. The purpose of this study was to determine whether biochemical changes of skeletal muscle that occur as a result of exercise in young rats persist into adulthood.
• 2.2. Littermates (10 days old) were assigned to a 3, 6 and 12 week control or training group. In addition, a rest-exercise group (R-E) and exercise-rest (E-R) group were included.
• 3.3. The rest-exercise and exercise-rest rats were maintained for the 12 weeks with the first 6 weeks being either rest or exercise and the condition reversed during the last 6 weeks of the experiment.
• 4.4. Myofibril ATPase activity of rat plantaris increased from the 10d to 12 week animals (P < 0.05). As anticipated, training resulted in a lowered activity at 6 and 12 weeks compared to controls.
• 5.5. The Ca²⁺ uptake and Ca²⁺-ATPase activity of the sarcoplasmic reticulum followed a similar pattern.
• 6.6. With regard to the exercise-rest rats, the myofibril and SR ATPase activities at 12 weeks were comparable to the 12 weeks control rats.
• 7.7. The rest-exercise group approximated the 12 week training group with regard to myofibril and SR ATPase activities (P > 0.05).
• 8.8. The results suggest that the training adaptations that occur during development of skeletal muscle return to normal, when training ceases in the adult rat.
• 9.9. Furthermore, animals that started to train prior to puberty do not have a greater capacity to adapt than animals which initiated training during adulthood.

     

Skin blood flow in the Wistar-Kyoto rat and the spontaneously hypertensive rat

• 1.1. Using laser Doppler techniques in man, we have previously demonstrated differences in skin blood flow properties at sites with primarily nutritive (NUTR) perfusion, such as the elbow or knee, as compared to sites such as the finger pulp, with predominantly arteriovenous anastomotic (AVA) perfusion.
• 2.2. Basal and heat stimulated flow is greater at AVA sites. In man, blood pressure changes are reflected primarily by changes at AVA rather than NUTR sites.
• 3.3. These blood pressure induced changes affect the red blood cell velocity (VEL) component at AVA sites more than microvascular volume (VOL).
• 4.4. Given these findings in man, we decided to compare skin blood flow properties in a suitable animal model.
• 5.5. We chose the Wistar-Kyoto (WKY) and Spontaneously Hypertensive Rat (SHR) strains, in view of the marked difference in systemic blood pressure in these two related strains.
• 6.6. Skin blood flow varied considerably at different skin sites in the rats. Skin sites with hair covering, on the back and at the base of the tail, showed low basal and heat stimulated blood flow.
• 7.7. In contrast, the plantar surface of the paw behaved similarly to the finger or toe pulps in man, with 3–4-fold higher basal flow than the hair covered areas and a 7–8-fold rise with local heating to 44°C.
• 8.8. Furthermore, there was a 25% greater blood flow at the plantar paw surface in the SHR rats as compared to the WKY rats, corresponding to the 25% higher systemic blood pressure in these animals.
• 9.9. The heat induced increase in flow at the plantar surface of the paw was primarily a result of a marked increase in VEL rather than VOL.
• 10.10. The higher flow at this site in SHR as compared to WKY rats was likewise ascribable to an increase in VEL, VOL being equivalent in the two strains.

     

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.

     

Effects of high dietary methionine on activities of selected enzymes in the liver of kittens (felis domesticus)

• 1.1. Growing male kittens were fed an 18% casein diet supplemented with 2, 3, or 4% l-methionine (MET) for 6 weeks.
• 2.2. Free MET concentration in liver increased 30-fold and cystathionine two- to three-fold; the activity of adenosyl-MET transferase and cystathionase also increased but remained lower than previously found in rats.
• 3.3. Taurine concentration in liver decreased in cats fed excess MET and appeared to depend on taurine intake.
• 4.4. Alanine aminotransferase activity was high in all groups while serine dehydratase activity was very low.
• 5.5. Pyruvate kinase and malic enzyme activities which are normally low in cat liver increased after excess MET. Also, glucose 6-phosphate and 6-phosphogluconate dehydrogenases increased.
• 6.6. Cat liver metabolism showed limited adaptation to an excess dietary intake of methionine compared to that found in rats.

     

Purification and characterization of elastase from the pyloric caeca of rainbow trout (Oncorhynchus mykiss)

• 1.1. An elastase-like enzyme was purified from the pyloric caeca of rainbow trout by hydrophobic interaction, cation exchange and gel-filtration chromatography.
• 2.2. The approximate molecular weight of the elastase was 27 kDa and the isoelectric point was remarkably basic.
• 3.3. The pH optimum of this enzyme was 8.0, when assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide.
• 4.4. When assayed with Succinyl-Ala-Ala-Ala-p-Nitroanilide, the enzyme activity had a temperature optimum of 45°C, and the enzyme was stable up to this temperature.
• 5.5. The trout elastase exhibited a higher specific activity than porcine elastase against Succinyl-Ala-Ala-Ala-p-Nitroanilide and elastin-orcein.
• 6.6. The trout elastase was inhibited by elastatinal, PMSF, TPCK, SBTI and Bowman-Birk inhibitor.

     

Allozyme variation in the stingless bee Trigona fuscobalteata (hymenoptera,apidae) from Peninsular Malaysia

• 1.1. A population of Trigona fuscobalteata from Peninsular Malaysia was analysed for genetic variation at 9 gene-enzyme systems comprising 13 loci.
• 2.2. Two gene-enzyme systems (phosphoglucomutase and isocitrate dehydrogenase) were polymorphic in the 20 colonies studied.
• 3.3. Isocitrate dehydrogenase was represented by duplicate genes.
• 4.4. The number of loci for several enzyme systems appeared to be different from that reported for the Australian stingless bees.

     

The regulation of glucose 6-phosphate dehydrogenase from Dicentrarchus labrax (bass) liver

• 1.1. Kinetic constant values of the reaction catalyzed by bass liver glucose 6-phosphate dehydrogenase show to be modified between 10 and 40°C.
• 2.2. The Arrhenius plot between 10 and 50°C shows two slopes with different activation energies.
• 3.3. These results suggest a regulation of this enzyme by environmental temperature.
• 4.4. Kinetics of ATP inhibition were examined between pH 6.2 and 7.8: patterns and K_i values obtained are affected by the pH variation.
• 5.5. NADH is an effective inhibitor of bass glucose 6-phosphate dehydrogenase but this enzyme does not show NAD-linked activity.
• 6.6. Kinetics of pyridoxal 5′-phosphate inhibition have indicated the presence of a lysine in the catalytic site for NADP⁺.

     

Secretory oedema in diabetic submandibular glands during parasympathetic nerve stimulation: Relationship to microvascular abnormalities in streptozotocin-treated rats

• 1.1. Submandibular secretion during parasympathetic stimulation (5 Hz) was examined in streptozotocin-diabetic and age-matched control rats.
• 2.2. At 3 weeks, but not 3 and 6 months, flow rate was initially greater than in controls, but it declined rapidly after 30 min.
• 3.3. The reduction in flow rate was associated with oedema of the gLond.
• 4.4. At 3 months, graded stimulation revealed a tendency to oedema at frequencies of 10 Hz and above.
• 5.5. Morphologically, submandibular capillary density was increased in diabetic rats.
• 6.6. Thus, in diabetes the submandibular gland appears less able to withstand continuous parasympathetic stimulation, due in part to an increase in tissue capillary area.

     

Purification and some properties of an atypical alkaline p-nitrophenylphosphate phosphatase activity from Halobacterium halobium

• 1.1. An alkaline p-nitrophenylphosphate phosphatase has been purified 440-fold from extracts of Hatobacterium halobium.
• 2.2. The enzyme has an apparent molecular weight of 24,000.
• 3.3. A K_m value for p-nitrophenylphosphate of 1.12mM has been found under optimal conditions.
• 4.4. The enzyme is selectively activated and stabilized by Mn²⁺.
• 5.5. It requires high salt concentrations for stability and maximum activity.
• 6.6. It displays an unusual restricted substrate specificity of 25 phosphate esters tested, only phosphotyrosine and casein were hydrolysed besides p-nitrophenylphosphate.

     

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.

     

Ostrich fructose-1,6-bisphosphatase: Kinetic characterization of the liver isoenzyme

• 1.1. Purified ostrich (Struthio camelus) liver fructose-1,6-bisphosphatase exhibited an absolute requirement for Mg²⁺.
• 2.2. The enzyme catalyzed the hydrolysis of fructose-1,6-bisphosphate, sedoheptulose-l,7-bisphosphate and ribulose-l,5-bisphosphate.
• 3.3. S_0.5 for substrate was 1.4 μM.
• 4.4. AMP was a potent non-competitive inhibitor with respect to substrate (K_i of 25 μM).
• 5.5. Fructose-2,6-bisphosphate was a potent competitive inhibitor of the enzyme (K_i of 4.8 μM).

     

Soluble and immobilized RP. palustris rhodanese—II. Some particular kinetic behaviour

• 1.1. Kinetic studies were carried out on the soluble and immobilized Rhodanese.
• 2.2. The soluble enzyme showed a typical Michaelis-Menten behaviour, an inhibitory effect was observed at high thiosulphate and cyanide concentrations.
• 3.3. The product sulphite was also an inhibitor, instead thiocyanate increased the enzyme velocity when it was added to the incubation mixture.
• 4.4. A ping-pong mechanism was proposed for Rp. palustris Rhodanese with a stable (free enzyme: E) and an unstable (sulfur substituted enzyme: ES) kinetic enzyme form.
• 5.5. The insolubilized Rhodanese presented an unusual kinetic behaviour, with sigmoid shape substrate profiles and non-linear double reciprocal plots.
• 6.6. From the empirical Hill equation, positive cooperativity (n>1) was found for both substrates.

     

A study of glycerolphosphate acyltransferase in rat liver mitochondria-submitochondrial localization and some properties of the solubilized enzyme

• 1.1. Glycerolphosphate acyltransferase (GPAT) was solubilized from the rat liver mitochondrial membranes using sodium cholate. Dithiothreitol was necessary to stabilize the solubilized enzyme on storage.
• 2.2. Unlike the enzyme in situ in mitochondrial membranes, the solubilized mitochondrial GPAT was susceptible to inhibition by N-ethylmaleimide; a property more characteristic of the distinct microsomal form of GPAT.
• 3.3. Solubilized mitochondrial GPAT retained its very high preference for saturated acyl-CoA substrate (palmitoyl-CoA) and had no activity whatever with any tested concentration of the unsaturated substrate oleoyl-CoA.
• 4.4. Solubilization increased the affinity of mitochondrial GPAT for palmitoyl-CoA whilst decreasing the K_m for glycerol phosphate.
• 5.5. After separation of liver mitochondrial outer and inner membranes and estimation of cross-contamination by appropriate markers it was concluded that the mitochondrial inner membrane contains significant GPAT activity. This was established with preparations from fed, 48 hr-starved and streptozotocin-diabetic rats.

     

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⁻.

     

Main kinetic characteristics of acetylcholinesterase from brain of Hypostomus punctatus,a Brazilian bentonic fish (cascudo)

• 1.1. A potentiometric method for the assay of cholinesterase has been proposed and compared with a colorimetric assay.
• 2.2. Main kinetic parameters of cholinesterase from Hypostomus punctatus brain were determined indicating that true acetylcholinesterase is by far the predominant enzyme in the brain of this fish.
• 3.3. We have compared our data with published results described from other fish species.
• 4.4. The enzyme inhibition achieved after 3 hr incubation of brain homogenates with ethyl-parathion have indicated that this enzyme shows a characteristic organophosphorous sensitive behavior.

     

The effect of γ-radiation on the NADP-MALIC enzyme from mango fruit,mangifera indica—I. Physico-chemical aspects

• 1.1. Malic enzyme purified from the fruit tissue of Mangifera indica was irradiated in dilute solution and the effect of γ-irradiation was investigated.
• 2.2. The activity of the enzyme decreased exponentially as a function of the applied dose under all conditions investigated. The inactivation yield (Go-value) in neutral solution and in air was 0.069.
• 3.3. The role of the radicals produced by water radiolysis in the inactivation of the enzyme was investigated by using different gas atmospheres and selective free radical-anions. The hydrogen atom and the hydrated electron (reducing species) were found to be important in the enzyme inactivation; as well as the possible destruction of cysteine and tryptophan residues.
• 4.4. The irradiated enzyme appears to adopt a more compact conformation as reflected in a slightly lower M_r, Stokes-radius and diffusion coefficient.
• 5.5. γ-Radiation does not lead to any heterogeneity in the charge and size properties of the enzyme and the pI and the M_r of the subunits were unaffected.
• 6.6. Some differences in the amino acid composition of the non-irradiated and irradiated enzyme were observed but specific amino acid residues were not preferentially destroyed.
• 7.7. These changes were also reflected in the ultraviolet spectrum of the enzyme which shifted to lower values.
• 8.8. The major cause of inactivation seem to be a change in conformation caused by chemical modification of amino acid side chains.