Evidence for two isoforms of carbonic anhydrase II in the leech (Hirudo medicinalis) central nervous system

• 1.1. We dissected, homogenized and prepared ganglia and connectives from the central nervous system of medicinal leeches for SDS gel electrophoresis. The isolated proteins were transferred to nitrocellulose and incubated with affinity column-purified antibodies.
• 2.2. The immunoblots showed a strong positive reaction of a bovine carbonic anhydrase standard at a molecular weight of 29 kDa, and a distinct double-bond at the same molecular weight in the analyzed material.
• 3.3. We demonstrated with rat antibodies that carbonic anhydrase II is detectable in the leech central nervous system as the main isoenzyme.
• 4.4. The biochemical knowledge of carbonic anhydrase reported here agrees well with the immunocytochemical locations, thus affirming the validity of specific staining.

     

Carbonic anhydrase III content in various equine muscles

• 1.1. In this study, carbonic anhydrase III (CA-III) content in 18 equine muscles was determined by enzyme immunoassay.
• 2.2. It was found to differ in several muscles.
• 3.3. That in external intercostal muscle, rectus abdominis muscle and splenius muscle from four horses was very high.
• 4.4. Although the masseter muscle had only type I fibers, CA-III content was similar to that in mixed-fiber type muscles such as the biceps femoris muscle.
• 5.5. It thus appear that equine type I fibers can be further subgrouped.

     

Red cell carbonic anhydrase levels in flounders,Platichthys flesus L., from salt water and fresh water

• 1.1. Carbonic anhydrase levels in flounder red cells were unchanged by adaptation to salt or fresh water.
• 2.2. Two major red cell isoenzymes were found in flounder red cells after electrophoresis. These patterns were identical in all fish studied, whether from salt or fresh water environments. Thus no inherited or adaptive changes were observed.
• 3.3. Both flounder red cell carbonic anhydrase isoenzymes split a range of ester substrates. Activity was abolished with acetazolamide.

     

The turnover characteristics of carbonic anhydrase in mouse tissues

• 1.1. An affinity chromatography technique for the determination of turnover parameters has been applied to the study of carbonic anhydrase in a variety of mouse tissues.
• 2.2. The methodology has allowed the establishment of the relative turnover characteristics in these tissues, and the evaluation of the half lives and degradation rate constants.
• 3.3. Considerable variation was evident between the individual tissues, with synthesis being indicated as most rapid in intestine while degradation was highest in liver.
• 4.4. These data have been discussed in relation to available comparisons in the literature, and the known physiological correlations of carbonic anhydrase in these tissues.

     

Properties and distribution of Mg2+-HCO−3-ATPase in brush border membranes isolated from rat small intestine

• 1.1. The small intestine was cut into seven segments and properties and distribution of brush border Mg²⁺-HCO⁻₃-ATPase activity in each segment were examined.
• 2.2. The optimal Mg²⁺ concentration was 1.0 mM.
• 3.3. The optimal HCO⁻₃ concentration was 100 mM in the first (duodenal), 50 mM in the 3rd and 40 mM in the 5th segment, respectively.
• 4.4. The optimal pH value was about 9.0.
• 5.5. l-phenylalanine (above 1 mM) and SCN⁻ (above 50 mM) significantly inhibited both Mg²⁺- and Mg²⁺-HCO⁻₃-ATPase activity.
• 6.6. The enzyme activity was found to be highest in the duodenal segment and then gradually decreased in consecutive segments as well as β-glycerophosphatase, Na⁺-K⁺-ATPase and supernatant carbonic anhydrase.
• 7.7. The functional significance of this ATPase and the relationship with carbonic anhydrase was discussed.

     

Developmental changes of carbonic anhydrase activity of parotid gland and stomach of goat

• 1.1. Carbonic anhydrase activities in the various tissues of ruminants and non-ruminants were compared.
• 2.2. The highest activity was found in the parotid gland of ruminants such as bovine and goat. However the activity in the kidney was not significantly different between the ruminants and non-ruminants.
• 3.3. The effect of development on the carbonic anhydrase activity was studied. The activities in both the parotid gland and kidney of the goat were found to increase with age whereas the activities of the pancreas, liver and submaxillary gland did not change significantly.
• 4.4. The activities in the abomasum of the goat also increased with age however the other stomachs did not vary prominantly.

     

Kinetic properties of primitive vertebrate carbonic anhydrases

• 1.1. Carbonic anhydrases from the red cells, ocular secretory tissues, and rectal gland of species of Myxine, elasmobranchii, and Teleostii, were examined using rates of CO₂ hydration. The enzyme is absent from corneal endothelium and lens of elasmobranchs and salt water teleosts, but present in fresh water fish.
• 2.2. The red cell carbonic anhydrase of the representative elasmobranch, Squalus acanthias, is a single enzyme of relatively low activity, K_cat = 2 × 10⁴ sec⁻¹ at 1°C. The ciliary folds and rectal gland of S. acanthias contain carbonic anhydrases with turnover numbers five times higher than the red cell enzyme.
• 3.3. Both red cell and secretory enzymes of S. acanthias are susceptible to inhibition by sulfonamides within a 10-fold range of mammalian secretory carbonic anhydrases. In general, they are moderately sensitive to anion inhibition; a notable exception is enzyme from rectal gland which is insensitive to halion inhibition.
• 4.4. In teleosts, both red cell and secretory carbonic anhydrases have a high turnover number, and are susceptible to sulfonamide inhibition. In red cells there appear isozyme(s) of lower activity, in considerable concentration.
• 5.5. Taken with earlier ion transport work in S. acanthias, the basic vertebrate pattern of aqueous humor formation, both chemically and physiologically, appears to be established in this “primitive” species.
• 6.6. The finding of at least three different types of carbonic anhydrases in S. acanhias suggests that separate loci for the enzyme have existed throughout most of vertebrate evolution.

     

Na+-K+ ATPase and carbonic anhydrase activities in larvae,postlarvae and adults of the shrimp Penaeus japonicus (Decapoda,Penaeidea)

• 1.1. Activities of Na⁺-K⁺ ATPase and carbonic anhydrase were measured through the early post-embryonic development of Penaeusjaponicus. In adults, only the Na⁺-K⁺ ATPase activity was measured.
• 2.2. ATPase activity was variable in the successive development stages. From zero in nauplii, the activity slightly increased in zoeae, and rose sharply in mysis stages 2 and 3.
• 3.3. A further significant increase in activity was noted at the transition from late mysis to early postlarvae, concomitant with a change from the larval osmoconforming pattern of osmoregulation to the postlarval and adult hyper-hyporegulating pattern.
• 4.4. The activity of Na⁺-K⁺ ATPase, measured in isolated cephalothorax, increased from PL3 to PL4 to its maximum value in PL5; at this stage, osmoregulatory capacity was fully efficient.
• 5.5. In young stages of P. japonicus, the variations in Na⁺-K⁺ ATPase activity appear correlated with the development of osmoregulatory ultrastructures, and with osmoregulation and salinity tolerance.
• 6.6. These results are discussed with regard to their ecological and physiological implications.
• 7.7. In adults, the activity of Na⁺-K⁺ ATPase was high in gills and epipodites and no activity was detected in branchiostegites. These results are related to the ultrastructure of these organs.
• 8.8. The activity of carbonic anhydrase did not change significantly in larval and postlarval stages.
• 9.9. From these results, it is proposed that the effector sites of osmoregulation are located in branchiostegites, pleurae and epipodites in postlarvae, and in epipodites and mainly in gills in adults.

     

Partial purification of rat liver cytoplasmic acetyl-CoA synthetase; Characterization of some properties

• 1.1. Rat liver cytoplasmic acetyl-CoA synthetase was partially purified (purification factor = 23, yield = 30%).
• 2.2. The apparent K_ms for acetate, coenzyme A, ATP and MgCl₂ were determined and found to be 52.5 μM, 50.5 μM, 570 μM and 1.5 mM, respectively.
• 3.3. The partially-purified enzyme showed a low affinity for short-chain carbon substrates other than acetate.
• 4.4. The properties of the partially-purified enzyme were compared with those of enzymes from other sources.

     

Aerobic and anaerobic respiration of adult Artemia salina L., acclimated to different oxygen concentrations

• 1.1. Adult male Atremia salina L. were acclimated to five different oxygen concentrations and their respiration in response to environmental oxygen concentrations was determined.
• 2.2. Anemia is a respiratory regulator over a wide range of partial O₂ pressures. A critical oxygen tension exists and decreases with acclimation to lower pO₂.
• 3.3. Hypoxic conditions induce the production of hemoglobin III.
• 4.4. Lactic acid is produced during anaerobiosis.
• 5.5. Production of Hb III and lactic acid, being inversely proportional to the acclimation level, has to be considered as a long term or short term adaptation to hypoxic conditions.

     

Characterization and purification of biliverdin reductase from the liver of eel,Anguilla japonica

• 1.1. Biliverdin reductase from the liver of eel, Anguilla japonica was characterized and purified with a novel enzymatic staining method on polyacrylamide electrophoretic gel.
• 2.2. This enzyme could use both NADPH and NADH as coenzyme. The K_m of NADPH was 5.2 μM, while that of NADH was 5.50 μM.
• 3.3. The optimum reaction pH for using HADPH as coenzyme was 5.3. That for NADH was 6.1. The optimum reaction temperature is 37°C.
• 4.4. When NADPH was used as coenzyme, the K_m of biliverdin was 0.6 μM. When NADH was used as coenzyme, the K_m of biliverdin was 7.0 μM.
• 5.5. The activity of the enzyme was inhibited by the concentration of biliverdin. Also, the potency of the enzyme was much less than that of the analogous enzyme isolated from mammals.
• 6.6. This is a fairly stable enzyme with a mol. wt around 67,000. Its estimated pI was pH 3.5–4.0.
• 7.7. This is the first time biliverdin reductase has been isolated and characterized from a vertebrate other than mammals. The property of it is quite different from that of mammals.

     

Purification and some particular kinetic properties of rat spleen cytosolic deoxynucleotidase

• 1.1. Rat spleen cytosolic deoxynucleotidase was purified 40,000-fold to almost homogeneity and had a specific activity of 3000 μmol/min per mg.
• 2.2. Molecular mass of the native enzyme was 45 kDa. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis indicated that the native enzyme comprises two identical 27-kDa subunits.
• 3.3. Specific enzyme activity increases with increasing concentration of enzyme protein and approaches a plateau at high enzyme concentrations.
• 4.4. Enzyme activity increases gradually and nonlinearly with increasing concentration of enzyme in the low concentration range. Above a certain concentration the increase attains a maximal and constant slope.
• 5.5. The kinetic properties can be explained by assuming dissociation of the enzyme into subunits with low or no activity.

     

Non-parallel secretion of pancreatic enzymes in sheep following hormonal or vagal stimulation

• 1.1. The flow of pancreatic juice and its composition of protein, amylase, trypsin and chymotrypsin were measured in sheep during treatments known to induce a response in nonruminants.
• 2.2. Intraduodenal peptone (100 or 200 μg/min) had no affect but intraduodenal hydrochloric acid (66 μequiv/min) or intravenous (iv) pentagastrin (10 μg/min) doubled the flow and enzyme output. Cholecystokinin (1.0 IDU/min iv) caused smaller changes in enzyme output but no change in flow, whereas, secretin (0.5 or 1.0 CU/min iv) caused a rapid, sustained, five- or six-fold increased in flow but only a transitory increase in enzyme output.
• 3.3. The largest increases in enzyme output occurred during stimulation of the vagus (10 Hz, 10 V); the outputs were sustained at 5–10 times control levels and the flow increased two- or three-fold.
• 4.4. A non-parallel response of amylase, trypsin and chymotrypsin occurred during administration of those treatments which significantly enhanced the enzyme output. Compared with periods of basal secretion the stimulated juice contained significantly more chymotrypsin and amylase than trypsin; the relationship between chymotrypsin and amylase did not change significantly.
• 5.5. The composition of the juice during stimulation approached and often equalled the enzyme composition of pancreatic tissue.
• 6.6. These results are compatible with the view that the mixture of enzymes in pancreatic juice is derived from at least two compartments with different enzyme compositions.

     

Human GMP synthetase

• 1.1. The specific activity of GMP synthetase was measured in several human tissues and found to be highest in cultured skin fibroblasts, followed by bone marrow, leukocytes, erythrocytes. placenta, and liver.
• 2.2. The enzyme from fibroblasts was purified approximately 50-fold by ammonium sulfate fractionation and gel filtration.
• 3.3. The K_m values were determined to be 4.9μM for XMP, 270μM for ATP. and 340 μM for glutamine.
• 4.4. Ammonium sulfate could replace glutamine as the amino donor but was much less efficient.
• 5.5. The enzyme was specific for ATP as the energy source.
• 6.6. Unlike the calf thymus enzyme, the human enzyme has no requirement for a reduced sulfhydryl compound.
• 7.7. Human GMP synthetase is inhibited by ATP, dATP, azaserine, and hydroxylamine.

     

Comparative study of hemoglobins from different Artemia populations. The influence of temperature on the oxygen equilibria

• 1.1. The P50 values of extracellular hemoglobin (Hb) of five Artemia populations from different geographical origin are affected by temperature.
• 2.2. The free oxygen binding energy is high for all the populations (ΔH between −34.7 and −56.2kj/mol).
• 3.3. A possible correlation between thermal sensitivity of Hb and the ambient temperature of the habitat must be considered very carefully.
• 4.4. The occurence of different quantities of Hb1 (αα chains) Hb2 (αβ chains) and Hb3 (ββ chains) in the different populations possibly influences thermal sensitivity.

     

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).

     

Purification and characterization of hatching enzyme of the pike (Esox lucius)

• 1.1. A choriolytic enzyme was isolated from the hatching medium of the pike, Esox lucius.
• 2.2. The enzyme is defined as hatching enzyme.
• 3.3. The molecular weight of the enzyme is 24,000.
• 4.4. The enzyme is a glycoprotein containing 2% carbohydrate.
• 5.5. Its isoelectric point is 6.5.
• 6.6. The pH optimum is around pH 8.
• 7.7. The enzyme molecule contains two disulfide bonds but no free cysteine.
• 8.8. Inhibitor studies and metal analysis show that the enzyme is a zinc-metalloprotease.

     

Inhibition of dihydrofolate reductase by palmitoyl coenzyme A

• 1.1. Palmitoyl-CoA was found to inhibit bovine liver dihydrofolate reductase.
• 2.2. 50% inhibition of the enzyme was obtained with 1.5 μM palmitoyl-CoA.
• 3.3. The inhibition was reversed by addition of bovine serum albumin, β-cyclodextrin or spermine.

     

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.

     

Accumulation,distribution and toxicity of selenium in the adult house fly,Musca domestica

• 1.1. Accumulation and distribution of dietary Se in relation to mortality was investigated in adult house flies.
• 2.2. The midgut preferentially accumulated Se and thereby limited toxicity.
• 3.3. Midgut Se concentrations were from 6- to 107-fold higher than in carcass, and from 15 to 71% of the total Se was associated with midgut.
• 4.4. When dietary levels of Se were raised the midgut saturated at 15 μg Se/g tissue, followed by a rise in carcass levels to greater than 0.5 μg Se/g tissue and increased mortality.
• 5.5. Se levels in lysosomal fractions were from 3- to 50-fold higher than in other subcellular fractions, suggesting that Se is sequestered in lysosomes.
• 6.6. Se added to drinking water was toxic at 4–8 ppm.