Distribution of enzymes of cGMP metabolism in glomeruli and tubules isolated from normal and nephrotic rat kidney cortex

• 1.1. Puromycin aminonucleoside (PA) nephrosis may constitute a good experimental model to investigate the involvement of the cGMP system in the regulation of several kidney functions and especially glomerular permeability.
• 2.2. After a single intravenous injection of PA we studied the evolution of the guanylate cyclase (GCase) and cGMP-phosphodiesterase (G-PDE) activities in pure preparations of glomeruli (Gl) and tubules (TU).
• 3.3. Both Gl and TU homogenates showed a strong increase of the GCase activity 12 days after PA injection.
• 4.4. In the presence of Triton X-100, TU homogenate GCase showed the same pattern as in absence of this detergent while the Gl enzyme decreased unexpectedly. On the other hand, the only G-PDE change was observed in the TU where this activity decreases progressively.
• 5.5. Gl pellets and TU supernatants showed similar changes as in total homogenates. But, compared to the total homogenate, both Gl and TU supernatant GCases were strongly activated and in the Gl these activation rates were not the same in normal and 12 days-nephrotic rats.
• 6.6. These results could be explained by the existence of a membrane bound GCase “effector” involved in the physiopathological evolution of the disease.
• 7.7. In conclusion it seems to be clear that the cGMP-system is involved in the evolution of PA-nephrosis. But the precise relation between variations in the cGMP-system and the disease remains unclear and needs further investigation.

     

Phenoloxidase activity of acridid grasshoppers from the subfamilies melanoplinae and oedipodinae

• 1.1. Phenoloxidase activity and wound melanization was studied in five species of grasshoppers representing the subfamilies Melanoplinae and Oedipodinae.
• 2.2. Most of the phenoloxidase activity was detected in the plasma fraction of grasshopper whole-body homogenates and supernatant fractions of the hemolymph. The species representing the Oedipodinae had 20–50% higher percentage of the total phenoloxidase activity associated with particulate matter from a whole-body homogenate when compared to the Melanoplinae.
• 3.3. Phenoloxidase activity could not be detected in sclerotized cuticle of adult grasshoppers.
• 4.4. The phenoloxidase existed as a zymogen which could be activated by chymotrypsin and inhibited by KCN and NaCN while EDTA showed no effect. It had optimum activity at 37°C and pH 7.3.
• 5.5. These findings are discussed in relation to wound repair and immune responses to infection in grasshopper species.

     

Role of antiproteolytic heparin-binding serum protein(s) in modulating the levels of sialyl- and galactosyltransferase activity released during the incubation of rat jejunal slices

• 1.1. Sialyltransferase released into the medium during the incubation of rat jejunal slices in serum-free buffer, was susceptible to proteolytic degradation. Heat inactivated horse serum or its antiproteolytic heparin-binding fraction was found to be necessary in determining the activity of sialyltransferase released (Nadkarni et al., 1991).
• 2.2. In the present study, we have shown that heat inactivated rat serum (HRS) or its antiproteolytic heparin-binding fraction (HBF) had a role in determining the sialyltransferase activity released during jejunal slice incubations.
• 3.3. Galactosyltransferase was also released during incubations, but was not proteolytically degraded and the presence of HRS or HBF in incubations did not alter the levels of galactosyltransferase activity released.
• 4.4. Trypsin activity in serum-free incubation medium was higher compared to medium containing HRS.
• 5.5. Addition of serum-free medium obtained from 4 hr incubations of the jejunal slices, to medium obtained from parallel incubations done in the presence of HRS, caused inhibition of sialyl- but not galactosyltransferase activity.
• 6.6. In jejunal homogenates stored at −20°C, sialyltransferase activity was decreased during 0–45 days of storage, whereas galactosyltransferase activity remained fairly stable for upto 56 days.
• 7.7. Inclusion of HRS or HBF in homogenates resulted in higher sialyl- but not galactosyltransferase activity compared to serum-free homogenate samples.
• 8.8. The results suggest that HRS or its antiproteolytic heparin-binding proteins have a role in determining the sialyltransferase activity released from the jejunal slices. In contrast galactosyltransferase released was not susceptible to proteolysis, and HRS or HBF was not required to express its activity.

     

In vitro Ca deposition by rat matrix vesicles: Is the membrane association of alkaline phosphatase essential for matrix vesicle-mediated calcium deposition?

• 1.1. Phosphatidylinositol phospholipase C (PI-PLC) treatment of rachitic rat matrix vesicles (MVs) released about 80% of membrane-bound alkaline phosphatase (ALP), AMPase, PPiase into the media.
• 2.2. About 20% hydrolytic activity was not released from MV membranes by PI-PLC treatment.
• 3.3. SDS-polyacrylamide gel electrophoresis and Western blot analysis showed only one immunoreactive protein corresponding to the molecular weight of ALP present in the soluble fraction after PI-PLC treatment.
• 4.4. The specific activity of the released ALP was at least 5-fold higher than the residual activity.
• 5.5. After PI-PLC treatment, MVs also demonstrated an 80% reduction of AMP- or βGP-dependent calcium deposition.
• 6.6. The soluble fraction containing 80% of ALP activity was unable to support calcium deposition. The mixing of the soluble and insoluble fractions after PI-PLC treatment failed to fully restore calcium-depositing activity.

     

Human seminal phosphatase: Properties and comparison with plant phosphatases

• 1.1. Phosphatase activities were determined in various seminal fluid and plant tissues.
• 2.2. Human semen showed a markedly higher phosphatase activity, and some plants and mushrooms exhibited a considerably higher phosphatase activity.
• 3.3. Phosphatase purified from human seminal fluid showed a pH optimum of 5–6 and was potently inhibited by l-(+)-tartrate.
• 4.4. Tartrate could not inhibit most plant phosphatases, but inhibited the mushroom phosphatase with a one-order lower affinity than that of the seminal enzyme.

     

The haemocytes and the activities of leucine aminopeptidase and alkaline phosphatase during development of Ceratitis capitata: Specific secretion of a leucine aminopeptidase isozyme

• 1.1. The types of haemocytes during larval development were studied.
• 2.2. The developmental profile of leucine aminopeptidase and alkaline phosphatase was studied. The maximum LAP activity was found to be in early larval development, while the maximum alkaline phosphatase during the white pupal stage.
• 3.3. These activities were compared with those determined in cell-free haemolymph.
• 4.4. Both hydrolytic enzymes have been found histochemically in the prohaemocytes and in the plasmatocytes.
• 5.5. In cultured haemocytes experiments it was found that 64% of the total LAP activity was secreted into the incubation medium, while electrophoretic analysis of released LAP activity demonstrated that only LAP A isozyme was secreted.
• 6.6. Based on the above results we suggest that both hydrolytic enzymes are functionally important throughout larval development.

     

Comparison of activities of several enzymes and protein constituents of oviducal and breast muscle of Japanese quail,Coturnix coturnix Japonica

• 1.1. Activities of several enzymes and protein constituents of magnum, isthmus, shell gland and breast muscle (pectoralis major) of Japanese quail, Coturnix coturnix japonica were compared.
• 2.2. The respective activity per g wet weight of lactate dehydrogenase, malate dehydrogenase and l-glycerol 3-phosphate dehydrogenase in pectoralis major was approx 20 times that of these enzymes in isthmus which showed the highest activity among oviducal tissues. On the other hand, the respective activity of lactate dehydrogenase was similar to that of malate dehydrogenase and was approx 10–20 times that of l-glycerol 3-phosphate dehydrogenase in each tissue.
• 3.3. Among NADPH-producing enzymes, NADP⁺-isocitrate dehydrogenase showed the highest activity in all tissues. The activity of malic enzyme was lowest in oviducal tissues, but was next to NADP⁺-isocitrate dehydrogenase in pectoralis major.
• 4.4. Sodium dodecyl sulfate polyacrylamide gel electrophoretic patterns of the whole homogenate, the supernatant and the precipitate fraction of magnum, isthmus, shell gland and pectoralis major showed tissue specific protein compositions. Proteins with molecular weight of 55,000, 70,000, 110,000 and 130,000 were observed in the respective precipitate (myofibrilar) fraction of magnum, isthmus and shell gland, but not in that of pectoralis major. It was noteworthy that the amount of myosin heavy chain of magnum was markedly less than that of other three tissues.

     

Purification and partial characterization of the alkaline phosphatase from Allolobophora caliginosa

• 1.1. A purification of the enzyme from the starting material was achieved by means of butanol and acetone fractionations and, successively, by DEAE cellulose and Sephadex G-200 chromatographies.
• 2.2. Two enzymatic forms were separated; they showed various similar characteristics but differed greatly in specific activity.
• 3.3. It is probable that in A. caliginosa a sole alkaline phosphatase form exists and the less active fraction is partly denatured enzyme.
• 4.4. It is not completely possible to exclude the existence of two isoenzymes.

     

Correlation of acid phosphatase but not alkaline phosphatase activity with myelination in the developing mouse brain

• 1.1. Neonatal mice received subcutaneous injections of buffer, thiourea (TU) or propylthiouracil (PTU).
• 2.2. The PTU-treated mice were sacrificed on postnatal day 14 (P14) and the TU-treated mice on P28.
• 3.3. Brain weights of the TU- and PTU-treated mice were not significantly different from the controls.
• 4.4. Acid but not alkaline phosphatase activity in the braistem decreased after TU and PTU treatment.
• 5.5. Myelination as indicated by intensity of luxol fast blue staining was weaker in drug-treated animals.
• 6.6. The level of myelin marker enzyme, 2′,3′-cyclic nucleotide 3′-phosphohydrolase, was lower in the brainstem of PTU-treated animals.
• 7.7. The results suggest a correlation between acid phosphatase but not alkaline phosphatase activity with myelination in the developing mouse brain.

     

γ-Glutamyltranspeptidase in echinoderm eggs and larvae: fertilization-dependent and developmentally-induced changes in specific activity

• 1.1. γ-Glutamyltranspeptidase is present in echinoderm eggs and larvae: in homogenates the level of activity is comparable to that of rat cerebral cortex.
• 2.2. In eggs of Lytechinus pictus, fertilization induces an early rapid and sustained (5 min–6 hr) 37% increase in the activity of γ-glutamyltranspeptidase in homogenate fractions.
• 3.3. Relative to these homogenate levels, the specific activity of γ-glutamyltranspeptidase are ≈60% lower in 40,000 g supernatant fractions and 2.7-fold higher in 40,000 g particulate fractions in both unfertilized and 15 min post-fertilized Lytechinus pictus eggs.
• 4.4. The subcellular distribution of γ-glutamyltranspeptidase is the same in both unfertilized and 15-min post-fertilized Lytechinus pictus eggs: 78% in 40,000 g particulate fractions, 22% in 40,000 g soluble fractions.
• 5.5. In both unfertilized and 15 min post-fertilized eggs of Lytechinus pictus the enzyme responds to heat (50 vs 37°C) by activation in a similar manner: 1.72- and 1.68-fold homogenates; 2.6- and 3.0-fold in supernatants; 1.97- and 1.90-fold in particulate fractions.
• 6.6. In homogenates of Pisaster ochraceous larvae, γ-glutamyltranspeptidase activity increases steadily during the course of larval development: relative to the low activity at day 5, activities exhibit an increase of 1.2-, 2.0-, 3.1- and 5.4-fold at days 10, 16, 22 and 28, respectively.

     

Prostaglandin E2/parathyroid hormone-induced suppression of alkaline phosphatase activity is mediated by protein kinase C

• 1.1. Bone resorptive factors, prostaglandin E2 and parathyroid hormone are shown to suppress alkaline phosphatase activity in a rat osteoblastic cell line.
• 2.2. Phorbol myristate acetate, but not dibutyryl cAMP or calcium ionophore can suppress alkaline phosphatase activity.
• 3.3. The protein kinase C inhibitors (H89, staurosporine) are able to block the suppression of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.
• 4.4. These data suggest that protein kinase C is involved in the inhibition of alkaline phosphatase activity induced by prostaglandin E2 and parathyroid hormone.

     

Transamination and transsulphuration of l-cysteine in ehrlich ascites tumor cells and mouse liver: The nonenzymatic reaction of l-cysteine with pyruvate

• 1.1. The activity of cysteine aminotransferase (CAT), 3-mercaptopyruvate sulfurtransferase (MPST) and rhodanese is much lower in Ehrlich ascites tumor cells (EATC) than in mouse liver.
• 2.2. Contrary to mouse liver homogenate, no synthesis of sulphane sulphur-containing compounds from L-cysteine is observed in EATC homogenate.
• 3.3. 2-Methyl-thiazolidine-2,4-dicarboxylic acid (CP), 2-methyl-thiazolidine-4-carboxylic acid (CA) and thiazolidine-4-carboxylic acid (CF) can be used as sources of low molecular-weight thiol compounds both in EATC and mouse liver homogenate.
• 4.4. Pyruvate formed from phosphoenolopyruvate (PEP) in EATC homogenates reacts with L-cysteine (l-CYS) to CP.

     

The isolation of a plasma membrane-rich fraction from the skeletal muscle of two species of marine crab,Carcinus maenas and Cancer pagurus

• 1.1. A rapid method for the isolation of a plasma membrane-rich fraction from crab leg muscle, with high purity and yield recovery was developed.
• 2.2. The method is based on sodium iodide extraction of the crude homogenate, followed by centrifugation on Percoll self-creating gradient.
• 3.3. (Na⁺K⁺)ATPase and alkaline phosphodiesterase I were used as marker enzymes for the plasma membrane and revealed levels of purification of approximately 13-fold and yields recovery of the total activity in the crude muscle homogenate of approximately 18%, for both species of crab studied.

     

A study on the rna polymerase activities in bovine thyroid nuclei isolated in isotonic sucrose,hypertonic sucrose or in anhydrous glycerol media: Influence of the isolation procedure on the electrophoretic pattern of acid soluble nuclear proteins

• 1.1. After differential pelleting of bovine thyroid bound RNA polymerase II was the more enriched enzyme activity in the nuclear fraction, and coincided best with the DNA profile.
• 2.2. The RNA polymerase I + III activity was compared in nuclear fractions isolated either in 0.25 M sucrose (wet tissue) or in anhydrous glycerol (lyophilized tissue) or in 2.4 M sucrose (lyophilized tissue).
• 3.3. Although the nuclei were more resistant to the isolation porcedure in glycerol, more proteins were extracted by that procedure than during the isolation in 2.4 M sucrose.
• 4.4. With the 2.4 M sucrose method a twofold enrichment of RNA polymerase I + III activity in respect to DNA occurred in the nuclei pointing to an exclusive localization of these activities within the nucleus.
• 5.5. Using the same isolation procedure the different classes of histones were better resolved upon polyacrylamide gelelectrophoresis.

     

Acid and alkaline phosphomonoesterase activities in snake venoms

• 1.1. Acid and alkaline phosphatase activities of eight different snake venoms were determined quantitatively by using synthetic substrates, o-carboxyphenylphosphate and p-nitrophenylphosphate respectively.
• 2.2. It was found that most of Elapidae venoms investigated had both acid and alkaline phosphatase activities.
• 3.3. Three Crotalidae venoms investigated did not show any alkaline phosphatase activity.
• 4.4. The strength of venom acid phosphatase activity is as follows: Agkistroden acutus > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.
• 5.5. The strength of venom alkaline phosphatase activity by using p-nitrophenylphosphate is in the order of Naja hannah > Naja haje > Naja naja samarensis > Naja naja atra > Naja melanoleuca.When o-carboxyphenylphosphate was used as a substrate, the order of enzyme activity is Naja hannah > Naja haje > Naja naja samarensis > Naja melanoleuca > Naja naja atra.
• 6.6. Acid phosphatase activity of all the Elapidae venoms was inhibited completely by fluoride. The alkaline phosphatase activity of Elapidae venoms was not inhibited by fluoride either using p-nitrophenylphosphate or o-carboxyphenylphosphate.
• 7.7. The acid phosphatase of all the Elapidae venoms was not inhibited by zinc ion. However, most of the venom alkaline phosphatases were inhibited by zinc ion.
• 8.8. Ethylenediaminetetraacetic acid (EDTA) had inhibitory action on venom phosphatase activity. However, tris-(hydroxymethyl)-aminoethane had a counter effect on the inhibitory action of EDTA.
• 9.9. Optimum pH studies of the snake venom phosphatases showed that the acid phosphatases of the snake venoms had their highest activity in the range of pH 4–5. The alkaline phosphatases of the snake venoms had their optimum pH at 9.
• 10.10. Comparable experiments were also conducted by using chicken intestine alkaline phosphatase and wheat germ acid phosphatase.

     

Proline synthesis from glutamate in mitochondria from the blowfly Aldrichina grahami

• 1.1. Glutamyl kinase in a homogenate of blowfly abdomens was not inhibited by proline and its analogues, and was found only in the mitochondrial fraction.
• 2.2. Proline was biosynthesized from glutamate only in a cell-free system prepared from blowfly abdomen.

     

The digestive proteases of langostilla (pleuroncodes planipes,decapoda): their partial characterization,and the effect of feed on their composition

• 1.1. Three methods of recuperating and preserving enzyme activity from freshly-caught langostilla were assessed. In the pressing and acetone extract methods, the recovered specific activity was similar.
• 2.2. Protease activity was higher between 6.5 and 8 pH, and was sensitive to high temperatures.
• 3.3. In PAGE and serine inhibition assays, one fraction resembled bovine trypsin.
• 4.4. The composition of proteins and molecules bearing protease activity from the hepatopancreas and stomach of both fed and starved animals was similar, indicating proteases are not induced but constitutive.

     

The effect of cortisol on stimulation of enzymatic activity and absorption of amino acids in the small intestine of adult hamsters

• 1.1. The s.c. administration of cortisol to hamsters (50 mg/kg body wt/day for 4 days) produces a significant increase in maltase sucrase, alkaline phosphatase and leucineaminopeptidase activity in intestinal mucosa.
• 2.2. Lactase activity is unaffected by cortisol.
• 3.3. Gamma-glutamyltranspeptidase activity increases slightly in females but remains unchanged in males.
• 4.4. Cortisol causes increase in proline and glycine absorption without changing the absorption of lysine.

     

Comparative hematology: Studies on cats including one with factor XII (hageman) deficiency

• 1.1. Cat plasma prothrombin and partial thromboplastin times are faster than human. Thromboplastin generation tests are very similar.
• 2.2. Factors VIII and V assay 24 and 13 times the human standard. Cat factors VII, X. IX, XI and XII assayed at 2.5 to 4 times human. Factors I, II and XIII fell within the human range and Fletcher was extremely low.
• 3.3. One cat lacked factor XII and showed a prolonged APTT and clotting time.
• 4.4. Cat profibrinolysin was activated by streptokinase but not by urokinase.
• 5.5. Cat platelets aggregated with the usual human aggregation agents with the exception of thrombin and ristocetin.
• 6.6. Cat erythrocytes were smaller and more numerous than human.
• 7.7. Leukocyte counts were quite variable.
• 8.8. Serum protein electrophoretic patterns differed from human in the greater migration of albumin and the presence of numerous unidentified bands.
• 9.9. Biochemical tests showed high sodium and chloride values.

     

Digestive enzymes in the exocrine pancreas of the frog Rana esculenta

• 1.1. The activity of some digestive enzymes has been investigated in a crude pancreas homogenate of frog Rana esculenta. The levels of amylase, trypsin and chymotrypsin depend on nutritional status being lower in fasted animals; ribonuclease and lipase levels do not seem to be affected by fasting.
• 2.2. Frog pancreatic enzymes show pH optima and thermostability similar to those reported for higher vertebrates.
• 3.3. The effects of PMSF and EDTA on proteolytic enzymes suggest that in the frog pancreas besides serine-proteases which represent the major proteolytic activity, other enzymes, possibly metalloenzymes, are present.