Activity of renal hydrolases in pre- and postnatal development of mice

The fetal and postnatal activity patterns of different hydrolytic enzymes (alkaline phosphatase, gamma-glutamyltransferase, trehalase, maltase, glucoamylase, lactase, and sucrase) have been examined in mouse renal homogenates. Alkaline phosphatase and gamma-glutamyltransferase activities presented approximately similar changes. They increased from 18 days of gestation up to 30 days after birth. These activities showed marked increases during the 3rd and 4th postnatal weeks. A similar important rise was observed for trehalase activity at the end of the suckling period. Maltase activity increased gradually after birth. Traces of lactase, sucrase, and glucoamylase activities were detected at each developmental stage.     

Purification of some glycoside hydrolases by affinity chromatography

Two glycoproteins have been isolated from the cell walls of baker's yeast. One is a glucan-protein complex which has been partially characterised as having a branched carbohydrate structure composed of chains of (1→3)-linked β-d-glucosyl residues, some of which are attached by (1→6)-linkages to the main chain. Immobilization of this glycoprotein was achieved by covalent attachment to Sepharose, and the product was used to isolate a number of (1→3)-β-d-glucan hydrolases from Helix pomatia, malted barley, and Basidiomycete QM806. The second glycoprotein, a mannan-protein complex, after immobilization, has been used in the purification of an α-d-mannosidase from jack-bean meal.     

Activity and flexibility of alcohol dehydrogenase in organic solvents

The oxidation of cinnamyl alcohol to cinnamaldehyde by horse liver alcohol dehydrogenase (LADH) was carried out in nearly anhydrous organic solvents and in solvents containing from 0.1 to 10% added water. In nearly anhydrous solvents containing less than 0.02% water, the oxidation rate increased as the water solubility in the solvent decreased, but the reaction did not require active LADH. Moreover, the highest activity in nearly anhydrous heptane was obtained by lyophilizing the enzyme from a solution of pH 2.0, even though LADH exhibits virtually no enzymatic activity in water at this pH. The catalytic activity of LADH was restored and increased dramatically as small amounts of water were added to each solvent. In conjunction with the activity measurements, electron paramagnetic resonance (EPR) spectroscopy and two active-site directed spin labels were used to examine solvent-dependent structural features of LADH. The EPR spectra indicated that LADH became more rigid as the dielectric constant of the solvent decreased. The degree of rigidity also depended on the pH from which the enzyme was lyophilized, indicating that the ionization state of the enzyme can have an important influence on its dynamics in organic solvents. Finally, adding 1% water to organic solvents had no apparent effect on the enzyme's conformation or flexibility near the spin label, even though enzyme activity was an order of magnitude higher when 1% water was present.     

Activity of murein hydrolases in synchronized cultures of Escherichia coli.

Murein hydrolase activities were analyzed in synchronized cultures of Escherichia coli B/r. Cell wall-bound murein hydrolase activities, including the penicillin-sensitive endopeptidase, increased discontinuously during the cell cycle and showed maximum activity at a cell age of 30 to 35 min (generation time, 43 min). Maximum activity was observed at the same time that the rate of cell wall synthesis reached its maximum. These oscillations depended on the termination of replication: no increase in hydrolase activity was found if deoxyribonucleic acid synthesis was inhibited at an early time in the life cycle. In contrast, the activity of another murein hydrolase that was not tightly bound to the membrane (transglycosylase) increased exponentially with time, even when deoxyribonucleic acid synthesis was inhibited.     

Histochemistry of some acid hydrolases in striated muscles of the rat

Summary The distribution of acid phosphatase, -n-acetylglucosaminidase, -glucuronidase, and acid -galactosidase was studied in mm. extensor digitorum longus, soleus, and diaphragm of rats. Using the technic of semipermeable membranes activities of these enzymes were demonstrated beside cells of the interstitial tissue in muscle fibers themselves as well. Acid phosphatase displayed the highest activity which appeared in many small dots dispersed in the fiber. The activity of acid phosphatase was about 1.2 x higher in the m. soleus than in the m. extensor digitorum longus. In the latter muscle a somewhat higher activity was often found in muscle fibers displaying a higher staining for NADH tetrazolium reductase. The activity of -n-acetylglucosaminidase was slightly lower, that of -glucuronidase very weak but still discernible. The activity of acid -galactosidase was not ascertained in the majority of fibers. The ratio of activities measured in an area of the same size in cells of the interstitial tissue and in muscle fibers amounted in average to 2.6: 1 in the case of acid phosphatase, 2.5:1 in the case of -n-acetylglucosaminidase, 5.7: 1 in the case of -glucuronidase, and 44.3:1 in the case of acid -galactosidase. The importance of the histochemical technic in studies concerned with acid hydrolases in striated muscle fibers in normal and pathological conditions is pointed out.     

Subcellular localisation of some acid hydrolases Triturus cristatus spermatozoa

Summary The histochemical localisation of acid -glycerophosphatase has been studied, by optical and electron microscopy, in the sperm cells of the newt. The enzymatic activity is restricted to some of the acrosomal structures, other parts of the cell being unreactive.The subcellular localisation of this enzyme, and of other four acid hydrolases (Proteinase, DNase, RNase and -glucuronidase) has been studied using biochemical techniques. The sedimentability of the before mentioned hydrolases with the acrosomes detached from the entire cells and their solubilisation by Triton X-100, have been examined.The evidence obtained was discussed in relation to the hypothesis of a lysosomal-like nature of the acrosome.     

The metabolism in vitro and hepatic microsomal interactions of some enantiomeric drug substrates

1. The metabolism in vitro and microsomal interactions of (+)-amphetamine, (-)-amphetamine, (+)-benzphetamine and (-)-benzphetamine were studied with hepatic microsomes from phenobarbitone-pretreated male rabbits. 2. (+)-Benzphetamine was N-demethylated 30-35% faster than (-)-benzphetamine, but the apparent Michaelis constants for the two enantiomers were similar. 3. (-)-Amphetamine was deaminated about 200% faster than (+)-amphetamine. 4. The benzphetamine enantiomers gave qualitatively and quantitatively identical type I microsomal difference spectra (peak, 390nm; trough, 425nm) indicating identical apparent binding affinities for microsomes and identical spectral changes at maxima (DeltaE(max.) values). 5. The amphetamine enantiomers gave qualitatively identical type II microsomal difference spectra (peak, 433nm; trough, 395nm). However, the type II spectral data indicated that (+)-amphetamine had a markedly higher apparent binding affinity than (-)-amphetamine for microsomes. The amphetamine enantiomers gave identical DeltaE(max.) values. 6. The benzphetamine enantiomers (0.5mm) enhanced the rate of microsomal cytochrome P-450 reduction by NADPH by 400-500%, (+)-benzphetamine enhancing the rate 20-25% more than (-)-benzphetamine. 7. The amphetamine enantiomers decreased the rate of microsomal cytochrome P-450 reduction by NADPH. At a concentration of 2mm, (+)-amphetamine decreased the rate more than (-)-amphetamine. 7. All four enantiomers enhanced microsomal NADPH oxidation.     

Oviposition site selectivity of some stream-dwelling caddisflies

Population dynamics depends upon the spatial distribution of individuals in heterogeneous environments. The various processes surrounding insect oviposition are central to understanding their population dynamics because the choice of oviposition site ultimately influences the survivorship and spatial distribution of their progeny. Aquatic insects are often assumed to have non-selective oviposition habits, but empirical data are scarce and selective oviposition may be quite common. We quantitatively sampled egg masses of stream-dwelling caddisflies (Trichoptera) that specialise in egg-laying on hard substrata underwater, in order to characterise oviposition site selectivity and test for communal oviposition. In a field survey of two Scottish streams, we sampled egg masses of three species, Polycentropus flavomaculatus, Hydropsyche siltalai, Rhyacophila dorsalis, with the aim of testing whether egg mass abundance varied with current (riffles vs. pools), location within the channel (margins vs. centre) and rock exposure (emergent vs. fully submerged). In one stream, we captured adults landing on emergent rocks and assessed whether females were modified morphologically for swimming. The egg masses of two species (P. flavomaculatus, H. siltalai) occurred primarily on submerged rocks in pool margins, and adult females had legs modified for swimming. In contrast, egg masses of R. dorsalis were most abundant on the underside of emergent rocks in riffles, and females were not modified for swimming. Communal oviposition was evident for all three species, with most egg masses aggregated on the minority of potential rocks. How females locate oviposition sites and the consequences of these highly specialised oviposition behaviours to the survival and spatial distribution of larvae now require investigation. The effects of these behaviours on population dynamics are likely to differ from terrestrial herbivores because oviposition sites are not food resources for these aquatic species.     

Distribution of some hydrolases in the rat kidney (author's transl)

Most of the available histochemical methods and techniques (azodye, metal salt and indigogenic methods, cryostat, free-floating and lyophilized section techniques) and different modifications of these methods (different substrate concentrations, pH, temperature, incubation time e.g.) were applied to study the distribution of acid phosphatase (AcPB = after Barka and Anderson; AcPG = after Gomori), beta-glucuronidase (beta-Glu), aryl sulfatase (AS), beta-N-acetylglucosaminidase (NAG), acid 5'-nucleotidase (a5-Nucl), non-specific esterase (NE) and alkaline phosphatase (AlP) in the kidneys of rats of both sexes. The optimal conditions for the demonstration of these enzymes were established. As most important proved: the incubation of free-floating sections cut from "standard"-fixed (2 h in formol-calcium continued for another 18-22 h in the same fixative plus 0.88 M sucrose at 4 degrees C) kidney slices - only for AcPB and NE material fixed after Holt had to be used; the incubation for AlP and NE at 4 degrees C; final pH of the incubation medium for AcPB 5.5, AcPG 5.0 and NE 6.5; the use of Fast Garnet GBC Salt as coupler in the NE azo-dye reaction. Sex differences and for the female rats an increased activity during oestrus were established for all hydrolases studied. In particular the following results were obtained: AcPB, a5-Nucl and A1P are more intensive in male and AcPG in female S1 segments of the juxtamedullary nephrons in relation to the nephrons of the other parts of the cortex. In the medullary rays the NE and the a5-Nucl show a higher activity in the S2 segments of female rats demonstrate a more intensive activity for NAG and NE. This is true for AcPG and A1P in male rats. In the inner medulla a stronger beta-Glu activity in male rats and a stronger NAG activity in female rats is observed. The AcPB activity of the cortical distal tubules is higher in male rats.