Biochemical characteristics and modulation by external and internal factors of aminopeptidase-N activity in the hepatopancreas of a euryhaline burrowing crab.

Strikingly, in spite of its physiological importance, information about occurrence, biochemical characteristics and mechanisms of regulation of aminopeptidase-N (APN) in the hepatopancreas of intertidal euryhaline crabs is still lacking. In this work, we determined the occurrence, biochemical characteristics, response to environmental salinity and dopamine of APN in the hepatopancreas of the euryhaline crab Neohelice granulata (Dana 1851) from the open mudflat of Mar Chiquita coastal lagoon (Buenos Aires province, Argentina). APN activity was maximal at pH and temperature range of 7.6-9.0 and 37-45 °C, respectively. APN activity exhibited Michaelis-Menten kinetics (apparent Km = 0.19 ± 0.10 mM) (pH 7.6, 37 °C) and appeared to be sensitive to bestatin (I 50 = 15 mM) and EDTA (I 50 = 9 mM). In crabs acclimated to 10 psu (hyper-regulation conditions) and 37 psu (hypo-regulation conditions), APN activity was about 45 and 160% higher, respectively, than in 35 psu (osmoconformation). APN activity in the hepatopancreas was stimulated in vitro (about 137%) by 10(-4) M dopamine. Higher dopamine concentrations produced a similar extent of increase. The responses of APN activity to salinity and dopamine in vitro suggest the role of APN in digestive adjustments upon hyper and hypo-regulatory conditions and its modulation via direct mechanisms on hepatopancreas by dopamine.

Salinity dependent Na+-K+ATPase activity in gills of the euryhaline crab Chasmagnathus granulata.

The occurrence and response of Na+-K+ATPase specific activity to environmental salinity changes were studied in gill extracts of all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). All of the gills exhibited a salinity dependent Na+-K+ATPase activity, although the pattern of response to environmental salinity was different among gills. As described in other euryhaline crabs highest Na+-K+ATPase specific activity was found in posterior gills (6 to 8), which, with exception of gill 6, increased upon acclimation to reduced salinity. However, a high increase of activity also occurred in anterior gills (1 to 5) in diluted media. Furthermore, both short and long term differential changes of Na+-K+ATPase activity occurred among the gills after the transfer of crabs to reduced salinity. The fact that variations of Na+-K+ATPase activity in the gills were concomitant with the transition from osmoconformity to ionoregulation suggests that this enzyme is a component of the branchial ionoregulatory mechanisms at the biochemical level in this crab.

Branchial carbonic anhydrase (CA) of gills of Chasmagnathus granulata (Crustacea Decapoda).

The occurrence, localization and response to environmental salinity of carbonic anhydrase (CA) activity were studied in all of the gills of the euryhaline crab Chasmagnathus granulata from Mar Chiquita coastal lagoon (Buenos Aires Province, Argentina). CA activity in all gills appeared to be dependent on salinity. The pattern of distribution of CA activity among gills was different upon transition of C. granulata from osmoionoconformity (more uniform distribution) to hyperregulation (highest activity in posterior gills 6-8). Upon abrupt salinity change a differential response of CA activity occurred among gills which could suggest a differential role of CA in ion transport process in different gills of this crab. Furthermore, CA activity in anterior and posterior gills was found in cytosolic and microsomal fractions, although highest activity appeared to be membrane-associated. Both pools of CA were also strongly influenced by salinity and very sensitive to sulfonamide acetazolamide. The results suggest a differential participation of branchial CA in ionoregulatory mechanisms of C. granulata.

Effect of histamine on soluble and membrane-associated carbonic anhydrase (CA) activity of pig and bovine gastric mucosa.

1. As part of their characterization, the effect of histamine (H) on both soluble and membrane-associated carbonic anhydrase (CA) activity of pig and bovine gastric light microsomal membranes was investigated. 2. H did not affect the activity of soluble CA purified from pig oxyntic mucosa, whereas 10(-7) and 10(-4) M H produced a significant enhancement of pig gastric firmly-membrane-associated CA activity at 20 and 30 degrees C (about 2-3-fold), but it failed at 0 degree C. The increase of activity produced by H occurred within 1 min and it was maintained for at least 15 min. 3. H also stimulated bovine gastric firmly-membrane-associated CA activity, the stimulation also being dependent on temperature.

pH-dependent association of carbonic anhydrase (CA) with gastric light microsomal membranes isolated from bovine abomasum. Partial characterization of membrane-associated activity.

1. The effect of pH on the association of carbonic anhydrase (CA) with bovine gastric light microsomal membranes (LMMs) was investigated (a) by washing LMMs containing CA activity with solutions of different pHs; (b) by studying the adsorption at various pHs of soluble bovine erythrocyte CA to washed gastric LMMs. In both cases, the association of CA with gastric LMMs was dependent on pH, being lower at neutral or alkaline pH. 2. The amount of soluble CA associated with gastric LMMs at pHs 8.0 and 9.0 was reduced when 140 mM K+/10 mM Na+ was added to the incubation medium. 3. Two sources of CA activity in bovine gastric LMMs were assumed: a loosely- and a firmly-membrane-associated activity. Both CA activities were dose-dependently inhibited by acetazolamide (I50: 3.6 x 10(-9) and 8.4 x 10(-9) M, respectively) and by chloride, acetate, iodide, bromide and nitrate at 100 mM. Firmly-membrane-associated activity appeared to be less sensitive to inhibition by acetazolamide, chloride and iodide. 4. Both activities exhibited different behavior and stability following treatment with alkaline Triton X-100. 5. The possible importance of a membrane-associated CA activity in gastric LMMs related to gastric acid secretion is discussed.

Studies on carbonic anhydrase (CA) of light microsomal membranes isolated from bovine and pig gastric mucosa.

1. The occurrence and characteristics of carbonic anhydrase (CA) activity were studied in light microsomal membranes (LMM) purified from bovine gastric mucosa. 2. Bovine gastric LMM contained a high activity of CA ranging from 170 to 400 mumol.H+/min/mg protein when assayed at 0 degree C by pH-stat technique. 3. The addition of 2mM EDTA to the assay mixture increased the enzyme activity. Lower concentrations (0.5-1 mM) had no effect. 4. The enzyme activity was dose-dependently inhibited by acetazolamide and furosemide (I50: 5 x 10(-10) M and 4.8 x 10(-7) M, respectively) and by chloride ion (Ki 85 mM) and appeared to be quite stable to treatment with alkaline Triton X-100. 5. Most of the CA activity is loosely associated with the LMM since it was removed by different washing treatments. Nevertheless, after extensive washes, gastric LMM still contained CA activity suggesting the existence of a firmly membrane-associated form of CA. 6. Values of CA activity higher than those reported previously were found in pig gastric LMM. Furthermore, the washing treatments described in this work were more effective in washing CA activity off pig gastric LMM than procedures described previously.