Brief description
The CCO and LDH activities in liver, white muscle and gill tissues in the black bream (Acanthopagrus butcheri) were measured to determine their aerobic and anaerobic metabolism. Black bream were collected from five sites within the Swan-Canning estuary under summer conditions in 2001 and 2002 and again under winter conditions in 2002. Enzyme activity levels in the tissues taken were compared to identify spatial, seasonal orannual variations.
Lineage
Maintenance and Update Frequency: notPlanned
Statement: - Fish and Sample Collection -
Feral black bream were captured from four sites in the Swan River: Helena, Ascot, Barrack Street and Freshwater Bay, and one site in the Canning River (Riverton) (See thumbnail) during autumn (April-May) in both the years 2001 and 2002 and again in late winter (August?September) 2002. Water samples were collected approximately 45 cm below
the draft of the fishing vessel, and temperature, pH and salinity measured using a TPS WP-81 Conductivity-Salinity-pH-Temp Meter. Dissolved oxygen (DO) measurements were taken with an OXI320 Oximeter. None of the sites could be considered true control sites, as all areas in the Swan-Canning estuary have been impacted by human activity.
The black bream were captured by a commercial
fisherman using a 120 m monofilament haul net with a mesh size of 100 mm. Each fish was sacrificed within 2 h of capture, 1 g each of gill, liver and white muscle tissue collected, placed in cryovials and immediately immersed in liquid nitrogen. The white muscle sample was collected
from the left side of the fish just below the anterior end of the dorsal fin. The samples were later stored in a -80 degrees C freezer until analysis.
Feral black bream were captured from four sites in the Swan River: Helena, Ascot, Barrack Street and Freshwater Bay, and one site in the Canning River (Riverton) (See thumbnail) during autumn (April-May) in both the years 2001 and 2002 and again in late winter (August?September) 2002. Water samples were collected approximately 45 cm below
the draft of the fishing vessel, and temperature, pH and salinity measured using a TPS WP-81 Conductivity-Salinity-pH-Temp Meter. Dissolved oxygen (DO) measurements were taken with an OXI320 Oximeter. None of the sites could be considered true control sites, as all areas in the Swan-Canning estuary have been impacted by human activity.
The black bream were captured by a commercial
fisherman using a 120 m monofilament haul net with a mesh size of 100 mm. Each fish was sacrificed within 2 h of capture, 1 g each of gill, liver and white muscle tissue collected, placed in cryovials and immediately immersed in liquid nitrogen. The white muscle sample was collected
from the left side of the fish just below the anterior end of the dorsal fin. The samples were later stored in a -80 degrees C freezer until analysis.
Statement: - Reaction media -
Samples to be analysed for metabolic enzyme activities were randomly selected from each tissue type collected at each site for each collection period. Gill tissues were compared for seasonal differences only due to limited gill tissue mass for metabolic enzyme analysis in summer 2001 to
enable comparison with summer 2002. Each tissue sample was first thawed on ice, approximately 0.5 g of tissue was weighed out, and homogenised with 4.5 mL of 50 mM imidazole buffer pH 8.0 using a Diax 900 Ultrasonic homogeniser. The homogenate was centrifuged at 4000 xg for 5 min at 4 degrees C and the supernatant collected for immediate use.
Samples to be analysed for metabolic enzyme activities were randomly selected from each tissue type collected at each site for each collection period. Gill tissues were compared for seasonal differences only due to limited gill tissue mass for metabolic enzyme analysis in summer 2001 to
enable comparison with summer 2002. Each tissue sample was first thawed on ice, approximately 0.5 g of tissue was weighed out, and homogenised with 4.5 mL of 50 mM imidazole buffer pH 8.0 using a Diax 900 Ultrasonic homogeniser. The homogenate was centrifuged at 4000 xg for 5 min at 4 degrees C and the supernatant collected for immediate use.
Statement: - Determination of enzyme activities -
Assay conditions for enzyme activity were based on the methods of Sidell et al. (1987), which were optimised for black bream.
-Cytochrome c oxidase
(Cytox; EC 1.9.3.1), reaction medium: 50 mM Imidazole buffer pH 8.0, 70 AM cytochrome c. The reduction of cytochrome was carried out by the addition of sodium hydrosulphite. The excess reducing agent was removed by gently bubbling with air (Pelletier et al., 1993). A reference was run with the assay mixture plus 0.33% (w/v) K3Fe(CN)6. Enzymatic activity was calculated using a molar extinction coefficient of 29.5 for cytochrome c
reduced.
-Lactate dehydrogenase
(LDH; EC 1.1.1.27); reaction medium: 100 mM phosphate buffer pH 7.4, 0.15 mM NAOH, 0.8 mM pyruvate, 50 mM Imidazole buffer pH 7.4. Enzymatic activity was calculated using a molar extinction coefficient of 6.22 for LDH. Enzyme activity was measured on a Pharmacia UV?Visible Spectrophotometer following the rate of oxidation of
cytochrome c for CCO (550 nm) and the rate of oxidation of NADH for LDH (340 nm). Protein content of the homogenate was determined according to Lowry et al. (1951). Enzymatic activity was calculated in international units (U) normalised per milligram of protein. One U is equivalent to the conversion of 1 mol of substrate to product per minute, and is expressed as units per milligram of protein per minute (U mg protein-1 min-1).
Assay conditions for enzyme activity were based on the methods of Sidell et al. (1987), which were optimised for black bream.
-Cytochrome c oxidase
(Cytox; EC 1.9.3.1), reaction medium: 50 mM Imidazole buffer pH 8.0, 70 AM cytochrome c. The reduction of cytochrome was carried out by the addition of sodium hydrosulphite. The excess reducing agent was removed by gently bubbling with air (Pelletier et al., 1993). A reference was run with the assay mixture plus 0.33% (w/v) K3Fe(CN)6. Enzymatic activity was calculated using a molar extinction coefficient of 29.5 for cytochrome c
reduced.
-Lactate dehydrogenase
(LDH; EC 1.1.1.27); reaction medium: 100 mM phosphate buffer pH 7.4, 0.15 mM NAOH, 0.8 mM pyruvate, 50 mM Imidazole buffer pH 7.4. Enzymatic activity was calculated using a molar extinction coefficient of 6.22 for LDH. Enzyme activity was measured on a Pharmacia UV?Visible Spectrophotometer following the rate of oxidation of
cytochrome c for CCO (550 nm) and the rate of oxidation of NADH for LDH (340 nm). Protein content of the homogenate was determined according to Lowry et al. (1951). Enzymatic activity was calculated in international units (U) normalised per milligram of protein. One U is equivalent to the conversion of 1 mol of substrate to product per minute, and is expressed as units per milligram of protein per minute (U mg protein-1 min-1).
Notes
PurposeTo determine the usefulness of metabolic enzyme activity in black bream as a biomarker of effect in environmental monitoring of contaminants within the Swan-Canning estuary.
Issued: 27 07 2005
Data time period: 2001-04 to 2002-09
text: westlimit=115.75; southlimit=-32.1; eastlimit=116; northlimit=-31.85
Subjects
37 353003 |
ANIMALS/VERTEBRATES |
Acanthopagrus butcheri |
BIOLOGICAL CLASSIFICATION |
Black bream |
COASTAL PROCESSES |
Contaminants |
EARTH SCIENCE |
Estuaries |
Fish |
OCEANS |
TERRESTRIAL HYDROSPHERE |
WATER QUALITY/WATER CHEMISTRY |
aquatic toxicology |
bioindicators |
biomarkers |
oceans |
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Other Information
(PhD Thesis)
uri :
http://adt.curtin.edu.au/theses/available/adt-WCU20061204.135553/
global : d5aac340-5c41-11dc-af0d-00188b4c0af8
Identifiers
- global : e0be9990-5f67-11dc-a47f-00188b4c0af8
