D. A. Holwerda

Characterization of the adipokinetic and hyperglycaemic substances from the locust corpus cardiacum

Abstract An extract of locust corpus cardiacum exhibits adipokinetic and hyperglycaemic activities. For purification and characterization of the hormones extracts were subjected to gel filtration, electrofocusing, ultrafiltration, and paper chromatography. Examination of the presence of hormonal activities in the experimental fractions was by bioassay. Throughout the purification procedure both hormone activities remained connected. Acid hydrolysis of the purified fraction yielded the following amino acids in roughly equimolar amounts: aspartic acid, glutamic acid, glycine, leucine, phenylalanine, proline, serine, and threonine. Tryptophan is virtually absent.

Involvement of adenosine 3′:5′-cyclic monophosphate in lipid mobilization in Locusta migratoria

The role of cyclic AMP in hormone-induced lipid mobilization in Locusta migratoria was investigated. Injection of a corpus cardiacum extract into adult female locusts resulted in an increased level of cyclic AMP in the fat body. The cAMP concentration is maximal at about 5 min of incubation and returns to the resting level after about 10 min. The dose-response curve is linear up to about 0.01 corpus cardiacum pair equivalents. Dibutyryl-cyclic AMP mimics the lipid mobilizing effect of corpus cardiacum extract. After flight the cyclic AMP concentration in fat body increased. Injection of corpus cardiacum extract had no effect on flight muscle cyclic AMP concentration.

Cadmium kinetics in freshwater clams. I. The pattern of cadmium accumulation inAnodonta cygnea

Freshwater clams (Anodonta cygnea) were exposed to cadmium under laboratory conditions. Time courses of Cd accumulation were determined for whole animal and the separate organs. At 5 Μg/L, Cd accumulation proceeded mainly linearly. At 25 Μg/L, the accumulation pattern was biphasic for whole animals and most of the organs: during the first four weeks Cd concentrations increased linearly, remaining at a constant level for the next two weeks. Thereafter, metal concentrations increased strongly until, after 10 weeks, a saturation level was reached. It is argued that the biphasic course of accumulation is not caused by an advancing selection through mortality or by the gradually decreasing mean animal weight, nor by the typical laboratory conditions of absence of food and substratum. The partition of total Cd among the organs gradually changed over the first eight weeks, remaining constant from that time. The ultimate order of Cd concentrations was: gills > labial palps > mantle-edge > mantle, kidney, whole animal > midgut gland > guts/gonads complex > foot. A possible relation between the time course of Cd accumulation and behavioral responses is presented.

The effect of phosphoenolpyruvate, fructose 1,6-diphosphate and pH on allosteric pyruvate kinase in muscle tissue of the bivalve Mytilus edulis L

1. 1.|Pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) in muscle tissue of the sea mussel Mytilus edulis possesses properties which are similar to allosteric L-type pyruvate kinase of the rat liver with respect to stimulation by phosphoenolpyruvate and Fru-1,6-P2. The action of these modulators is pH dependent. In contrast to L-type there is an increasing stimulation by phosphoenolpyruvate and Fru-1,6-P2 by lowering the pH within the range from 8 to 6. 2. 2.|A possible regulatory role of the pH influence on enzyme activity is discussed.

Kinetic and molecular characteristics of allosteric pyruvate kinase from muscle tissue of the sea mussel Mytilus edulis L

Abstract 1. 1.At low phosphopyruvate concentrations, fructose 1,6-diphosphate enhances the activity of pyruvate kinase (ATP:pyruvate phosphotransferase, EC 2.7.1.40) from the adductor muscle of sea mussels. At pH 7.6 [phosphopyruvate] - 0.2 mM and [ADP] = 5 mM, half-maximal stimulation is reached with [ Fru -1,6-P 2 ] = 2 μM; at ph 6.7 it is reached with [ Fru -1,6-P 2 ] = 15 μM 2. 2.Glucose 1,6-diphosphate enhances enzyme activity but less so than Fru -1,6-P 2 . Cyclic AMP stimulates to a even lesser extent 3. 3.The reaction rate is inhibited when the concentration of the co-substrate ADP is substantially higher than phosphopyruvate. The Lineweaver-Burk plots for ADP at fixed concentrations of phosphopyruvate show no cooperative effects, whilst similar plots for phosphopyruvate at fixed concentrations of ADP all show positive cooperativity 4. 4.Alanine strongly inhibits enzyme activity. At pH 6.7 [phosphopyruvate] = 0.2 mM and [ADP] - 2 mM, 50% inhibition is reached with [alanine] = 0.1 mM; at pH 7.6 it is reached at [alanine] - 1 mM. 0.1 mM Fru -1,6-P 2 counteracts this inhibition giving a hyperbolic substrate-saturation curve 5. 5.ATP inhibition is strongly pH-dependant. At pH 6.7, [phosphopyruvate] = 0.2. mM and [ADP] = 2 mM, 50% inhibition is reached at [ATP] = 0.62 mM. At pH 7.6 there is a little influence of ATP. Fru -1,6-P 2 also cancels this inhibition. Other trinucleotides and AMP inhibit to a less extent 6. 6.The molecular mass of the enzyme was found to be 220 000 ± 10%. On electrofocusing, one main peak is found with pI 6.70. Three smaller peaks have pI values of 6.44, 6.59 and 6.83.

A field study on stress indices in the sea mussel, Mytilus edulis: application of the "stress approach" in biomonitoring.

Sea mussels,Mytilus edulis, collected from a relatively unpolluted area of the Eastern Scheldt, were transplanted along contaminated sites of the Western Scheldt for 21/2z and 5 months. Several established stress indices were determined such as accumulation of pollutants, adenylate energy charge (AEC), and condition index. Following field exposure, mussels were also subjected to an additional lethal or sublethal stress. The data show that environmental exposure alters the mussels response to stress,viz., aerial exposure and increased temperature, at the organismal (anoxic survival time), biochemical (AEC), and molecular (heat shock protein synthesis) level. The “stress approach” to assessment of environmental contamination appears to be a promising method to disclose early changes in the organism at a stage when conventional parameters (condition index, AEC) remain still unchanged.

Anoxic survival time and metabolic parameters as stress indices in sea mussels exposed to cadmium or polychlorinated biphenyls

Sea mussels,Mytilus edulis, were exposed to cadmium under laboratory and semi-field conditions and to polychlorinated biphenyls (PCBs) under semi-field conditions. After various exposure times, the animals were subjected to environmental anoxia by aerial exposure. The anoxic survival time was significantly reduced by short-time laboratory or semi-field exposure to cadmium. The effect of PCBs on the anoxic survival time was apparent after six months. In addition, during long-term semi-field exposure to cadmium or PCBs, also other potential stress indices, such as the adenylate energy charge, condition indices, glycogen and metabolite contents, and cadmium-binding proteins were examined.

ENERGY METABOLISM IN BIVALVES AND CEPHALOPODS

ABSTRACT Characteristics of anaerobic metabolic pathways in bivalves and cephalopods are described, especially with respect to energy demand. Except for octopine formation, pathways are not fixed for the whole period of anoxia. Both the duration of anaero-biosis and the season affect the proportional accumulation of end products. Relations between anaerobic metabolism and seasonal variations in exogenous factors, physiological activity, and biochemical composition are discussed. In the sea mussel, formation of succinate and propionate involves the functioning of a part of the electron transfer chain, including one ATP-synthesizing site. Reducing equivalents are delivered by the malic enzyme reaction and the pyruvate dehydrogenase, and by forward citric acid cycle reactions.

Cadmium Kinetics in Freshwater Clams. V. Cadmium-Copper Interaction in Metal Accumulation by Anodonta cygnea and Characterization of the Metal-Binding Protein

Freshwater clams were exposed for 61/2 weeks to cadmium chloride, at 48 ppb1 Cd, or to copper chloride, at 47 ppb Cu, or to a mixture of the salts, at 122 ppb Cd+139 ppb Cu. The Cd-accumulation factor (AF) for whole animal was reduced by 90% in the presence of Cu. Cu-AF was reduced by 50% in the presence of Cd. Copper did not influence thein vitro uptake of Cd by the excised gill but increased the elimination of previously accumulated Cd. However, decrease of the ventilation rate probably accounts for the greater part of the Cu effect on Cd accumulation. In the cytosol, the two metals were partitioned between two protein fractions in gel permeation. Except for hepatopancreas, Cu was for the greater part bound to the high-molecular-weight fraction. Cadmium was mainly bound to a specific metal-binding, carbohydrate-containing protein fraction of Mr≈11,000. Co-exposure to copper increased the portion of Cd bound to this fraction.

Effects of cadmium in freshwater clams. III. Interaction with energy metabolism inAnodonta cygnea

Freshwater clams,Anodonta cygnea, were exposed to cadmium, at 50 ppb (μg/L), to investigate the effects of semi-chronic exposure on energy metabolism. Parameters examined included: adenylate energy charge (AEC), glycogen content, blood glucose and protein concentration, the accumulation of anaerobic metabolic end products,viz. lactate and succinate, and mitochondrial NADH-oxidase activity. In all tissues, AEC was significantly lowered after 12 weeks. Glycogen contents of the separate organs, except for the gills, were diminished to one half those of control animals. Hemolymph glucose increased between 4 and 8 weeks, whereas protein in hemolymph steadily decreased, to about one half the concentration of controls at 12 weeks. Lactate increased in mantle and midgut gland, whereas in gill only an initial accumulation was found after 2 weeks of exposure. Succinate concentrations increased in all organs between 4 and 8 weeks of Cd exposure. Thereafter, no further accumulation occurred. From the onset of exposure to cadmium there was a gradual reduction of NADH-oxidase activity of gill mitochondria, down to 60% of the control value after 12 weeks. The data indicate an impairment of oxidative carbohydrate metabolism that is brought about by a metabolic blockade, rather than by (partial) anaerobiosis as a consequence of shell closure.