Manfred K. Grieshaber

Three enzymatic activities catalyze the oxidation of sulfide to thiosulfate in mammalian and invertebrate mitochondria

Hydrogen sulfide is a potent toxin of aerobic respiration, but also has physiological functions as a signalling molecule and as a substrate for ATP production. A mitochondrial pathway catalyzing sulfide oxidation to thiosulfate in three consecutive reactions has been identified in rat liver as well as in the body‐wall tissue of the lugworm, Arenicola marina. A membrane‐bound sulfide : quinone oxidoreductase converts sulfide to persulfides and transfers the electrons to the ubiquinone pool. Subsequently, a putative sulfur dioxygenase in the mitochondrial matrix oxidizes one persulfide molecule to sulfite, consuming molecular oxygen. The final reaction is catalyzed by a sulfur transferase, which adds a second persulfide from the sulfide : quinone oxidoreductase to sulfite, resulting in the final product thiosulfate. This role in sulfide oxidation is an additional physiological function of the mitochondrial sulfur transferase, rhodanese.

Pyruvate reductases catalyze the formation of lactate and opines in anaerobic invertebrates

Abstract The recent discovery of several enzymes, other than lactate dehydrogenase, with pyruvate reductase activity together with studies on the formation of end products of glycolysis during environmental and functional anaerobiosis have made it clear that anaerobic glycolysis in invertebrates is more important than previously thought. The presence of pyruvate reductase activity guarantees the continuous flux of glycolysis and, consequently, a constant supply of ATP by maintaining a low NADH/NAD+ ratio during exercise and hypoxia as well as in the subsequent recovery period. This review summarizes distribution, physicochemical, catalytic and regulative parameters of lactate-, octopine-, strombine- and alanopine dehydrogenase. In the second part, details are given on the formation of the end products lactate, octopine, strombine and alanopine as well as an evaluation of the biological role of the pyruvate reductases.

The biological role of octopine in the squid,Loligo vulgaris (Lamarck)

SummaryThe enzymatic activities of glyceraldehyde-3-phosphate dehydrogenase, octopine dehydrogenase and lactate dehydrogenase were determined fromLoligo vulgaris. Octopine dehydrogenase displays the highest activity yet recorded for this enzyme, exceeding glyceraldehyde-3-phosphate dehydrogenase sixfold and lactate dehydrogenase 365-fold (Table 1).During jet propulsion swimming octopine accumulates instead of lactate (Table 2), while phosphoarginine, the phosphagen of the squid, is depleted (Table 3).The formation of octopine is discussed in relation to anaerobic metabolism which might occur during burst activity in cephalopods.

Energy metabolism and valve closure behaviour in the Asian clam Corbicula fluminea

SUMMARY Since its invasion of Europe in the early 1980s, the Asian clam Corbicula fluminea has become very abundant in nearly all western river systems. Today this species is one of the most important biomass producers in the River Rhine. Monitoring the valve movements of C. fluminea over a period of 2 years revealed a circadian rhythm in summer, with extended periods (10-12 h) of valve closure, predominantly in the morning hours. Altogether valve movements were very scarce, frequently fewer than four movements per individual per day. Simultaneous measurements of heat dissipation and oxygen consumption (calorespirometry) revealed an intermittent metabolism in the clam. With the onset of valve closure, C. fluminea reduced its metabolic rate to 10% of the standard metabolic rate (SMR) measured when the valves were open. Nevertheless, this depressed metabolism remained aerobic for several hours, enabling the clam to save energy and substrates compared to the requirements of the tenfold higher SMR. Only during long-lasting periods of valve closure (more than 5-10 h) did the clams become anaerobic and accumulate succinate within their tissues (2 μmol g-1 fresh mass). Succinate is transported into the mantle cavity fluid, where it reaches concentrations of 4-6 mmol l-1. Because this succinate-enriched fluid must pass the gills when the valves open again, we suggest that this anaerobic end product is at least partly reabsorbed, thus reducing the loss of valuable substrates during anaerobiosis. Propionate was also produced, but only during experimental N2-incubation, under near-anoxic conditions. The intermittent metabolism of C. fluminea is discussed as an adaption to efficiently exploit the rare food supply, saving substrates by the pronounced metabolic depression during valve closure.

Breakdown and formation of high-energy phosphates and octopine in the adductor muscle of the scallop,Chlamys opercularis (L.), during escape swimming and recovery

Summary1.The scallopChlamys opercularis uses jet propulsion swimming as a means of escape if attacked by the star fishMarthasterias glacialis. The energy for these rapid movements is supplied by the depletion of the phosphagen phospho-L-arginine. During escape swimming the energy charge of the adductor muscle drops from 0.93 to 0.42. Due to the decrease of phospho-L-arginine from 20.4 to 1.5 μmol/g fresh wt there is a concomitant increase of L-arginine from 14.7 to 34.7 μmol/g fresh wt. No octopine, D- or L-lactate was accumulated in exhausted animals.2.During the first 30 min of recovery in aerated sea water, octopine, instead of lactate, is synthesized in adductor muscles. The energy charge rises to 0.92. Within 60 min the phospho-L-arginine pool is replenished and half of the accumulated octopine again metabolized.3.During anaerobic revovery, i.e. when the scallops are kept exposed to air, octopine synthesis is pronounced (7.5 μmol/g fresh wt/h). The energy charge increases to 0.85. However, under these conditions no phospho-L-arginine is formed.4.There is no evidence for transport of octopine from the adductor muscle to other tissues.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells

It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive. We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized. In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

Anaerobiosis and acid-base status in marine invertebrates: a theoretical analysis of proton generation by anaerobic metabolism

SummaryIn animals, various organic acids are accumulated during hypoxia or anoxia as products of anaerobic energy metabolism. The diversity of such acids is largest in marine invertebrates where succinate, propionate, acetate, lactate, alanine, octopine, strombine, and alanopine, are produced mainly from glycogen and aspartate. The effect of these substances on the acid-base status was assessed by a theoretical analysis of the respective metabolic pathways. This resulted in a general rule which was applied to evaluate the proton balance of the reactions in energy metabolism: net changes in the number of carboxyl groups or changes in the degree of dissociation of other groups (e.g. phosphate or ammonia) determine the net amount of H+ ions released or bound by the substrates and the metabolic end products.For marine invertebrates the results of the analysis can be summarized as follows: In glycogenolysis one mol of protons per mol of end products is released during cytosolic glycolysis, independent of the type of metabolic end product (lactate, octopine, alanopine, strombine, or alanine). The same applies for mitochondrial production of propionate and acetate, whereas formation of succinate results in dissociation of two mol H+ per mol. Fermentation of aspartate, however, diminishes the amount of protons which is produced in the succinate-propionate pathway. Net metabolisation of Mg ATP2− yields extra protons, whereas the cleavage of phosphagens (e.g. creatine phosphate, arginine phosphate) consumes protons.Additionally the break-down of energy-rich phosphates to inorganic phosphate has to be taken into account because of the shift of the intracellular buffer curve caused by changes of the respective effective pK values.

Energy supply and the formation of octopine in the adductor muscle of the scallop, Pecten jacobaeus (Lamarck)

Abstract 1. 1. The accumulation of octopine in the adductor muscle of actively swimming scallop Pecten jacobaeus was demonstrated. In the same tissue, phosphoarginine was depleted. 2. 2. Lactate was almost completely absent although glycolysis was enhanced. 3. 3. In working muscle, the energy charge decreased to 75% as compared to non-working muscle.

Metabolic adaptation of the intertidal worm Sipunculus nudus to functional and environmental hypoxia

The scope of anaerobic metabolism of Sipunculus nudus L. was assessed from the maximal activities of some enzymes of the intermediary metabolism and from the concentration of some metabolites accumulated during enhanced muscular activity and during prolonged experimental hypoxia.(1)Maximal enzyme activities demonstrate that the scope of anaerobic glycolysis, as indicated by maximal activities of glycogen phosphorylase (0.84 U g-1 fresh wt), far exceeds the aerobic capacity, which is assumed not to surpass the activity of succinate dehydrogenase (0.09 U g-1 fresh wt). Three pyruvate reductase activities (alanopine-, strombine- and octopine dehydrogenase) can possibly terminate anaerobic glycolysis.(2)During muscular activity, energy is provided by the degradation of phospho-L-arginine and by anaerobic glycolysis. Octopine is the major endproduct during functional anaerobiosis while the formation of strombine is less pronounced.(3)During exposure to a nitrogen atmosphere, several anaerobic endproducts are found to accumulate. Anaerobic glycolysis is terminated by strombine synthesis. This opine accumulates in concentrations much higher than octopine. In addition the concentrations of succinate, propionate and acetate are found to increase in tissues, and/or in the coelomic fluid and the incubation water.(4)The relative contribution of energy by the different anaerobic metabolic pathways are estimated during functional and environmental hypoxia.

Oxygen consumption and mode of energy production in the intertidal worm Sipunculus nudus L.: Definition and characterization of the critical PO2 for an oxyconformer

Oxygen consumption, anaerobic metabolism, and oxygen supply of inner tissues were analysed in Sipunculus nudus at different oxygen tensions. Oxygen consumption, energy expenditure, and the PO2 in the coelomic fluid decreased linearly with declining ambient PO2. Below a certain range of PO2, which was a function of the size of the animals, the rate of oxygen consumption deviated progressively from the linear PO2/MO2 function. In the same range of ambient PO2 the coelomic PO2 levelled off. Anaerobic glycolysis, phosphagen degradation, and the succinate-propionate pathway became apparent with concentration changes of anaerobic metabolites first occurring in inner tissues. In extension of the conventional definition (Prosser, 1973; Dejours, 1981) the term critical PO2 (Pc) is applied to the oxyconforming Sipunculus nudus. The Pc is redefined as the steady-state PO2 below which environmental oxygen availability becomes insufficient for complete aerobic metabolism (as indicated by the onset of anaerobic energy production). It is discussed to be closely linked to the oxygen supply of inner tissues. This redefined critical PO2 is shifted to higher partial pressures with increasing size of the animals because of the diffusion distance related decrease in coelomic PO2. Accordingly, with decline of ambient PO2, oxygen starts to be released from haemerythrin at higher ambient PO2 values in larger animals. The pigment, which is likely to function as an oxygen store, defers anaerobiosis and, thereby, supports compensation of a higher Pc in large individuals by means of an increased haematocrit. The Pc is discussed as crucial factor for survival of individual animals in intertidal oxygen-depleted environments.