Metabolic and functional characteristics of erythrocytes from Glycera and Noetia

• 1.1. Annelid and molluscan red blood cells (RBC) may de differentiated metabolically from vertebrate RBC by their increased permeability to substrate, their magnitude of amino acid catabolism and their higher aerobic metabolism.
• 2.2. At 22°C, Glycera and Noetia RBC oxidize glucose and glutamate to CO₂ without accumulation of either d- or l-lactate. By comparison, the oxidation of glutamate by rat and chicken RBC is negligible at this temperature despite its incorporation into the cells.
• 3.3. At 37°C, chicken RBC oxidize glutamate at a rate 4 times greater than at 22°C, with oxygen uptake still lower than that in Noetia RBC at 32°C. At 37°C, rat RBC do not increase their oxidation of glutamate above that at 22°C, but oxygen uptake increases to slightly more than half that of chicken RBC.
• 4.4. Our finclings indicate that RBC of these two invertebrate species have both a higher aerobic metabolism and lower anerobic capacity than vertebrate RBC.
• 5.5. Moreover, the annelid and molluscan RBC have a relatively lower activity of the pentose phosphate (PPO₄) pathway than vertebrate RBC, as evidenced by their higher thermal sensitivity of oxygen uptake and their higher *C₁O₂/*C₆O₂ isotope ratio.

     

Blood of the common indian weaver birds Ploceus philippinus and Ploceus benghalensis (Linnaeus).

The blood contents of two common Indian weaver birds, Ploceus philippinus and Ploceus benghalensis have been studied. Morphology of RBC and WBC, total differential count, the number of RBC and WBC, Hb contents, PCV, MCV, MCH, MCHC, ESR, haemin crystal structures, coagulation and prothrombin time, glucose and cholesterol contents were calculated and compared with the haematological data recorded for other birds and vertebrates. Variation with respect to length and breadth of the erythrocytes and length of the nucleus of the RBC were also discussed. Males have higher RBC number and Hb content in these birds. The mean RBC number and Hb content were also more than in fishes, amphibians and reptiles but little less than in man. The RBC number for males of both the species do not differ significantly but the Hb contents in males and females of P. benghalensis differ significantly but this was not found in P. philippinus. Again the Hb content in male of both the species differ significantly but this was not true in females. Like Passer and Columba large number of polymorphs and lymphocytes were present but only a few basophils was observed in most of the slide preparations. No special type of haemin crystals were observed in any sex of both the species. The coagulation time was more than Passer and Columba but less than in man. There was also no marked difference in both the species with respect to prothrombin time. In both the species blood glucose was more in female than male but there was no marked difference in both the species.     

Cardiac metabolism in the Myxinidae: physiological and phylogenetic considerations

Cardiac muscle hearts of Atlantic hagfish continuously function under hypoxic conditions that would lead to cardiac failure in most other vertebrates. Contractile performance of hagfish systemic hearts is resistant to anoxia and respiratory poisons but shows a significant decrement when carbohydrate catabolism is blocked by 0.5 mM iodoacetic acid. Enzyme activity profiles of hagfish ventricle reveal a robust capacity for glycolysis of carbohydrate in comparison to that for general aerobic metabolism and catabolism of alternate metabolic fuels. Isolated working hagfish ventricles preferentially oxidize radiolabeled glucose even when fatty acid fuels are present in the incubation medium. Work output of the isolated ventricular preparation is maintained only in the presence of exogenous glucose. The results indicate that energy metabolism of the hagfish myocardium is predominantly carbohydrate-based and that energy demand of the tissue can be sustained by anaerobic glycolysis during extended periods of extreme hypoxia. Cardiac metabolism of this primitive species is compared with that of hearts from higher vertebrates and an evolutionary hypothesis relating cardiac workload to preferred metabolic fuel is discussed.     

Ultrastructure of the integumental melanophores of the South American lungfish (Lepidosiren paradoxa) and the African lungfish (Protopterus sp.)

The integumental melanophores of two genera of lungfish, Lepidosiren paradoxa and Protopterus sp. were examined by light and electron microscopy. Both species possess both epidermal and dermal melanophores with fine structural characteristics basically similar to those of other vertebrates. The epidermal melanophores of both species are located in the intermediate epidermis, and possess thin perikarya containing round nuclei, and slender dendrites extending into the nearby intercellular spaces. The dermal melanophores occur immediately beneath the basement membrane, and possess flat perikarya and dendrites running horizontally between the collagen fibers of the dermis. The integument of both species does not possess an epidermal melanin unit or a dermal chromatophore unit. As in other vertebrates, each melanophore contains numerous oval, electron-opaque melanosomes, relatively large mitochondria, vacuolar endoplasmic reticula, and groups of RNP particles. Although micro filaments running randomly between other organelles occur regularly, microtubules were not demonstrated. Premelanosomes at various stages of differentiation were best illustrated in the dermal melanophores of Protopterus, and it is concluded from the observation of their fine structure that the morphological development of lungfish melanosomes closely parallels that of higher vertebrates. On the basis of melanophore morphology, Lepidosiren and Protopterus appear to be more closely related to each other than to Neoceratodus.     

Pituitary glycoprotein hormone beta subunits in the Australian lungfish and estimation of the relative evolution rate of these subunits within vertebrates

The beta subunits of the two pituitary gonadotropins LH and FSH and of thyroid-stimulating hormone (TSH) were cloned from Australian lungfish (Neoceratodus forsteri) pituitary glands. These three glycoprotein hormone beta subunits possess the main characteristics common to their counterparts in other vertebrates. Taking advantage of the phylogenetic position of the lungfish, close to the root of tetrapods, a maximum parsimony tree was inferred from these new sequences and sequences from representatives of the diversity of vertebrates. The topology of the tree was imposed so that it reflected as closely as possible the real evolutionary history of the subunits. This tree was used to estimate the relative evolution rate of the three subunits in vertebrates. Cumulated amino acid substitutions from the basal subunit node (ancestral subunit sequence) to the species node were calculated and compared. It showed that a burst in evolutionary rate occurred for the LHbeta subunit in the tetrapod lineage sometime after the emergence of amphibians. The rate of evolution of the LHbeta subunit was particularly high throughout the radiation of mammals while FSH and TSHbeta subunits kept quite stable in this lineage. A burst in evolutionary rate was also observed for the FSHbeta subunit in the lineage leading to teleosts sometime after the emergence of chondrosteans and the dynamic of evolution was high throughout the radiation of teleosts. These results were consistent with data obtained from pairwise comparisons.     

Anion exchange in the giant erythrocytes of African lungfish

Carbon dioxide transport in African lungfish Protopterus aethiopicus blood conformed to the typical vertebrate scheme, implying a crucial and rate-limiting role of erythrocyte Cl^–/HCO₃^– exchange. The rate coefficient for unidirectional Cl^– efflux via the anion exchanger ( k , s⁻¹) increased with temperature in African lungfish, but values were well below those reported in other species. The erythrocytes of African lungfish were, however, very large (mean cellular volume = 6940 µm³), and the ratio of cell water volume to membrane surface area was high ( V _w A _m⁻¹ = 1·89). Hence, the apparent Cl^– permeability ( P _Cl =  kV _w A _m⁻¹, µm s⁻¹) was close to that in other vertebrates. The plot of ln P _Cl against the inverse absolute temperature was left-shifted in the tropical African lungfish compared to the temperate rainbow trout Oncorhynchus mykiss , which supports the idea that P _Cl is similar among animals when compared at their preferred temperatures. Also, Q ₁₀ for anion exchange calculated from P _Cl values in African lungfish was 2·0, supporting the idea that the temperature sensitivity of erythrocyte anion exchange matches the temperature sensitivity of CO₂ production and transport in ectothermic vertebrates.     

Studies on the endogenous metabolism and senescence of starved Sarcina lutea

1. When washed suspensions of Sarcina lutea are starved aerobically in phosphate buffer at the growth temperature of 37 degrees , the rate of endogenous oxygen consumption decreases to very low values after 10hr., although many of the cells survive for 40hr. If starvation is prolonged further, the bacteria die at a rate of approximately 1.5% of the initial viable population per hour. 2. Oxidation of intracellular free amino acids accounts for most of the observed endogenous oxygen uptake but RNA is also utilized and a portion of the component bases and pentose is degraded and presumably oxidized. Ammonia appears in the supernatant and some pentose and ultraviolet-absorbing nucleotide are released from the cells. DNA, protein and polysaccharide are not measurably degraded. 3. Survival can be correlated with the ability of aerobically starved bacteria to oxidize exogenous l-glutamate and glucose. When starved under nitrogen for 40hr. cells continue to oxidize their endogenous reserves at undiminished rates when transferred to aerobic conditions; on prolonging anaerobic starvation the rate of oxidation declines during the period of most rapid loss of viability. 4. In the presence of Mg(2+), RNA degradation during aerobic starvation is almost completely suppressed without affecting the period for which the bacteria survive. 5. Cells grown in peptone supplemented with glucose accumulate reserves of polysaccharide which are metabolized in aerobic starvation, together with free amino acids. Ammonia is evolved and RNA is degraded to a greater extent than in peptone-grown suspensions. Bacteria rich in polysaccharide survive less well than those which are deficient in the polymer; the reason for this phenomenon has yet to be established. 6. In peptone medium, endogenous oxygen uptake and the concentration of intracellular free amino acids decline as growth progresses and they continue to decrease when the organism is held in stationary phase. Under the conditions used, the endogenous Q(o2) and free amino acid pool of cells grown in peptone with 2% (w/v) glucose did not decline so markedly and the bacteria contained large amounts of polysaccharide at all stages of growth.     

Association of stomatin (band 7.2b) with Glut1 glucose transporter

Employing a monoclonal antibody directed against the C-terminal peptide of glucose transporter molecule 1 (Glut1), we identified a approximately 30-kDa polypeptide which coimmunoprecipitated with Glut1 from sample of human red blood cells (RBC) membranes. The approximately 30-kDa polypeptide reacted with an antibody directed against stomatin, an integral plasma membrane protein which is also present at a high abundance in the human RBC plasma membrane. Likewise, employing anti-stomatin antibody, we found that Glut1 coimmunoprecipitated with stomatin from samples of RBC membranes. However, neither band 3, which is the most abundant integral membrane protein in the RBC, nor actin coimmunoprecipitated with Glut1, indicating a specific interaction between Glut1 and stomatin. Similar to the results obtained in the RBC, Glut1 and stomatin immunoprecipitated with each other in lysates of Clone 9 cells, a rat liver cell line in which Glut1 is expressed at approximately 1/200 the level present in RBC. Employing conditions that resulted in immunoprecipitation of approximately 10% of Glut1 in RBC membranes led to a approximately 3% coimmunoprecipitation of stomatin. A mixed population of Clone 9 cells stably transfected with a plasmid overexpressing the mouse stomatin exhibited 30 +/- 3% reduction in the basal rate of glucose transport compared to control cells or cells stably transfected with the empty vector. The above results suggest that stomatin is closely associated with Glut1 in the plasma membrane and that overexpression of stomatin results in a depression in the basal rate of glucose transport.     

Antioxidant functions for the hemoglobin β93 cysteine residue in erythrocytes and in the vascular compartment in vivo

The β93 cysteine (β93Cys) residue of hemoglobin is conserved in vertebrates but its function in the red blood cell (RBC) remains unclear. Because this residue is present at concentrations more than 2 orders of magnitude higher than enzymatic components of the RBC antioxidant network, a role in the scavenging of reactive species was hypothesized. Initial studies utilizing mice that express human hemoglobin with either Cys (B93C) or Ala (B93A) at the β93 position demonstrated that loss of the β93Cys did not affect activities nor expression of established components of the RBC antioxidant network (catalase, superoxide dismutase, peroxiredoxin-2, glutathione peroxidase, GSH:GSSG ratios). Interestingly, exogenous addition to RBCs of reactive species that are involved in vascular inflammation demonstrated a role for the β93Cys in hydrogen peroxide and chloramine consumption. To simulate oxidative stress and inflammation in vivo, mice were challenged with lipopolysaccharide (LPS). Notably, LPS induced a greater degree of hypotension and lung injury in B93A versus B93C mice, which was associated with greater formation of RBC reactive species and accumulation of DMPO-reactive epitopes in the lung. These data suggest that the β93Cys is an important effector within the RBC antioxidant network, contributing to the modulation of tissue injury during vascular inflammation.     

Lungfish Axial Muscle Function and the Vertebrate Water to Land Transition

The role of axial form and function during the vertebrate water to land transition is poorly understood, in part because patterns of axial movement lack morphological correlates. The few studies available from elongate, semi-aquatic vertebrates suggest that moving on land may be powered simply from modifications of generalized swimming axial motor patterns and kinematics. Lungfish are an ideal group to study the role of axial function in terrestrial locomotion as they are the sister taxon to tetrapods and regularly move on land. Here we use electromyography and high-speed video to test whether lungfish moving on land use axial muscles similar to undulatory swimming or demonstrate novelty. We compared terrestrial lungfish data to data from lungfish swimming in different viscosities as well as to salamander locomotion. The terrestrial locomotion of lungfish involved substantial activity in the trunk muscles but almost no tail activity. Unlike other elongate vertebrates, lungfish moved on land with a standing wave pattern of axial muscle activity that closely resembled the pattern observed in terrestrially locomoting salamanders. The similarity in axial motor pattern in salamanders and lungfish suggests that some aspects of neuromuscular control for the axial movements involved in terrestrial locomotion were present before derived appendicular structures.     

外源精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下生长和发酵特性的影响

【目的】探究外源添加不同氨基酸和相容性溶质对谷氨酸棒杆菌(Corynebacterium glutamicum)在高糖胁迫环境下生长的影响及可能的作用机理。【方法】通过在培养基中外源添加各种氨基酸和相容性溶质,研究其对谷氨酸棒杆菌在高葡萄糖和高蔗糖胁迫下生长的影响,并分析添加精氨酸对高葡萄糖胁迫下菌株糖转运和代谢途径中关键酶转录水平的影响,以及对菌株发酵产氨基酸的影响。进一步探究了碱性氨基酸在其它棒状杆菌属中抵御高葡萄糖胁迫的潜在作用。【结果】在高葡萄糖胁迫条件下,外源添加赖氨酸、精氨酸和组氨酸后谷氨酸棒杆菌的生物量分别提高54.7%、50.0%和37.6%;而在高蔗糖胁迫条件下,添加脯氨酸和四氢嘧啶后菌株生物量增加20%以上。进一步研究表明,在高葡萄糖胁迫下,外源添加精氨酸后谷氨酸棒杆菌的葡萄糖利用速率提高约2.5倍,谷氨酸的发酵产量也增加了127.5%。此外,碱性氨基酸对其它4种棒状杆菌也具有一定的渗透保护效应。【结论】精氨酸对谷氨酸棒杆菌在高葡萄糖胁迫下具有良好的渗透保护作用,可能归因于其能促进葡萄糖的转运和代谢能力,同时发现碱性氨基酸的渗透保护效应对棒状杆菌属具有一定的普遍性。     

Characteristics of the oxidative metabolism in strains with varying levels of fucidin biosynthesis]

Oxidative capacity of the fusidin-producing strains with various biosynthetic activity was studied comparatively. The studies showed that by their capacity to oxidize pyruvate and some metabolites of the tricarboxylic acid cycle (acetate, succinate, malate) the strains were arranged in the order reverse to their antibiotic activity. Such regularity was observed during the whole fermentation process and was most pronounced by the 3rd and 4th days (beginning of the idiophase). The rate of glucose oxidation was higher in more active strains. The same regularity was noted in the 2nd phase of the strain development associated with beginning of fusidin biosynthesis. In the 1st phase (the 1st and 2nd days) the strains almost did not differ by their capacity to oxidize glucose. By oxidation of phosphorylated ethers of carbohydrates (glucose-6-phosphate and fructoso-6-phosphate) the strains did not differ. Various fusidin-producing strains oxidized NAD-N and NADP-N approximately with the same rate. It is supposed that mutations leading to increased antibiotic production are associated with changes in acetate metabolism in the direction of more intensive biosynthesis of isoprenoid compounds, potential precursors of the fusidin molecule.     

The Development of Aerobic Spoilage Flora on Meat Stored at Chill Temperatures

In meat juice medium, aerobic spoilage bacteria utilized the following substrates in the order shown: Pseudomonos , glucose, amino acids, lactic acid; Acinetobacter , amino acids, lactic acid: Enterobacter , glucose, glucose-6-phosphate, amino acids; Microbacterium thermosphactum , glucose, glutamate. All the bacteria grew at their maximum rate utilizing the first and second substrates, but the growth rates declined when these were exhausted. The growth rate of Acinetobacter was reduced at pH 5·7 and below. All other species grew at their maximum rate within the pH range 5·5–7·0. On meat pseudomonads grew faster than the other species at all temperatures between 2° and 15°C. Interactions between any two species were observed only when one organism had attained its maximum cell density. Substrate exhaustion at the meat surface did not limit bacterial growth and it is suggested that the maximum cell density of aerobic spoilage cultures is determined by oxygen limitation of growth.     

Control of pulmonary surfactant secretion: an evolutionary perspective

Pulmonary surfactant, a mixture consisting of phospholipids (PL) and proteins, is secreted by type II cells in the lungs of all air-breathing vertebrates. Virtually nothing is known about the factors that control the secretion of pulmonary surfactant in nonmammalian vertebrates. With the use of type II cell cultures from Australian lungfish, North American bullfrogs, and fat-tailed dunnarts, we describe the autonomic regulation of surfactant secretion among the vertebrates. ACh, but not epinephrine (Epi), stimulated total PL and disaturated PL (DSP) secretion from type II cells isolated from Australian lungfish. Both Epi and ACh stimulated PL and DSP secretion from type II cells of bullfrogs and fat-tailed dunnarts. Neither Epi nor ACh affected the secretion of cholesterol from type II cell cultures of bullfrogs or dunnarts. Pulmonary surfactant secretion may be predominantly controlled by the autonomic nervous system in nonmammalian vertebrates. The parasympathetic nervous system may predominate at lower body temperatures, stimulating surfactant secretion without elevating metabolic rate. Adrenergic influences on the surfactant system may have developed subsequent to the radiation of the tetrapods. Furthermore, ventilatory influences on the surfactant system may have arisen at the time of the evolution of the mammalian bronchoalveolar lung. Further studies using other carefully chosen species from each of the vertebrate groups are required to confirm this hypothesis.     

Lungfish albumin is more similar to tetrapod than to teleost albumins: purification and characterisation of albumin from the Australian lungfish, Neoceratodus forsteri

Lobe-finned fish, particularly lungfish, are thought of as the closest extant relatives to tetrapods. Albumin, the major vertebrate plasma protein, has been well studied in tetrapods, but there exists no comparative study of the presence and characteristics of albumin in lobe-finned fish versus other vertebrates. There is a controversy over the presence of albumin in fish, although it is present in salmonids and lamprey. The presence of albumin in lungfish has also recently been documented. We identified albumin in plasma of the Australian lungfish, Neoceratodus forsteri, using a combination of agarose gel electrophoresis, [(14)C]palmitic acid binding and SDS-PAGE. Lungfish albumin was purified using DEAE-ion exchange chromatography, and has a mass of 67 kDa, is present at approximately 8 g/L in plasma and like other fish albumins, does not bind nickel. However, like tetrapod albumins, it is not glycosylated. N-terminal and internal peptide sequencing generated 101 amino acids of sequence, which showed a high degree of identity with tetrapod albumins. Despite the similarity in sequence but congruent with the evolutionary distances separating them, lungfish albumin did not cross-react with anti-chicken or anti-tuatara A albumin antisera. Lungfish albumin has characteristics more akin with tetrapod albumin and less like those of other fish.     

NfCR1, the first non-LTR retrotransposon characterized in the Australian lungfish genome, Neoceratodus forsteri, shows similarities to CR1-like elements

The genomes of lungfish, together with those of some urodele amphibians, are the largest of all vertebrate genomes. It has been assumed that the bulk of the DNA making up these large genomes has been derived from repeat elements, like the noncoding DNA of those genomes that have been sequenced (e.g., human). In an attempt to characterize repeat sequences in the lungfish genome, we have isolated, by restriction enzyme digestion of genomic DNA, sequences of a repeat element in Neoceratodus forsteri, the most primitive of the living lungfishes. The fragments sequenced from the EcoRI and BglII digests were used to perform genome walking PCR in order to obtain the full sequence of the repeat element. This element shares homology with the non-LTR (LINE) element, Chicken Repeat 1 (CR1), described for several vertebrates and some invertebrates; we have called it N. forsteri CR1 (NfCR1). NfCR1 shares all the domains of other CR1 elements but it also has several unique features that suggest it may no longer be active in the lungfish genome. It occurs in both full-length and 5'-truncated versions and in its present "inactive" form represents approximately 0.05% of the lungfish genome.     

Glutamine and α-ketoglutarate as glutamate sources for glutathione synthesis in human erythrocytes

Glutathione (GSH) is an intracellular antioxidant synthesized from glutamate, cysteine and glycine. The human erythrocyte (red blood cell, RBC) requires a continuous supply of glutamate to prevent the limitation of GSH synthesis in the presence of sufficient cysteine, but the RBC membrane is almost impermeable to glutamate. As optimal GSH synthesis is important in diseases associated with oxidative stress, we compared the rate of synthesis using two potential glutamate substrates, α-ketoglutarate and glutamine. Both substrates traverse the RBC membrane rapidly relative to many other metabolites. In whole RBCs partially depleted of intracellular GSH and glutamate, 10 mm extracellular α-ketoglutarate, but not 10 mm glutamine, significantly increased the rate of GSH synthesis (0.85 ± 0.09 and 0.61 ± 0.18 μmol·(L RBC)(-1) ·min(-1), respectively) compared with 0.52 ± 0.09 μmol·(L RBC)(-1) ·min(-1) for RBCs without an external glutamate source. Mathematical modelling of the situation with 0.8 mm extracellular glutamine returned a rate of glutamate production of 0.36 μmol·(L RBC)(-1) ·min(-1), while the initial rate for 0.8 mM α-ketoglutarate was 0.97 μmol·(L RBC)(-1) ·min(-1). However, with normal plasma concentrations, the calculated rate of GSH synthesis was higher with glutamine than with α-ketoglutarate (0.31 and 0.25?μmol·(L RBC)(-1) ·min(-1), respectively), due to the substantially higher plasma concentration of glutamine. Thus, a potential protocol to maximize the rate of GSH synthesis would be to administer a cysteine precursor plus a source of α-ketoglutarate and/or glutamine.     

Astrocyte activation in working brain: energy supplied by minor substrates

Glucose delivered to brain by the cerebral circulation is the major and obligatory fuel for all brain cells, and assays of functional activity in working brain routinely focus on glucose utilization. However, these assays do not take into account the contributions of minor substrates or endogenous fuel consumed by astrocytes during brain activation, and emerging evidence suggests that glycogen, acetate, and, perhaps, glutamate, are metabolized by working astrocytes in vivo to provide physiologically significant amounts of energy in addition to that derived from glucose. Rates of glycogenolysis during sensory stimulation of normal, conscious rats are high enough to support the notion that glycogen can contribute substantially to astrocytic glucose utilization during activation. Oxidative metabolism of glucose provides most of the ATP for cultured astrocytes, and a substantial contribution of respiration to astrocyte energetics is supported by recent in vivo studies. Astrocytes preferentially oxidize acetate taken up into brain from blood, and calculated local rates of acetate utilization in vivo are within the range of calculated rates of glucose oxidation in astrocytes. Glutamate may also serve as an energy source for activated astrocytes in vivo because astrocytes in tissue culture and in adult brain tissue readily oxidize glutamate. Taken together, contributions of minor metabolites derived from endogenous and exogenous sources add substantially to the energy obtained by astrocytes from blood-borne glucose. Because energy-generating reactions from minor substrates are not taken into account by routine assays of functional metabolism, they reflect a "hidden cost" of astrocyte work in vivo.     

Phylogenetic aspects of carbamoyl phosphate synthetase in lungfish: a transitional enzyme in transitional fishes

Carbamoyl phosphate synthetase (CPS) catalyses the formation of carbamoyl phosphate from glutamine or ammonia, bicarbonate and ATP. There are three different isoforms of CPS that play vital roles in two metabolic pathways, pyrimidine biosynthesis (CPS II) and arginine/urea biosynthesis (CPS I and CPS III). Gene duplication has been proposed as the evolutionary mechanism creating this gene family with CPS II likely giving rise to the CPS I/III clade. In the evolutionary history of this gene family it is still undetermined when CPS I diverged from CPS III on the path to terrestriality in the vertebrates. Transitional organisms such as lungfishes are of particular interest because they are capable of respiring via gills and with lungs and therefore can be found in both aquatic and terrestrial environments. Notably, enzymatic characterization of the mitochondrial CPS isoforms in this transitional group has not led to clear conclusions. In order to determine which CPS isoform is present in transitional animals, we examined partial sequences for liver CPS amplified from five species of lungfish, and a larger fragment of CPS from one lungfish species (Protopterus annectens) and compared them to CPS isoforms from other fish and mammals. Enzyme activities for P. annectens liver were also examined. While enzyme activities did not yield a clear distinction between isoforms (virtually equal activities were obtained for either CPS I or III), CPS sequences from the lungfishes formed a monophyletic clade within the CPS I clade and separate from the CPS III clade of other vertebrates. This finding implies that the mitochondrial isoform of CPS in lungfish is derived from CPS I and is likely to have a physiological function similar to CPS I. This finding is important because it supports the hypothesis that lungfish employ a urea cycle similar to terrestrial air-breathing vertebrates.