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1. Leishmania major promastigotes showed a large decrease in ATP and increases in ADP and AMP contents after 4 min of anaerobiosis. 2. When ADP was added to intact promastigotes, it was completely metabolized, apparently by its conversion to adenosine extracellularly followed by adenosine uptake, further metabolism intracellularly, and release of hypoxanthine. Under anaerobic conditions, adenosine uptake was strongly inhibited and ADP degradation was stopped at adenosine. 3. Under both aerobic and anaerobic conditions, ATP was released into the medium. ATP release was specific, since ADP and AMP were not detectable extracellularly even when their external degradation was inhibited with molybdate.

The pellet height response (a measure of the increase in height of the pellet of cilia obtained by brief centrifugation in the presence of ATP as compared to the absence of ATP) of Tetrahymena cilia prepared by deciliation in the presence of Ca2+ is sensitive to the concentration of free Ca2+ during the pellet height assay. The magnitude of the increase in pellet height and the sharpness of the pellet boundary both increase markedly with increasing [Ca2+]. The half-maximal effect is attained at a free [Ca2+] of about 1.5 x 10(-7) M. The pellet height assay thus measures a Ca2+-sensitive component of the ciliary motile system. The possibility that this is the Ca2+-sensitive orientation system is discussed.

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, beta-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that alpha-mannosidase, beta-fucosidase, and beta-galactosidase are secreted into the salt solution and the secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for alpha-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.

The starting and stopping transients observed in sea urchin sperm flagella in the presence of high Ca2+ are believed to begin with an influx of Ca2+ into the axoneme and to end, as indicated by resumption of normal beating, when the Ca2+ has been reduced to very low levels by an active extrusion process. If the influx and efflux processes were uniformly distributed along the length of the flagellum, it is not likely that the starting and stopping transients would occur as a well defined sequence of events that always proceeds from the proximal to the distal end. Theoretical analysis of the concentration profiles of Ca2+ expected if Ca2+ influx occurred along the length of the flagellum but efflux was restricted to the proximal end shows that the time required to reduce [Ca2+] in the distal portion of the flagellum would generally be longer than the observed recovery times. Localization of both the influx and efflux processes near the proximal end, however, yields concentration profiles consistent with observations on the duration of starting and stopping transients.

Leishmania braziliensis panamensis, L. donovani, L. major, and L. mexicana amazonensis promastigotes, Trypanosoma lewisi bloodstream forms, and T. brucei gambiense procyclic forms were incubated with glucose as sole carbon source. All species consumed glucose more rapidly under aerobic than anaerobic conditions. All produced glycerol under anaerobic conditions, though the rate of glycerol production by T. lewisi was markedly lower than that by the other species. The four Leishmania species produced D-lactate, but not L-lactate, whereas T. b. gambiense procyclic forms produced L-lactate, but not D-lactate, and T. lewisi produced both isomers.

Tetrahymena grown overnight in deep cultures were incubated for 1 hr with [1-14C]labeled substrates in the presence or absence of 3-mercaptopicolinic acid (3MPA). 3-MPA inhibited appearance of label in glycogen from bicarbonate, acetate, pentanoate, octanoate, and succinate, but not from glycerol or glucose. In vitro assays of phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase activity showed that both enzymes were about equally distributed between the particulate and cytosol fractions. 3-MPA inhibited phosphoenolpyruvate carboxykinase from both the cytoplasmic and particulate fractions, but had no effect on phosphoenolpyruvate carboxylase from either location. These results suggest that the in vivo effects of this drug are due to inhibition of glyconeogenesis at this site.

A method is presented for determining the retardation of diffusion of particles inside cells owing to cytoskeletal barriers. The cytoskeletal meshwork is treated as a repeating periodic two-dimensional or three-dimensional lattice composed of elements of given size, shape, and spacing. We derive an analytic expression for the diffusion coefficient relative to that of the cytosol. This expression is evaluated by solving numerically an appropriate boundary-value problem for the Laplace equation. For the two-dimensional case, e.g., diffusion in a membrane, the results are quantitatively similar to those obtained by Saxton (1987. Biophys. J. 52:989-997) using Monte Carlo methods. The three-dimensional results are quantitatively similar to experimental results reported by Luby-Phelps et al. (1987. Proc. Natl. Acad. Sci. USA. 84:4910-4913) for the diffusion of dextran and Ficoll particles in Swiss 3T3 cells. By accounting for geometrical factors, these results allow one to assess the relative contributions of geometrical hindrance and of binding to the cytoskeletal lattice from measurements of intracellular diffusion coefficients of proteins.

A metabolic scheme of glycolysis and the pentose phosphate pathway has been constructed, assuming that the reactions occur in a single compartment. From this scheme, equations are written for a system in metabolic and isotopic steady state. These allow computation of the specific activity of every carbon atom of all the intermediates of the glycolytic and pentose phosphate pathways and consequently of the flux of carbon along each step of these pathways. A sufficiently large number of well distributed measurements of incorporation of radioactive label from different positions of several substrates into intermediates or products must be made to determine all the fluxes. This is done by choosing a set of metabolic fluxes, calculating incorporation with the aid of a computer, and then manipulating the flux rates until the computed incorporations match the data. The model is used in this paper to analyze the metabolism of the protozoan Tetrahymena pyriformis. The metabolic scheme of the model is consistent with all available information on the enzyme complement of this ciliate. Cells grown to transition phase in proteose/peptone medium were inoculated into a mixture of glucose (6 mM), fructose (6 mM), ribose (3 mM), and glycerol (3 mM) and incubated for 1 h. In each of these experiments, one of the following labeled substrates was present: [1-, 2-, 6-, or U-14C]glucose; [1- or U-14C]fructose; [1- or U-14C]ribose; [1(3)-or 2-14C]glycerol. The incorporation of label from these substrates into CO2, lipid, glycogen, and RNA was measured. In contrast to earlier studies on the metabolism of 2- and 3-carbon substrates by Tetrahymena, the rate of incorporation of label from some substrates into some products (e.g. from [1-14C]glucose into CO2) changed during the incubation. To treat these time-dependent data within the framework of the steady state model, the 1-h incubation was divided into three 20-min intervals; within each of these, the rates of incorporation were approximately constant, as required for a steady state system. Measurements of the pool sizes of glucose-6-P and fructose-6-P showed that only slow changes in pool sizes occurred after the first 5 min of incubation and indicated that the system was effectively in a metabolic and isotopic steady state throughout most of the incubation. The finding that a low concentration of cycloheximide prevented the acceleration of 14CO2 production from labeled glucose suggests a role for protein synthesis in the slow adaptation to carbohydrate addition and supports the quasi-steady state treatment of this system. The expected incorporation into each product was computed for trial sets of 1, independent flux rates. A set of flux values was found which yielded a good fit to the 29 measurements made for each interval. These flux values therefore constitute a quantitative description of temporal changes in carbon flow along the glycolytic and pentose phosphate pathways during the 1st h of adaptation to the carbohydrate mixture...

Tetrahymena pyriformis were grown to early-stationary phase and resuspended in a defined mixture containing glucose, fructose, ribose, glycerol, acetate, pyruvate, bicarbonate, glutamate, and hexanoate, with only one substrate labeled with 14C in any flask. Incorporation of label into CO2, glycogen, RNA, alanine, glutamate, glycine, lipid glycerol, and lipid fatty acids was measured 20, 40, and 60 min after the start of the incubation. To develop a model suitable for quantitative analysis of the data, it was necessary to join two preceding models, one for carbohydrate-metabolizing cells and one for acetate-metabolizing cells, eliminating the over-simplified sections of each. Equations were written and programmed for a digital computer to allow computation of the amount of label expected to be incorporated into any of the products measured for any given set of steady state flux values in the metabolic network. The model formed by simply joining the two preceding models did not yield satisfactory agreement with the complete data obtained in the present study, although each partial set of data could be fit well by the appropriate partial model. Analysis of the ways in which the model failed to yield good fits to the data indicated that another pool of P-enolpyruvate, of pyruvate, and of acetyl-CoA had to be added at the junction of the two models. The presence of such poolte into fatty acids as compared to the incorporation of label from glucose into fatty acids. A new model was therefore constructed which differed from the preceding model only in its structural organization at the level of P-enolpyruvate, pyruvate, and acetyl-CoA. The model is consistent with all known information on the compartmental structure of metabolism in Tetrahymena, on enzyme localization, and on the enzyme complement of this cell. Over 70 measurements of label incorporation into products were made at each time. These, plus a large number of "limit" measurements which constrain any possible solutions, were in sufficient excess of the 39 independent flux values to permit a stringent assessment of the model. A set of flux values was found which yielded a good fit to the data. These flux values therefore provide a quantitative description of metabolite flux in the intact cell during the slow adaptation to the nine-substrate mixture. The rates of utilization of glucose, fructose, glycerol, and ribose were in the ratio of about 10:1:0.33:0.16, i.e. fairly similar to the ratio observed with carbohydrate-metabolizing cells. Initial flux through phosphofructokinase is about 160 nmol/10(6) cells.h, increasing over 3-fold during tje jpir incubation. Initial flux through fructose-1,6-diphosphatase is about 110 nmol/10(6) cells.h and also increases almost 3-fold during the incubation. Thus net flux is glycolytic and increases 4-fold during the hour with a large amount of futile cycling at this step...

Plasmodium falciparum-infected human erythrocytes grown in vitro do not release 14CO2 when incubated in the presence of [1-14C]glutamate, despite the presence of glutamate dehydrogenase, implying the absence of alpha-ketoglutarate dehydrogenase activity and the lack of functional tricarboxylic acid cycle in the human malaria parasite. Cultures incubated with [14C]bicarbonate, however, fix CO2 into acid-stable metabolites; CO2 fixation proceeds linearly for up to two hours after an initial brief lag and may contribute appreciably to the metabolism of the parasite.

Hepatocytes were isolated from the livers of fed rats and incubated, in the presence and absence of 100 nM-glucagon, with a substrate mixture containing glucose (10 mM), fructose (4 mM), alanine (3.5 mM), acetate (1.25 mM), and ribose (1 mM). In any given incubation one substrate was labelled with 14C. Incorporation of 14C into glucose, glycogen, CO2, lactate, alanine, glutamate, lipid glycerol and fatty acids was measured after 20 and 40 min of incubation under quasi-steady-state conditions [Borowitz, Stein & Blum (1977) J. Biol. Chem. 252, 1589-1605]. These data and the measured O2 consumption were analysed with the aid of a structural metabolic model incorporating all reactions of the glycolytic, gluconeogenic, and pentose phosphate pathways, and associated mitochondrial and cytosolic reactions. A considerable excess of experimental measurements over independent flux parameters and a number of independent measurements of changes in metabolite concentrations allowed for a stringent test of the model. A satisfactory fit to the data was obtained for each condition. Significant findings included: control cells were glycogenic and glucagon-treated cells glycogenolytic during the second interval; an ordered (last in, first out) model of glycogen degradation [Devos & Hers (1979) Eur. J. Biochem. 99, 161-167] was required in order to fit the experimental data; the pentose shunt contributed approx. 15% of the carbon for gluconeogenesis in both control and glucagon-treated cells; net flux through the lower Embden-Meyerhof pathway was in the glycolytic direction except during the 20-40 min interval in glucagon-treated cells; the increased gluconeogenesis in response to glucagon was correlated with a decreased pyruvate kinase flux and lactate output; fluxes through pyruvate kinase, pyruvate carboxylase, and phosphoenolpyruvate carboxykinase were not coordinately controlled; Krebs cycle activity did not change with glucagon treatment; flux through the malic enzyme was towards pyruvate formation except for control cells during interval II; and 'futile' cycling at each of the five substrate cycles examined (including a previously undescribed cycle at acetate/acetyl-CoA) consumed about 26% of cellular ATP production in control hepatocytes and 21% in glucagon-treated cells.

Hepatocytes isolated from fasted male rats were incubated with a mixture of glucose, ribose, mannose, glycerol, and acetate and then treated with vasopressin (ADH), glucagon, or vasoactive intestinal polypeptide (VIP). Each of these hormones causes a rapid transient increase in the fluorescence signal arising from NADH and a sustained increase in the rate of metabolic oxygen consumption (QO2). The NADH transient was largest in response to glucagon, followed by ADH and VIP, respectively. For each hormone the responses were prevented by addition of a calcium-chelating agent. These results show that a transient, Ca2+-dependent redox shift in NADH and stimulation of QO2, perhaps resulting from an increase in the rate of delivery of reducing equivalents to the mitochondrion, occur early in the sequence of events by which several hormones that increase gluconeogenesis act.

The cross-bridge formalism of T. Hill has been incorporated into the nonlinear differential equations describing planar flagellar motion in an external viscous medium. A stable numerical procedure for solution of these equations is presented. A self-consistent two-state diagram with curvature-dependent rate functions is sufficient to generate stable propagating waves with frequencies and amplitudes typical of sperm flagella. For a particular choice of attachment and detachment rate functions, reasonable variation of frequency and wave speed with increasing viscosity is also obtained. The method can easily be extended to study more realistic state diagrams.

13C-nuclear magnetic resonance (NMR) spectroscopy was used to investigate the products of glycerol and acetate metabolism released by Leishmania braziliensis panamensis promastigotes and also to examine the interaction of each of these substrates with glucose or alanine. The NMR data were supplemented by measurements of the rates of oxygen consumption and substrate utilization, and of 14CO2 production from 14C-labeled substrate. Cells incubated with [2-13C]glycerol released acetate, succinate and D-lactate in addition to CO2. Cells incubated with acetate released only CO2. More succinate C-2/C-3 than C-1/C-4 was released from both [2-13C]glycerol and [2-13C]glucose, indicating that succinate was formed predominantly by CO2 fixation followed by reverse flux through part of the Krebs cycle. Some redistribution of the position of labeling was also seen in alanine and pyruvate, suggesting cycling through pyruvate/oxaloacetate/phosphoenolpyruvate. Cells incubated with combinations of 2 substrates consumed oxygen at the same rate as cells incubated with 1 or no substrate, even though the total substrate utilization had increased. When promastigotes were incubated with both glycerol and glucose, the rate of glucose consumption was unchanged but glycerol consumption decreased about 50%, and the rate of 14CO2 production from [1,(3)-14C]glycerol decreased about 60%. Alanine did not affect the rates of consumption of glucose or glycerol, but decreased 14CO2 production from these substrates by increasing flow of label into alanine. Although glucose decreased alanine consumption by 70%, it increased the rate of 14CO2 production from [U-14C]- and [l-14C]alanine by about 20%.(ABSTRACT TRUNCATED AT 250 WORDS)

Egestion of carmine particle-containing food vacuoles from the cytoproct of Tetrahymena pyriformis has been analyzed by high-speed cinemicrography. The vacuole may enter into position in the cytoproct approximately 7 sec before ejection, and forms a distinct bulge beyond the outline of the cell surface for over 2 sec prior to ejection. The ejection process itself requires 20-80 msec.

Tetrahymena pyriformis were grown in proteose-peptone medium and then washed and incubated in a dilute salt solution for one hour. The cells were then discarded and the lysosomal hydrolases that had been secreted were subjected to DEAE cellulose column chromatography. At least three isoenzymes of acid phosphatase, three of acid protease, and two of beta-N-acetylhexoseaminidase were found, as well as single peaks of alpha-mannosidase, beta-galactosidase, and beta-fucosidase. The latter two activities were not resolved by the DEAE column and could not be separated in a second chromatographic step on CM-cellulose. Cells were also grown under identical conditions and homogenized in 0.25 M sucrose in order to allow comparison of some of the intracellular lysosomal hydrolases with their secreted counterparts. Two lysosomal populations were resolved by sucrose density gradient sedimentation, a heavy lysosomal fraction, contered at a density of about 1.25 gm/cm3, and a light lysosomal fraction, centered at a density of about 1.16 gm/cm3. These two populations differed in that the light lysosomes did not appear to contain significant amounts of beta-fucosidase, beta-galactosidase, or acid protease, whereas all six of the hydrolase activities studied were present in the heavy lysosomes. The light lysosomal peak occurred in cells grown to transition phase, but was markedly reduced in cells from cultures grown to stationary phase. In addition to these two fractions a third very light particle, containing only alpha-mannosidase activity, was detected just inside the gradient. Measurements were made of the effect of heat (10 minutes at 66 degrees) and of a change in pH from 4.5 (standard assay condition) to 6.0 on the three acid phosphatases and two beta-N-acetylhexoseaminidase isoenzymes resolved by DEAE column chromatography of the secreted hydrolases and on these hydrolyases in the heavy and light lysosomal fractions on the sucrose gradient. Use of the thermostability and pH criteria permitted computation of the expected properties of the intralysosomal acid phosphatase and hexoseaminidase activities if these consisted of the respective isoenzymes in the proportions secreted. It was found that neither the intralysosomal acid phosphatase nor the intralysosomal hexoseaminidase had the properties expected if they consisted of the secreted mixture of the respective isoenzymes, indicating that modification of some of these isoenzymes may have occurred during the 1-hour starvation period or after secretion.

The growth of Tetrahymena pyriformis strain HSM was strongly inhibited by 4-pentenoic acid. Supplementing the medium acetate reversed the growth inhibition, but pyruvate was ineffective. Glycogen content was much lower in cells grown with 4-pentenoic acid than in controls; this effect was not reversed by acetate or by pyruvate. There was little effect of 4-penteonic acid on the in incorporation of label from [1-14C]acetate, [2-14C]glycerol, [1-14C]ribose, [U-14C]fructose, or [1-14C]glucose into CO2 but incorporation of label into glycogen was inhibited, the strongest inhibition being on acetate and the weakest approximately 20%) on ribose, fructose, and glucose. A 3-compartment model for quantitation of labeled acetyl CoA fluxes was shown to be applicable to Tetrahymena grown in the presence of 4-pentenoic acid, and experiments were performed to establish the flux of [1-14C]acetyl CoA into glycogen, lipids, CO2, glutamate, and alanine. It was evident from the results of these experiments that 4-pentenoic acid did not appreciably inhibit beta-oxidation or lipogenesis, but markedly decreased the glyconeogenic flux of labeled acetyl-CoA from the peroxismal and outer mitochondrial compartments.

Murase & Shimizu (1986, J. theor. Biol. 119, 409) introduced an excitable dynein-microtubule system based on a three-state mechanochemical cycle of dynein to demonstrate bend propagation in the absence of a curvature control mechanism. To examine the essential behavior of this class of models in a viscous fluid, we have represented the force generated by the complex dynein mechanochemistry by a formal model consisting of "force" and "activation" functions vs. sliding distance. Since the model has excitable properties with threshold phenomena and hysteresis switching between two opposed subsystems, it closely resembles the more realistic dynein kinetic scheme in its overall properties but is specified by fewer parameters. This model displays both bend initiation and bend propagation when the filaments at the basal end are either fixed or free to slide. A passive region is necessary at one end of the axoneme in order to obtain stable wave propagation; bends propagate towards the end with the passive region. Stable bend propagation is highly sensitive to small perturbations in external force distribution.

A detailed model of intermediary metabolism has been constructed which is consistent with all known information on the compartmental structure of metabolism in Tetrahymena, on the enzyme complement of this cell, and on the localization of the enzymes. The model allows computation of the specific activity of every carbon atom of all metabolites and thus of the flux of carbon along the major pathways of metabolism under steady state conditions. To test the model, data were required from cells grown under standard conditions and then suspended in a dilute salt solution and incubated for 1 hour in a mixture of acetate, pyruvate, hexanoate, bicarbonate, and glutamate labeled in a total of 10 positions, but with only one substrate labeled in any given flask. Twenty-seven measurements of label incorporation into CO2, lipids, glycogen, glutamate, and alanine were made, plus measurements of label distribution into fatty acid and glycerol moieties for 4 of the substrates and of oxygen consumption and of glycogenolysis, yielding 33 independent measurements. These, plus about 18 "limit" measurements which also constrain any possible solutions, were in sufficient excess of the 23 independent parameters to permit a stringent assessment of the model. Equations derived directly from the structure of the model and from the known stereochemistry of the reactions were programmed on a PDP-15 computer and values of the Qo2 and of label expected to be incorporated into the various products actually measured were computed for any given set of flux rates. A set of flux rates was found which yielded an excellent fit to the observed data. The ability to achieve a fit to the data for an overdetermined system constitutes strong support for this structural model of intermediary metabolism and the computed flux rates therefore provide a quantitative description of metabolite flow in the intact cell. Despite the redundancy of measurements relative to parameters to be determined, it was not possible to define a unique set of values for the flux through phosphoenolpyruvate carboxylase and phosphoenolpyruvate carboxykinase, although the relationship between these fluxes is specified by the model. The analysis allows estimation of the recycling of phosphoenopyruvate through pyruvate kinase under conditions of net glyconeogenesis and an apparently futile exchange of acetyl-CoA between the inner and outer mitochondrial compartments. Carbon flow through the glyoxylate bypass under these conditions is about one-third of that through the Krebs cycle. The analysis also shows a net transport of malate from the peroxisomes to the mitochondria, consistent with the anaplerotic role of the peroxisomal glyoxylate bypass in Tetrahymena.

Glycerol-extracted cilia from Tetrahymena pyriformis were demembranated by treatment with Triton X-100 and then heated for up to 30 min at temperatures between 34-38 degrees C. Heat treatment caused an uncoupling of the ATPase from motility as indicated by an increase in ATPase activity and a loss of pellet height response. After heat treatment, the ATPase activity of the dynein in situ differed from that in unheated cilia as shown by an increased sensitivity to a lower temperature of assay (0 degrees C) and by a loss of the activation normally observed upon reaction with N-ethylmaleimide or p-phenylenedimaleimide. Upon extraction of the heat-treated cilia by Tris-EDTA, there was a large loss in ATPase activity so that the heat-treated cilia yielded a crude dynein fraction with a lower specific activity compared with that obtained from unheated controls. The difference was not due to a change in the amount of protein recovered or in the amount of ATPase activity which remained unextracted. Resolution of the crude dynein by sucrose density sedimentation indicated that activity was lost from both the 14S and 30S peaks but more so from the latter than from the former. Thus dynein in situ in cilia in which the ATPase has been uncoupled from motility by gentle heat treatment differs in several important respects from dynein inside unheated cilia.

In a preceding study [Blum, J.J., and Reed, M.C. (1985): Cell Motil. 5:507-527], factors responsible for the shape and velocity of the leading edge of the radiolabeled organelle profile were analyzed, but processes that might influence the shape of the plateau-like region behind the advancing wave were ignored. It is now shown that deposition of material from the fast transport system into membrane-associated structures, degradation of such deposited material and its return to the soma by the retrograde transport system, or leakage of radiolabeled material from the axon can account for the shape of the plateau. Furthermore, these processes are compatible with the maintenance of such structural inhomogeneities as the nodes of Ranvier.

The products released by Leishmania major promastigotes incubated with [1-13C]glucose as sole exogenous carbon source were identified using nuclear magnetic resonance (NMR). Under aerobic (95% O2/5% CO2) conditions, acetate, succinate, and small amounts of pyruvate, D-lactate, and glycerol were released in addition to CO2. Under anaerobic (95% N2/5% CO2) conditions, the relative amounts of products formed changed and alanine was also released. The changes in the rates of glucose consumption and product formation during the aerobic to anaerobic transition were measured. Under hypoxic conditions (O2 less than 0.2%), glucose consumption was decreased by about 50%. Under completely anaerobic conditions (100% N2), glucose consumption almost ceased (a total reverse Pasteur effect). The inclusion of 5% CO2 in the gas phase restored hypoxic and anaerobic glucose consumption to the aerobic rate, and increased production of succinate, pyruvate, and D-lactate. Thus, CO2 and very low concentrations of O2 have strong regulatory effects on L. major glucose metabolism. A quantitative carbon balance showed that the NMR-identified products accounted for only about 25% of the glucose carbons consumed under aerobic conditions. CO2, measured as the release of 14CO2 from [U-14C]glucose, accounted for an additional 25% of the glucose consumed. About 11% of the glucose carbon was incorporated into trichloroacetic acid-insoluble products, mostly lipid. Large amounts of label from [U-14C]glucose were incorporated into the intracellular pools of alanine, glutamate, glutamine, and aspartate, indicating that CO2 from unlabeled amino acids contributed to the carbon balance. Under anaerobic conditions, all the glucose carbons consumed could be accounted for solely by the NMR-identified products.

The dose-response curves for pituitary luteinizing hormone (LH) release in response to gonadotropin-releasing hormone and its agonists are unusually broad. It appears, however, that these ligands bind to a single class of receptors. It is shown that these dose-response data can be explained by either of two models in which ligand-receptor complexes stimulate LH secretion by interacting with either of two different effector systems or by interacting with a single effector system but forming monomeric and dimeric active effector complexes. A combination of these two basic models can account for the very broad, biphasic dose-response curve reported for LH release in response to leukotriene C4.

A model for fast axonal transport is developed in which the essential features are that organelles may interact with mechanochemical cross-bridges that in turn interact with microtubules, forming an organelle-engine-microtubule complex which is transported along the microtubules. Computer analysis of the equations derived to describe such a system show that most of the experimental observations on fast axonal transport can be simulated by the model, indicating that the model is useful for the interpretation and design of experiments aimed at clarifying the mechanism of fast axonal transport.

We report here that a precipitating antibody prepared against Tetrahymena pyriformis calmodulin recognizes calcium-dependent determinants in the native protein. The ability of the antibody to precipitate 35S-labeled Tetrahymena calmodulin in direct radioimmunoassays was enhanced at least 3-fold in the presence of calcium. Competitive radioimmunoassay using homogeneous preparation of endogenously 35S-labeled Tetrahymena calmodulin and protein A-Sepharose-purified immunoglobulin G demonstrated that this antibody preparation is specific for protozoan calmodulin. Homogeneous vertebrate, invertebrate, and plant calmodulins, as well as rabbit skeletal muscle troponin C, did not show significant competition with the 35S-labeled Tetrahymena protein at concentrations 100-fold greater than that at which the homologous unlabeled Tetrahymena calmodulin produced 50% competition. A cyanogen bromide digest of Tetrahymena calmodulin also showed partial competition with the intact 35S-labeled protein, but only in the presence of calcium. The major antigenic determinants were localized to the carboxyl-terminal half of the molecule by immunoassay of limited trypsin fragments of Tetrahymena calmodulin. The antibody bound native calmodulin complexed to bovine brain phosphodiesterase (EC 3.1.4.17) but failed to recognize the Tetrahymena calmodulin carboxyl-terminal fragment (76-147) when complexed to the enzyme.

Tetrahymena pyriformis strain HSM was found to have monomine oxidase (MAO) and a catechol-3-methyl transferase-like (COMT) activity. As in mammalian tissues, the MAO activity is predominantly localized in the mitochondrial pellet and COMT in the cytosol. The COMT-like activity was present in amounts comparable to several mouse tissues and was inhibited by tropolone. MAO activity was much lower than in any of the mouse tissues tested, and its activity varied greatly from preparation to preparation. The substrate preference of Tetrahymena MAO was tryptamine greater than serotonin greater than dopamine, and activity increased with increasing pH from pH 6.5 to pH 7.8, as does that of mouse liver MAO. Teh Km of Tetrahymena MAO for tryptamine was approximately 4 micrometer, an order of magnitude lower than that of mouse liver MAO. Sensitivity of inhibition by MAO inhibitors was variable. In some preparations, no inhibition was observed. In others clear inhibition was obtained, harmine and clorgyline being among the most potent inhibitors.

Tetrahymena pyriformis, strain HSM, do not have glucose-6-phosphate dehydrogenase or 6-phosphogluconate dehydrogenase, but contain transaldolase, transketolase, ribose 5-phosphate isomerase, ribulose-5-phosphate 3-epimerase, and ribokinase. The nonoxidative enzymes of the pentose phosphate shunt function in metabolism as indicated by the incorporation of label from [1-14C]ribose into CO2 and glycogen and by the increase in total glycogen content of cultures supplemented with ribose.

Hepatocytes were isolated from the livers of fed rats and incubated with a mixture of glucose (10 mM), ribose (1 mM), mannose (4 mM), glycerol (3 mM), acetate (1.25 mM), and ethanol (5 mM) with one substrate labelled with 14C in any given incubation. Incorporation of label into CO2, glucose, glycogen, lipid glycerol and fatty acids, acetate and C-1 of glucose was measured at 20 and 40 min after the start of the incubation. The data (about 48 measurements for each interval) were used in conjunction with a single-compartment model of the reactions of the gluconeogenic, glycolytic and pentose phosphate pathways and a simplified model of the relevant mitochondrial reactions. An improved method of computer analysis of the equations describing the flow of label through each carbon atom of each metabolite under steady-state conditions was used to compute values for the 34 independent flux parameters in this model. A good fit to the data was obtained, thereby permitting good estimates of most of the fluxes in the pathways under consideration. The data show that: net flux above the level of the triose phosphates is gluconeogenic; label in the hexose phosphates is fully equilibrated by the second 20 min interval; the triose phosphate isomerase step does not equilibrate label between the triose phosphates; substrate cycles are operating at the glucose-glucose 6-phosphate, fructose 6-phosphate-fructose 1,6-bisphosphate and phosphoenolpyruvate-pyruvate-oxaloacetate cycles; and, although net flux through the enzymes catalysing the non-oxidative steps of the pentose phosphate pathway is small, bidirectional fluxes are large.

Equations are derived describing the dispersion of a permeable solute during Poiseuille flow in a capillary model. It is shown that for the normal range of physiological parameters such as capillary radius, capillary length, blood flow, permeability coefficients, and diffusion constants, the center of mass of a bolus of solute moves at a speed very close to the mean speed of flow and that the solute leaves the capillary with an exponential time course depending on the permeability but not on the diffusion constant. There is no appreciable difference in the dispersion of the solute or in its rate of permeation from the capillary whether one considers piston flow or Poiseuille flow. A bolus of arbitrary radial shape tends to become radially uniform very close to the arterial end of the capillary.

Cilia were isolated from Tetrahymena pyriformis by a glycerol method. The addition of low concentrations of ATP, but not of other nucleoside triphosphates, caused an increase of up to twofold in the amount of cilia pelleted in a low-speed centrifugation assay and decreased the density of the pellets compared to control pellets. Pellet size and density depend on pH, both in the absence and in the presence of low concentrations of ATP. High concentrations (5 mM and above) of ATP and of other nucleoside triphosphates tend to "dissolve" the cilia. Heating the cilia for 11 min at 40 degrees C abolishes the increase in pellet size and the decrease in pellet density caused by low ATP, but slightly increases the ATPase activity of the cilia. Heat treatment, however, does not prevent the dissolving effect of high ATP. There are, thus, two independent effects of ATP on the hydrodynamic properties of cilia suspensions.