Jacob J. Blum

Presence of calmodulin in Tetrahymena

Ca-dependent affinity chromatography on phenothiazine-Sepharose 4B has been used to isolate a pure protein from the ciliate Tetrahymena pyriformis. This protein has been identified as calmodulin by demonstrating three of the Ca-dependent activities attributed to calmodulins. Tetrahymena calmodulin also has physicochemical properties similar to those of the previously characterized mammalian, coelenterate, and plant proteins, except for a lower molecular weight (15,000) and slightly different CNBr fragments compared to bovine brain calmodulin. Calmodulin is a constituent of demembranated Tetrahymena cilia from which it can be extracted with the crude dynein fraction. Sucrose density gradient fractionation indicated its presence in fractions containing the 14S dynein ATPase. It is concluded that the essential properties of calmodulin have been highly conserved during much of eukaryotic evolution, and it is suggested that calmodulin plays a role in the control of ciliary motility in Tetrahymena.

A model for the computation and encoding of azimuthal information by the lateral superior olive

A structural model is proposed for the processing of interaural intensity differences by the lateral superior olive. One fundamental assumption is that the incoming excitatory projections from the ipsilateral anteroventral cochlear nucleus innervate columns of LSO neurons serially according to threshold. A second fundamental assumption is that the inhibitory innervation from the ipsilateral medial nucleus of the trapezoid body is also serially arranged according to threshold but in the opposite direction along the LSO column. Using neurophysiological and neuroanatomical data for neuronal response curves, connectional patterns, and cell and synapse numbers, the model was formulated quantitatively and implemented for machine computation. Azimuthal location is encoded by the position along the LSO column where LSO cell firing first goes to zero. Accuracy of coding was tested for three different connectional schemes, for variations in neuronal parameters, and for cell and synapse death. Encoding is shown to be independent of absolute sound level and to vary linearly with interaural intensity difference.

Existence of a breaking point in cilia and flagella

Abstract Data in the literature indicate that the transitional region between the kinetosome and the flagellar or ciliary shaft of many Protozoan species is specialized as a breaking point. Only two Metazoan species have been examined from this point of view, and in both of them the transition region is also a breaking point. A similar phenomenon may occur in some sperm. Breakage occurs in response to a wide variety of chemical, osmotic, physical and mechanical treatments. Possible implications of this phenomenon are discussed.

Approximate traveling waves in linear reaction-hyperbolic equations

Linear reaction-hyperbolic equations of a general type arising in certain physiological problems do not have traveling wave solutions, but numerical computations have shown that they possess approximate traveling waves. Using singular perturbation theory, it is shown that as the rates of the chemical reactions approach

On the Analysis of Metabolic Networks

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Effects of oxygen concentration on the intermediary metabolism of Leishmania major promastigotes.

, solutions approach traveling waves. The speed of the limiting wave and the first term in the asymptotic expansion are computed in terms of the underlying chemical mechanisms.

On the regulation of glycogen metabolism in Tetrahymena

In the past quarter of a century, it has become evident that the major pathways of intermediary metabolism are regulated by a remarkable variety of interlocking control mechanisms. These include covalent modification of enzymes: acetylation, methylation, adenylylation, phosphorylation by cAMP-sensitive and -insensitive protein kinases, changes in SH and S-S redox states, among others. Regulation by noncovalent modification is virtually ubiquitous, and includes sensitivity to various ions, especially Ca2+, to substrates and to end products, to ratios of modifier concentrations such as [ATP]/[ADP] · [Pi], [NADH]/ [NAD+], [acyl CoA]/[CoA], etc. While elucidation of regulatory mechanisms is clearly prerequisite to an understanding of the controls acting in vivo, it is not in itself sufficient to allow prediction of the flux of metabolite through any particular metabolic step, even if one were to perform a “complete” analysis of all the known regulatory effectors in the cell. This lack of certainty comes about not only because of the ever-present possibility that compounds which were not measured are important regulatory substances, but also because frequently the enzymes and/or putative regulatory metabolites are in different subcellular environments, whether these be readily indentifiable organelles (such as mitochondria, peroxisomes, glycogen particles) or microenvironments formed by multienzyme complexes (Srere and Mosbach, 1974). The situation is further complicated by much evidence suggesting that proteins interact with one another in the relatively concentrated intracellular or intraorganellar environments where they function, while most of the in vitro studies are performed on partially purified enzymes and at unphysiologically low protein concentrations. The development of techniques for rapid separation of mitochondria from the rest of the cytosol (Siess et al.,1977; Tischler et al.,1977) considerably reduces the ambiguity inherent in measurements of cell metabolite content, but even if it were possible to separate the peroxisomes and other organelles at the same time, it would remain very difficult to assess all the factors determining the metabolite flux through any particular step in vivo. Metabolic network analysis addresses a much simpler question than those we have just discussed—namely, what is the flux of metabolite through the steps of a metabolic pathway in the living cell? Even assuming that the question may be answered by the methods to be described below, it is clear that such information by itself contributes very little to an understanding of how metabolism is regulated. Flux values obtained by network analysis may be compared with the enzyme activity for each step measured in vitro under “optimal” conditions. Frequently the in vitro activity exceeds the in vivo flux, and this information may reinforce accepted views on regulated steps or may indicate new steps where control may be sought. In some cases, however, the apparent in vivo flux exceeds the enzyme activity assayed under “optimal” conditions. This reemphasizes the difficulty of extrapolating from in vitro measurements of enzyme activity to in vivo behavior. When flux values from network analysis are interpreted in the light of information on the in vitro behavior of the enzymes involved and on the concentrations of ligands in the relevant subcellular compartments, one may expect to acquire a deeper understanding of the regulation of metabolism in the living cell.

ATPase activity of Tetrahymena cilia before and after extraction of dynein

Leishmania major promastigotes grown in late log phase were incubated with glucose as sole exogenous carbon source in the presence of 5% CO2 and the amounts of glucose consumed and of the major products formed--succinate, pyruvate, alanine, acetate, glycerol, and D-lactate--were measured as a function of pO2. Glucose consumption increased as pO2 was lowered to 6% (a positive Pasteur effect) and then declined to the same level at 95% N2 as at 95% O2. The production of D-lactate and of glycerol increased as pO2 dropped from 95%, reaching a maximum at about 2% O2. Succinate production, however, increased dramatically when pO2 was reduced to 6% and remained at that level with further reduction of pO2. The amount of succinate produced relative to the amount of glucose carbon consumed suggests utilization of an endogenous carbon source. Acetate production did not change between 95% O2 and 6% O2 and then declined with decreasing pO2. These observations suggest the presence of two sensors, one with a high and one with a low affinity for oxygen. When glycerol or alanine were the only exogenous sources of carbon, the primary products released were acetate and succinate. Acetate production from alanine declined slightly as pO2 was reduced to 2%, and then dropped markedly when pO2 was reduced to 0%. Acetate production from glycerol increased over 4-fold when the pO2 was reduced from 95% to 4%, and then declined with further reduction in pO2. No succinate was formed from either substrate until complete anaerobiosis. This pattern of response, while differing from that when glucose was sole exogenous carbon source, is also consistent with the regulation of metabolism by a high and a low affinity O2 sensor. Cells from cultures in early stationary phase, before the appearance of metacyclic forms, consumed glucose at about the same rate as log phase promastigotes, but did not show a Pasteur effect. Stationary cells also consumed glycerol at the same rate as did log phase promastigotes, but consumed alanine at a much lower rate. Reduction of pO2 affected product formation from each of these substrates differently than for log phase promastigotes, demonstrating the sensitivity of several pathways of intermediary metabolism to regulation by pO2 during the transition from log to stationary phase.

A possible role for adenylate kinase in cilia: Concentration profiles in a geometrically constrained dual enzyme system☆

Abstract The glycogen phosphorylase and glycogen synthetase activities of Tetrahymena pyriformis were assayed after growth of cells under various conditions. Cells exposed to low concentrations of theophylline or to low oxygen tension during growth in proteose-peptone medium had increased synthetase and phosphorylase activities. Supplementing the medium with glucose increased the synthetase activity. Growth in the presence of both glucose and theophylline markedly increased the synthetase activity and decreased the phosphorylase activity. Neither reserpine nor dichloroisoproterenol affected the synthetase activity, but either or both drugs increased the phosphorylase activity. Triiodothyronine did not affect the phosphorylase activity but inhibited the synthetase activity. It was also found that Tetrahymena had 3′,5′-cyclic AMP phosphodiesterase activity and that caffeine and theophylline inhibited this activity. The effects of glucose supplementation, low oxygen tension, and of the drugs mentioned above were correlated with their effects on cell glycogen content, and the implications of these findings with respect to the control of glycogen metabolism are discussed.

Properties of the induced acid phosphatase and of the constitutive acid phosphatase of Euglena

Abstract Cilia from Tetrahymena pyriformis were extracted twice with Tris-EDTA. The first extraction increased the total ATPase activity by about one-third. No increase in activity occurred as a result of the second extraction, but 40% of the original ATPase activity remained in the pellet. The activity remaining in the pellet differed in its substrate specificity, its thermostability, and its sensitivity to monovalent cation chlorides from the solubilized dynein. Several of the properties of the ATPase activity of whole cilia differed from those computed for a mixture of 40% pellet ATPase + 60% solubilized dynein ATPase. From these differences it was deduced that dynein in situ is more thermostable than is solubilized dynein and, in contrast to solubilized dynein, is slightly inhibited by KCl, NaCl, LiCl, and NH4Cl. The increase in total activity upon solubilization of the dynein and the changes in thermostability and in sensitivity to monovalent cations indicates that dynein ATPase in situ is modified by interaction with other components of the axonemal bend generating system. The pellet remaining after extraction of dynein by two dialyses against Tris-EDTA was treated with 0.4% Triton X-100 to solubilize ciliary membranes. Less than half of the ATPase activity was solubilized by this treatment. The possibility that the activity remaining in the Tris-EDTA- and Triton X-100-extracted residue represents an additional ATPase of cilia is discussed.