Eating, drinking and temperature responses to intracerebroventricular cholecystokinin in the chick

The central effect of cholecystokinin-octapeptide (CCK), SQ 19,844 or sincalide, on the intake of food and water and on colonic temperature (Tc) was investigated using the broiler cockerel. Four-week old chicks were maintained in a thermoneutral environment of 23-24 degrees C. After food was removed for a 24 hr interval, CCK was infused in a volume of 10.0 microliters into the lateral cerebral ventricle (ICV) in doses ranging from 10-150 ng. Although lower doses of CCK had no effect on food intake, 100 or 150 ng of CCK significantly reduced consumption of food in a dose-dependent manner; water drinking was significantly decreased by 100 ng of CCK. In addition, CCK at doses of 100 and 150 ng prevented the slow rise in Tc observed following infusions of control CSF. This latter effect appeared to be a result of feeding activity associated with caloric intake and the heat increment in the control birds rather than a specific thermoregulatory effect. Overall, our results suggest that CCK may comprise a part of the central mechanism underlying the neural control of short term satiety in an avian species similar to that proposed for the mammal.     

Evidence for a two-state mobile carrier mechanism in erythrocyte choline transport: Effects of substrate analogs on inactivation of the carrier by N-ethylmaleimide

Summary Choline transport in erythrocytes is irreversibly inhibited by N-ethylmaleimide. The hypothesis that the carrier alternates between outwardfacing and inward-facing forms and that only the latter reacts with the inhibitor (Martin, K. (1971)J. Physiol. (London) 213:647–667; Edwards, P.A. (1973)Biochim. Biophys. Acta 311:123–140) is here subjected to a quantitative test. In this test the effects of a series of substrate analogs upon rates of inactivation and rates of choline exit are compared. By hypothesis the effect of an analog in the external solution on the inactivation rate depends only on how it affects the proportion of the inward-facing carrier. Since¹⁴C-choline efflux is necessarily proportional to the concentration of free carrier in the inward-facing form, the analogs should have related effects on the two rates. In every case the observed effects were identical, whether the analogs accelerated transport or inhibited it. Analysis of the results demonstrates that (1) the transport mechanism depends on the operation of a mobile element; (2) distinguishable inward-facing and outward-facing conformations of the free carrier, carrier-substrate complex, and carrier-inhibitor complex exist, and only the inwardfacing forms react at a significant rate with N-ethylmaleimide; (3) carrier mechanisms involving a single form of free carrier or a single form of carriersubstrate complex are ruled out; and (4) dissociation of the carrier-substrate complex is a rapid step with all substrate analogs.     

Malate synthase from Gossypium hirsutum

Malate synthase was purified 2000-fold from cotyledons of dark-germinated cotton, Gossypium hirsutum. The purified enzyme had a pH optimum of 8.2, and an absolute requirement for a divalent cation. Only glyoxylate and acetyl-CoA served as condensation partners. Results obtained with functional-group directed inhibitors suggest the presence of lysine, tyrosine and histidine residues in the active site. Temperature optimum was 40°, and energy of activation was 3.3 kcal/mol. The MW of cotton malate synthase, determined by rate-zonal density gradient sedimentation, was 750 000. Initial-rate studies indicated Michaelis-Menten kinetics. Inhibition by substrate analogs, plus substrate-interaction kinetics gave results consistent with a sequential bireactant mechanism.     

Cytoplasmic streaming inParamecium

Summary Certain species ofParamecium demonstrate rotational cytoplasmic streaming, in which most cytoplasmic particles and organelles flow along permanent route, in a constant direction. By means of novel methods of immobilization, observation and recording, some dynamic properties of cytoplasmic streaming have been described. It was found that the velocity profiles of coaxial layers of cytoplasm have a (parabolic) paraboidal shape and the mean output of cytoplasm flow in different examined zones of streaming is constant. As the consequence of randomly distributed elementary propulsion units within the cytoplasm, particles, which serve as markers of movement, exhibit movements of a saltatory nature; this form of movement is seen inParamecium streaming only in cases of error due to polarization of the saltating particles. Interaction of actin filaments and myosin is likely to occur under specific conditions in microcompartments of cytoplasm where local solations are generated eventually leading to contractions which might propagate on gelated neighbouring areas. Places of elementary contractions are scattered. Therefore the motile effect appears as streaming. Rotational cytoplasmic streaming inParamecium may serve as a convenient model for the study of the dynamics and function of cytoplasmic motility.     

Mechanism of activation of cyclic GMP-dependent protein kinase from rat liver by multiple modulators

A number of polyanionic compounds, including DNA, RNA and polyglutamate, were shown to exhibit protein kinase stimulatory modulator activity as they were required for cyclic GMP to stimulate the phosphorylation of various cationic substrates by rat liver cyclic GMP-dependent protien kinase. Anionic proteins (casein, phosvitin) were phosphorylated poorly by the enzyme and their phosphorylation was not stimulated by the stimulatory modulators. Studies of the mechanism of action suggest that the modulators interact directly with the substrates to form a complex which is a better substrate than free histone. The observed effect of modulator is complex as it depends on the ratio of modulator to histone and the resultant state of the complex formed (better or poorer substrate than free histone). The observed effect is also dependent on the properties of the histone substrate as Michaelis-Menten kinetics are not observed in the phosphorylation of arginine-rich histone in the absence or presence of cyclic GMP.     

Inactivation and reactivation of proteins (enzymes)

The molecular mechanisms of protein inactivation, i.e. aggregation, thiol-disulphide exchange, alteration of the primary structure, dissociation of cofactor molecules from the active centre, dissociation of the oligomeric proteins into subunits and conformational changes have been analysed. All these mechanisms are closely interrelated during inactivation of proteins. However, in many cases, the conformational changes accompany and trigger other inactivation processes. Reactivation of irreversibly inactivated proteins is·discussed. Reactivation can be successful when inactivation has been caused by aggregation, modification of SH-groups (or S-S bonds) or as a consequence of irreversible conformational changes.     

Interaction of high-affinity nucleotide binding sites in mitochondrial ATP synthesis and hydrolysis

The present study contributes to the problem of the dynamic structure of mitochondrial F1-ATPase and the functional interrelation of so-called tight nucleotide binding sites. Nucleotide analogs are used as a tool to differentiate two distinct functional states of the membrane-bound enzyme, proposed to reflect corresponding conformational states; they reveal F1-ATPase as a dual-state enzyme: ATP-synthetase, and ATP-hydrolase. The analogs used are 3-naphthoyl esters of AD(T)P, and 2(3)-O-trinitrophenyl ethers of AD(T)P. Both types of analogs act inversely to each other with respect to their relative effects on oxidative phosphorylation and on ATPase in submitochondrial vesicles. The respective ratios ofK _i versus both processes are 250/1 compared to 1/170. It is also shown that in the presence of the inhibitory 3-esters oxidative phosphorylation deviates from linear kinetics and that these inhibitors induce a lag time of oxidative phosphorylation depending on the initial pattern of nucleotides available to energized submitochondrial vesicles. The duration of the lag time coincides with the time course of displacement of the analog from a tight binding site. The conclusions of the study are: (a) the catalytic sites of F₁-ATP-synthetase are not operating independently from each other; they rather interact in a cooperative manner; (b) F₁-ATPase as a dual-state enzyme exhibits highly selective responses to tight binding of nucleotides or analogs in its energized (membrane-bound) state versus its nonenergized state, respectively.Abbreviations used: N-AD(T)P, 3-O-naphthoyl(1)-AD(T)P; DMAN-AD(T)P, 3-O-(5-dimethylaminonaphthoyl(1))-AD(T)P, also termed F-AD(T)P in previous papers because of its fluorescence; TNP-AD(T)P, 2(3)-O-(2,4,6-trinitrophenyl)-AD(T)P; FCCP,p-trifluoromethoxycarbonylcyanide phenylhydrazone.     

Hormonal mechanisms for differentiation in plant tissue culture

Summary Cells possess extraordinary powers to organize their molecular processes not only to maintain a cell in a given steady state but also to recognize that state during differentiation. Regulation of these organizational forces appears to be under the control of chemical factors, and a hormonal concept of regulation has evolved. Hormones have been considered to act by reacting with a specific target site. This may be part of their mode of action, but I would like to suggest that a hormone enters and becomes part of a total molecular resonance system. In so doing, the entire molecular system of the cell is modified. Of the known plant hormones, the cytokinins, because of their role in experimentally induced cell division and differentiation, serve as a probe of hormonal involvement in differentiation. Cultured somatic cells of tobacco plants can be induced to undergo differentiation by addition of cytokinin and auxin to the medium. Studies of the cytokinin hormones show a series of diverse molecular involvements. The archetype cytokinin, N⁶-(Δ²-isopentenyl) adenosine (i⁶Ado), occurs in some molecular species of tRNA where it plays a vital role in the codon-anticodon interaction of tRNA and m-RNA. i⁶Ado under-goes extensive metabolism in the tobacco tissue. It is either degraded to adenosine or converted to derivatives that possess biological activity. It is perhaps, therefore, more correct to consider the hormone function as being derived from this total metabolic web. The normal somatic cells of tobacco cultures spontaneously change occasionally into an autonomous form that requires no external growth factors. This line of cells synthesizes i⁶Ado. The metabolic web of the hormone-dependent strain can be perturbed by added auxin but such is not the case in the autonomous strain. These data provide some insight into the altered state of cytokinin activity in which a cell line changes into an autonomous form. Curiously, in become independent of the requirement for exogenous cytokinin, the autonomous tissue becomes sensitive to added cytokinin. i⁶Ado also inhibits the growth of lines of mammalian cancer cells grown in culture. Presented in the formal symposium on Information Transfer in Eukaryotic Cells, at the 26th Annual Meeting of the Tissue Culture Association, Montreal, Quebec, June 2–5, 1975.     

Glutamine-dependent asparagine synthetase from Lupinus luteus

Asparagine synthetase (glutamine-hydrolyzing [l-aspartate: l-glutamine amido-ligase (AMP-forming), E.C. 6.3.5.4] was purified over 500-fold from cotyledon extracts of 1-week-old yellow lupin seedlings. The enzyme was labile and required protection by high levels of thiols; glycerol and the substrates also stabilized it. The reaction products were shown to be asparagine, AMP, PPi and glutamate. The limiting K_m values were for aspartate 1·3 mM, for MgATP 0·14 mM and for glutamine 0·16 mM. Positive homotropic cooperativity was observed for MgATP only, and gel filtration studies indicated that the substrate-free enzyme (MW 160 000) associated to a dimer (MW 320 000 in the presence of MgCl₂ and ATP. The purified enzyme, which had some glutaminase activity, catalyzed an aspartate- and glutamine-independent ATP-PPi exchange reaction at a rate 5–7-fold higher than the rate of asparagine synthesis. Initial velocity studies and exchange data indicated an overall ping-pong mechanism. Compared to similar enzymes isolated from mammalian tumor cells, the lupin enzyme appears to be unique with respect to MW, reaction mechanism and regulatory properties. The allosteric properties observed suggest an important role for this enzyme in the regulation of asparagine biosynthesis.