1-Carboxyallenyl phosphate, an allenic analogue of phosphoenolpyruvate

1-Carboxyallenyl phosphate, the allenic homologue of phosphoenolpyruvate, has been synthesized in six steps. The key step in the synthesis is the isomerization of methyl 2-hydroxy-3-butynoate to the corresponding allenol and phosphorylation of this material. The allene is an excellent substrate for pyruvate kinase, undergoing reaction at more than half the rate of phosphoenolpyruvate. The allene is also a substrate for phosphoenolpyruvate carboxylase, being hydrolyzed by the enzyme rather than carboxylated. With both enzymes, the organic product is 2-oxo-3-butenoate, which gradually inactivates the enzymes by reaction with one or more sulfhydryl groups not at the active site.     

Monocyte requirement for mitogen-induced aggregation of human peripheral mononuclear leukocytes in vitro

Formation of distinct multicellular aggregates is one of the phenomena associated with activation of quiescent human mononuclear leukocytes in vitro. Aggregate formation involves active cell motility and enhances cell-cell interactions required for an optimal proliferative response of T-cells stimulated with agents like phytohemagglutinin. We have developed an assay to quantitate the rate at which motile cells form aggregates on a flat surface. This assay follows the time rate of deviation of cells in undisturbed culture away from an initial random distribution using an "aggregation index." We used this assay to establish minimal culturing conditions required to observe an aggregation response for a partially purified mononuclear leukocyte population. We also studied the ability to aggregate of various subpopulations enriched for T- and B-lymphocytes and monocytes and found evidence for a monocyte requirement for lymphocyte aggregation. In a second assay, we followed the rate of entry of esterase positive monocytes into aggregates and compared this to the rate of entry of mononuclear cells in toto. We found that monocytes are preferentially associated with non-esterase positive cells within one hour of PHA stimulation. The results support the conclusion that monocytes play a central role in directing the motility of human T-lymphocytes leading to their aggregation response in tissue culture.     

Characterization of cross-bridge elasticity and kinetics of cross-bridge cycling during force development in single smooth muscle cells

Force development in smooth muscle, as in skeletal muscle, is believed to reflect recruitment of force-generating myosin cross-bridges. However, little is known about the events underlying cross-bridge recruitment as the muscle cell approaches peak isometric force and then enters a period of tension maintenance. In the present studies on single smooth muscle cells isolated from the toad (Bufo marinus) stomach muscularis, active muscle stiffness, calculated from the force response to small sinusoidal length changes (0.5% cell length, 250 Hz), was utilized to estimate the relative number of attached cross-bridges. By comparing stiffness during initial force development to stiffness during force redevelopment immediately after a quick release imposed at peak force, we propose that the instantaneous active stiffness of the cell reflects both a linearly elastic cross-bridge element having 1.5 times the compliance of the cross-bridge in frog skeletal muscle and a series elastic component having an exponential length-force relationship. At the onset of force development, the ratio of stiffness to force was 2.5 times greater than at peak isometric force. These data suggest that, upon activation, cross-bridges attach in at least two states (i.e., low-force-producing and high-force-producing) and redistribute to a steady state distribution at peak isometric force. The possibility that the cross-bridge cycling rate was modulated with time was also investigated by analyzing the time course of tension recovery to small, rapid step length changes (0.5% cell length in 2.5 ms) imposed during initial force development, at peak force, and after 15 s of tension maintenance. The rate of tension recovery slowed continuously throughout force development following activation and slowed further as force was maintained. Our results suggest that the kinetics of force production in smooth muscle may involve a redistribution of cross-bridge populations between two attached states and that the average cycling rate of these cross-bridges becomes slower with time during contraction.     

Regulation of malic-acid metabolism in Crassulacean-acid-metabolism plants in the dark and light: In-vivo evidence from 13C-labeling patterns after 13CO2 fixation

The labeling patterns in malic acid from dark ¹³CO₂ fixation in seven species of succulent plants with Crassulacean acid metabolism were analysed by gas chromatography-mass spectrometry and ¹³C-nuclear magnetic resonance spectrometry. Only singly labeled malic-acid molecules were detected and on the average, after 12–14 h dark ¹³CO₂ fixation the ratio of [4-¹³C] to [1-¹³C] label was 2:1. However the 4-C carboxyl contained from 72 to 50% of the label depending on species and temperature. The ¹³C enrichment of malate and fumarate was similar. These data confirm those of W. Cockburn and A. McAuley (1975, Plant Physiol. 55, 87–89) and indicate fumarase randomization is responsible for movement of label to 1-C malic acid following carboxylation of phosphoenolpyruvate. The extent of randomization may depend on time and on the balance of malic-acid fluxes between mitochondria and vacuoles. The ratio of labeling in 4-C to 1-C of malic acid which accumulated following ¹³CO₂ fixation in the dark did not change during deacidification in the light and no doubly-labeled molecules of malic acid were detected. These results indicate that further fumarase randomization does not occur in the light, and futile cycling of decarboxylation products of [¹³C] malic acid (¹³CO₂ or [1-¹³C]pyruvate) through phosphoenolpyruvate carboxylase does not occur, presumably because malic acid inhibits this enzyme in the light in vivo. Short-term exposure to ¹³CO₂ in the light after deacidification leads to the synthesis of singly and multiply labeled malic acid in these species, as observed by E.W. Ritz et al. (1986, Planta 167, 284–291). In the shortest times, only singly-labeled [4-¹³C]malate was detected but this may be a consequence of the higher intensity and better detection statistics of this ion cluster during mass spectrometry. We conclude that both phosphoenolpyruvate carboxylase (EC 4.1.1.32) and ribulose-1,5-biphosphate carboxylase (EC 4.1.1.39) are active at this time.Abbreviations CAM Crassulacean acid metabolism - GCMS gas chromatography-mass spectrometry - MS mass spectrometry - NMR nuclear magnetic resonance spectrometry - PEP phosphoenolpyruvate - RuBP ribulose 1,5-bisphosphate     

Role of head group structure in the phase behavior of amino phospholipids. 1. Hydrated and dehydrated lamellar phases of saturated phosphatidylethanolamine analogues

Analogues of dimyristoylphosphatidylethanolamine (DMPE) have been prepared with head groups modified by N-alkylation, alkylation of carbon 2 of the ethanolamine group, or interposition of extra methylene segments between the phosphoryl and amino groups. The phases formed by these lipids in aqueous dispersions have been examined by high-sensitivity differential scanning calorimetry and Raman spectroscopy. All of the DMPE analogues examined, excepting N-methyl-DMPE but including N-ethyl-DMPE, form hydrated gel phases that are metastable with respect to a dehydrated "high-melting" solid phase that has been observed previously for DMPE itself. The properties and the conditions of formation of this high-melting phase are qualitatively distinct from those of the "subgel" phase, which is observed for dipalmitoylphosphatidylcholine and for some of the DMPE analogues examined in this study. The high-melting phases of different DMPE analogues all exhibit similarly tight packing of the acyl chains, which however do not pack according to a single type of subcell that can be universally and specifically associated with this phase. Increasing the size of the PE head group invariably decreases the melting temperature of the hydrated gel phase, even when the normal hydrogen-bonding capability of the head group is preserved. By contrast, addition of larger alkyl substituents to either the amino group or carbon 2 of the ethanolamine moiety substantially increases the transition temperature of the high-melting solid phase, indicating that the contributions of the head group to the energies of the hydrated gel and the high-melting phases are fundamentally different. Our results suggest that the head group structural requirements for a neutral phospholipid to form stable hydrated bilayers are rather stringent, a fact that may explain the overwhelming predominance of only a few such head group structures in most natural membranes.     

Carbon isotope effect on dehydration of bicarbonate ion catalyzed by carbonic anhydrase

The carbon-13 kinetic isotope effect on the dehydration of HCO3- by bovine carbonic anhydrase has been measured. To accomplish this, bicarbonate was added to a buffer solution at pH 8 containing carbonic anhydrase under conditions where purging of the product CO2 from the solution is rapid. Measurement of the isotopic composition of the purged CO2 as a function of the concentration of carbonic anhydrase permits calculation of the isotope effect on the enzymic reaction. The isotope effect on the dehydration is k12/k13 = 1.0101 +/- 0.0004. This effect is most consistent with a ping-pong mechanism for carbonic anhydrase action, in which proton transfer to or from the enzyme occurs in a step separate from the dehydration step. Substrate and product dissociation steps are at least 2-3-fold faster than the hydration/dehydration step.     

Mode of neural control mediating rat tail vasodilation during heating

The purpose of this investigation was to delineate the mode of efferent neural control mediating rat tail vasodilation during body heating. Tail blood flow (venous occlusion plethysmography), tail skin temperature over the ventral vascular bundle, and arterial pressure were measured in Sprague-Dawley rats anesthetized with pentobarbital sodium (45 mg/kg). Three protocols were followed: anesthesia of the lumbar sympathetic chain, bilateral lumbar sympathectomy, and sympathetic nerve stimulation during varying degrees of alpha-adrenergic receptor blockade. Mean tail blood flow and tail vascular conductance (TVC) during body heating were 40.3 +/- 8.7 ml X 100 ml-1 X min-1 and 39.2 +/- 9.2 ml X 100 ml-1 X min-1 X 100 mmHg-1, respectively. Interruption of sympathetic nerve activity by sympathetic nerve anesthetization or sympathectomy during heat stress caused a nonsignificant increase in TVC to 112.7 +/- 1.8 and 121.12 +/- 6.3%, respectively, of the values achieved with body heating. Sympathectomy performed in normothermic animals that had recovered from prior heating caused an increase in TVC to 128.4 +/- 14.0% of the levels achieved during the previous heating period. In addition, sympathetic nerve stimulation after complete alpha-adrenergic receptor blockade failed to produce a vasodilation [control TVC = 10.2 +/- 3.9 vs. TVC during nerve stimulation = 10.4 +/- 3.9 (P greater than 0.05)]. It is concluded that the increase in TVC during body heating occurs solely via a reduction in vasoconstrictor nerve activity.