Long-lived tryptophan fluorescence in phosphoglycerate mutase

Phosphoglycerate mutase (PGM; EC 2.7.5.3) isolated from rat and rabbit muscle has been shown to possess an unusually long-lived fluorescence component when excited by ultraviolet light below 310 nm. On the basis of spectral and physical measurements, this 16.4 (+/- 0.2) ns fluorescence lifetime at room temperature is assigned to a tryptophan residue in an unusual environment. The emission profile of this long-lived tryptophan is red shifted from the other tryptophans of PGM by approximately 25 nm. PGM has been crystallized and sequenced from yeast where it has been shown to be a tetramer with 29K subunits. However, we have not been able to detect the existence of an unusually long-lived fluorescence component in the yeast isomer. The long fluorescence lifetime is lost upon denaturation of rabbit PGM and is partially restored upon introduction of the protein to a nondenaturing environment, suggesting the long lifetime is not the result of a covalent modification. The PGM molecule was studied by a number of techniques including time-resolved tryptophan fluorescence, quenching studies of tryptophan fluorescence, and enzyme activity studies. The long-lived fluorescence has been shown to be statistically quenched by Br-, I-, and Cu2+ in the submillimolar region while the acrylamide quenching shows the tryptophan is marginally accessible to solvent. Characterization of the long-lived fluorescence and its possible sources are discussed.     

Inhibition of proteinase K by phosphorylated sugars.

Proteolysis of lactate dehydrogenase, aldolase and the synthetic substrate N-succinylalanylalanylalanyl-p-nitroanilide by proteinase K is inhibited by glucose-6-phosphate and fructose-1,6-biphosphate. Analysis of the kinetic data obtained with the synthetic substrate indicates that the inhibition is a mixed-type and that more than one inhibitor molecule binds to proteinase K. Glucose and fructose are ineffective as inhibitors. In the presence of 0.2-4 mM fructose-1,6-biphosphate, aldolase becomes more susceptible to proteolysis, probably as a result of a conformational change induced by the substrate.     

Denaturation of glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride; identification of inactive, partially unfolded, dimeric intermediates.

The denaturation of the dimeric enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides by guanidine hydrochloride has been studied using enzymatic activity, intrinsic fluorescence, circular dichroism, and light scattering measurements. Equilibrium experiments at 25 degrees C revealed that between 0.9 and 1.2 M denaturant the enzyme underwent a conformational change, exposing tryptophan residues to solvent, with some loss of secondary structure and a complete loss of enzymatic activity but without dimer dissociation to subunits. This inactive, partially unfolded, dimeric intermediate was susceptible to slow aggregation, perhaps due to exposure of 'sticky' hydrophobic stretches of the polypeptide chain. A second equilibrium transition, reflecting extensive unfolding and dimer dissociation, occurred only at denaturant concentrations above 1.4 M. Kinetics experiments demonstrated that in the denaturant concentration range of 1.7-1.9 M the fluorescence change occurred in two distinct steps. The first step involved a large, very rapid drop in fluorescence whose rate was strongly dependent on the denaturant concentration. This was followed by a small, relatively slow rise in the emission intensity, the rate of which was independent of denaturant concentration. Enzymatic activity was lost with a denaturant-concentration-dependent rate, which was approx. 3-times slower than the rate of the first step in fluorescence change. A denaturation mechanism incorporating several unfolding intermediates and which accounts for all the above results is presented and discussed. While the fully unfolded enzyme regained up to 55% of its original activity upon dilution of denaturant to a concentration that would be expected to support native enzyme, denaturation intermediates were able to reactivate only minimally and in fact were found to aggregate and precipitate out of solution.     

Isolation of a kaurene synthetase inhibitor from castor bean seedlings and cell suspension cultures

Biosynthesis of ent-kaurene was investigated in extracts of cell suspension cultures and seedlings of castor bean. Both cell-free extracts contain an inhibitor of kaurene synthetase. The inhibition affects mainly the cyclization of geranylgeranyl pyrophosphate to copalyl pyrophosphate (activity A) and has little or no effect on the further cyclization of copalyl pyrophosphate to ent-kaurene (activity B) in both castor bean and Fusarium moniliforme cell-free enzyme preparations. In castor bean cell suspension cultures, the inhibitor diffuses out of the cells to the growth medium. The inhibitor is stable to 100 C heat treatment for 10 minutes and exposure to pH values of 2.0 or 13.0, and it diffuses through a dialysis bag (10⁴-dalton cutoff). Gel filtration chromatography of the inhibitor on a calibrated Bio-Gel P-10 column indicated a molecular weight of 7,500. Kinetic studies indicate that the inhibition of activity of A of kaurene synthetase is noncompetitive and reversible.     

A model for processing of movement in the visual system

Processing of spatio-temporal information in the human visual system has been investigated thoroughly during the past decade, but is still far from being properly understood. Moreover, the theory of separation of information by means of sustained and transient channels already at the retinal level is not satisfactory, as experimental results indicate that these two types of channels span a continuum of temporal characteristics. It is however obvious, that the process of pattern recognition and velocity perception calls for their separation at some level of the hierarchy. In this communication, we extend our model of three-dimensional spatio-temporal frequency expansion in the visual system (Gafni and Zeevi, 1977) to show how velocity-information extraction channels, sensitive to direction and velocity exclusively, can be formed by simple summation of signals from well-defined sets of channels representing points in the frequency space. Correspondence of these channels to characteristics of the cortical neurons is discussed.     

Cooperativity of lectin binding to lymphocytes,and its relevance to mitogenic stimulation

The relationship between the binding patterns of soybean agglutinin, peanut agglutinin (both in their native (unaggregated) form and in their polymerized form), and of Phaseolus vulgaris leucoagglutinin, to neuraminidase-treated lymphocytes from different sources, and the mitogenic activity of these lectins, was studied. In all cases investigated, binding of a lectin to lymphocytes which resulted in stimulation was a positive cooperative process. Our findings support the assumption that clustering of receptors and conformational changes in membrane structure are prerequisites for mitogenic stimulation.     

Apparent microviscosity of intact and post-lipolysis ("remnant") very low density lipoprotein particles.

The apparent microviscosity of intact rat plasma very low density lipoprotein (VLDL) and post-lipolysis very low density lipoprotein was determined by fluorescence depolarization measurements and flurorescence decay measurements using 1, 6-diphenylhexatriene. Post-lipolysis very low density lipoprotein was prepared in vitro after incubation of the intact lipoprotein with either purified bovine milk lipoprotein lipase or lipoprotein lipase rich (post-heparin) plasma. During lipolysis, an average of 88% of the triglycerides were hydrolyzed, and the lipoprotein became depleted in phospholipids, cholesterol and apolipoprotein C. The apparent microviscosity of the lipoprotein increased by three-fold from 0.63 to 1.88 poise. It is concluded that the compositional changes occurring during lipolysis affect the physical properties of the lipoprotein, as measured here by the fluidity (microviscosity) of the particles.     

A systematic review of the literature examining the diagnostic efficacy of measurement of fractionated plasma free metanephrines in the biochemical diagnosis of pheochromocytoma

BACKGROUND: Fractionated plasma metanephrine measurements are commonly used in biochemical testing in search of pheochromocytoma. METHODS: We aimed to critically appraise the diagnostic efficacy of fractionated plasma free metanephrine measurements in detecting pheochromocytoma. Nine electronic databases, meeting abstracts, and the Science Citation Index were searched and supplemented with previously unpublished data. Methodologic and reporting quality was independently assessed by two endocrinologists using a checklist developed by the Standards for Reporting of Diagnostic Studies Accuracy Group and data were independently abstracted. RESULTS: Limitations in methodologic quality were noted in all studies. In all subjects (including those with genetic predisposition): the sensitivities for detection of pheochromocytoma were 96%-100% (95% CI ranged from 82% to 100%), whereas the specificities were 85%-100% (95% CI ranged from 78% to 100%). Statistical heterogeneity was noted upon pooling positive likelihood ratios when those with predisposition to disease were included (p < 0.001). However, upon pooling the positive or negative likelihood ratios for patients with sporadic pheochromocytoma (n = 191) or those at risk for sporadic pheochromocytoma (n = 718), no statistical heterogeneity was noted (p = 0.4). For sporadic subjects, the pooled positive likelihood ratio was 5.77 (95% CI = 4.90, 6.81) and the pooled negative likelihood ratio was 0.02 (95% CI = 0.01, 0.07). CONCLUSION: Negative plasma fractionated free metanephrine measurements are effective in ruling out pheochromocytoma. However, a positive test result only moderately increases suspicion of disease, particularly when screening for sporadic pheochromocytoma.     

Neuronal assemblies: single cortical neurons are obedient members of a huge orchestra

Spontaneous cortical activity of single neurons is often either dismissed as noise, or is regarded as carrying no functional significance and hence is ignored. Our findings suggest that such concepts should be revised. We explored the coherent population activity of neuronal assemblies in primary sensory area in the absence of a sensory input. Recent advances in real-time optical imaging based on voltage-sensitive dyes (VSDI) have facilitated exploration of population activity and its intimate relationship to the activity of individual cortical neurons. It has been shown by in vivo intracellular recordings that the dye signal measures the sum of the membrane potential changes in all the neuronal elements in the imaged area, emphasizing subthreshold synaptic potentials and dendritic action potentials in neuronal arborizations originating from neurons in all cortical layers whose dendrites reach the superficial cortical layers. Thus, the VSDI has allowed us to image the rather illusive activity in neuronal dendrites that cannot be readily explored by single unit recordings. Surprisingly, we found that the amplitude of this type of ongoing subthreshold activity is of the same order of magnitude as evoked activity. We also found that this ongoing activity exhibited high synchronization over many millimeters of cortex. We then investigated the influence of ongoing activity on the evoked response, and showed that the two interact strongly. Furthermore, we found that cortical states that were previously associated only with evoked activity can actually be observed also in the absence of stimulation, for example, the cortical representation of a given orientation may appear without any visual input. This demonstration suggests that ongoing activity may also play a major role in other cortical function by providing a neuronal substrate for the dependence of sensory information processing on context, behavior, memory and other aspects of cognitive function.