Fluorescence spectrophotometry of reduced phosphopyridine nucleotide in intact cells in the near-ultraviolet and visible region

Abstract

TRUE SUBSTRATES FOR ALKALINE PHOSPHATASE 19 o.o16 M/~-glycerophosphate where it might be assumed that substrate formation is fast enough so that it is no longer rate-controlling. In addition to the cases above, Ross and his associates 3 have reported variation in pH optima for alkaline phosphatase activity with substrate concentration for phenyl disodium phosphate, adenylic acid, fructose-I,6-diphosphate, fructose-6-phosphate, and ribonucleic acid. The same analysis may be applied to these; and the "true" substrate may be selected as that which demonstrates an optimum concentration invariant with pH change. It was found that the optimum concentration was constant when the un-ionized acid form was picked as the substrate for all cases but ribonucleic acid, where the monovalent ion showed invariance. SUMMARY In the enzymic hydrolysis of/~-glycerophosphate, the species on which the alkaline phosphatase acts is the un-ionized acid. Supporting evidence for this includes: (a) 82p specific activity of the hydrolysis product corresponding to the specific activity of the un-ionized acid in the reaction medium; (b) an increase in reactivity with enzyme dilution which disappears with lower pH; and (c) experimental data from the literature.

Subcellular metabolic transients and mitochondrial redox waves in heart cells

“…Early observations of metabolic oscillations in yeast (3)(4)(5) and our own investigation of oscillations of NADH and sarcolemmal K,ATP current in substrate-deprived heart cells (6) provide evidence that such phenomena can be observed in intact cells. In our previous work, modulation of the amplitude and͞or frequency of the oscillations by external glucose led to the hypothesis that they were driven by a glycolytic oscillator, but the predominantly mitochondrial origin of the NADH f luorescence in cardiomyocytes (7) suggested that metabolic transients in oxidative phosphorylation were also present.…”

State of Oxidation-Reduction and State of Binding in the Cytosolic NADH-System as Disclosed by Equilibration with Extracellular Lactate/Pyruvate in Hemoglobin-Free Perfused Rat Liver

“…Since its introduction by the groups of Duysens [24] and Chance [25,26] fluorescence a t NADH-specific wavelengths has been abundantly used on a qualitative basis on a variety of intact tissues or cell preparations [26a]. The calibration of surface fluorescence derived from the cytosolic NADH system with the help of specific transitions and internal standards is presented here with the intention of providing an example.…”

Two-photon microscopy of cortical NADH fluorescence intensity changes: correcting contamination from the hemodynamic response

“…1 The NADH autofluorescence has been utilized extensively in the past for noninvasive assessment of tissue metabolic state and oxygenation. 2,3 In addition to linear excitation of NADH fluorescence, a relatively small but sufficient two-photon absorption cross section exists in the near-infrared spectrum from 700 to 750 nm, 4 which allows two-photon laser scanning microscopy (TPLSM) imaging of NADH fluorescence. [4][5][6][7][8][9] The advantages of TPLSM imaging for in vivo, depth-resolved high-resolution imaging in highly scattering brain tissue make TPLSM imaging of NADH fluorescence very attractive for investigating brain metabolism.…”