Anton Stier

Non-lamellar structure in rabbit liver microsomal membranes: A 31P-NMR study

Inhomogeneity of lipid structures has been demonstrated for rabbit liver microsomal and other biological membranes [ 1,2]. Part of the microsomal lipids, presumably that surrounding integral proteins like cytochrome P-450, exists in a physical state different in its fluidity thermotropism to that of the bulk membrane lipids. Due to the chemical shift anisotropy of the lipid phosphorous, 31P-NMR spectroscopy has been successfully applied to the characterisation of bilayer membranes [3] and polymorphic lipid mixtures [4]. Results obtained by application of this technique to rabbit liver microsomal membranes and a reconstituted cytochrome P-450 system show that part of the lipids exist in an inverted micellar state which may be indispensible for the structural integration of cytochrome P-450 into the bilayer membrane.

The site of cyclic AMP-dependent protein kinase catalyzed phosphorylation of cytochrome P-450 LM2

The phenobarbital‐inducible form of cytochrome P‐450 purified from rabbit liver microsomes is phosphorylated by cAMP‐dependent protein kinase at a single site, the serine residue in position 128 of the amino acid sequence. The serine is located in a characteristic recognition sequence for cAMP‐dependent protein kinase and is part of a primary structure which is conserved during evolution, present also in phenobarbitalinducible rat cytochrome and cytochrome P‐450 CAM from Pseudomonas putida. The contribution of these findings to our understanding of the structure and membrane topology of cytochrome P‐450 LM2 and its turnover regulated by phosphorylation is discussed.

Conversion of hepatic microsomal cytochrome P-450 to P-420 upon phosphorylation by cyclic AMP dependent protein kinase

Cytochrome P-450, purified from liver microsomes of phenobarbital-induced rabbits, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Upon phosphorylation P-450 was found to be converted to its denatured form, P-420, as verified spectroscopically from the CO-bound form of the reduced cytochrome. The conversion was dependent on both kinase and ATP. Thus, cyclic AMP may regulate the biotransformation system through the control of the degradation rate of microsomal P-450 in vivo.

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity.

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.

NMR studies of the lipid metabolism of T47D human breast cancer spheroids.

The in vivo 31P NMR spectrum of T47D human breast cancer cells grown as spheroids shows changes in the phosphomonoester lipid precursors as a function of spheroid size. The ratio of phosphorylethanolamine (PE) to phosphorylcholine (PC) was 1.0±0.3 for 3‐day‐old, 150 μm spheroids. This ratio increased to 2.4±0.4 for spheroids 7 days and older and which were at least 300 μm in diameter. To investigate the phosphatidylethanolamine to phosphatidylcholine (PdyIE/Pdy1C) ratio in the membranes, chloroform/methanol extracts of spheroids were performed. The 31P spectrum of these extracts showed no change with spheroid size, namely the PdyIE/Pdy1C ratio was 0.5±0.06 for spheroids of all ages suggesting that membrane composition is strongly regulated at the precursor level.

Anisotropic propagation of Ca2+ waves in isolated cardiomyocytes

Digital imaging microscopy of fluor-3 fluorescence was used to study the propagation of intracellular Ca2+ waves in isolated adult rat cardiomyocytes from 17 to 37 degrees C. Ca2+ waves spread in both transverse and longitudinal direction of a myocyte. Transverse propagation was pronounced in waves starting from a focus at the edge of a myocyte and in waves following an irregular, curved path (spiral waves). For the former type of waves, propagation velocities were determined. Both transverse and longitudinal wave components propagated at constant velocity ranging from 30 to 125 micron/s. Myocytes were anisotropic with respect to wave propagation: waves propagated faster in the longitudinal than in the transverse direction. The ratio between longitudinal and transverse velocity increased from 1.30 at 17 degrees C to 1.55 at 37 degrees C. Apparent activation energies for transverse and longitudinal wave propagation were estimated to be -20 kJ/mol, suggesting that these processes are limited by diffusion of Ca2+. Direction-dependent propagation velocities are interpreted to result from the highly ordered structure of the myocytes, especially from the anisotropic arrangement of diffusion obstacles such as myofilaments and mitochondria.

Transient increases of intracellular Ca2+ induced by volatile anesthetics in rat hepatocytes

The affects of volatile anesthetics on mobilization of intracellular Ca2+ was monitored in primary cultures of rat hepatocytes using the fluorescent Ca2+ probe Fura-2. The use of Fura-2 was limited by several factors which complicated the quantitative analysis of the results, such as: (i) a high rate of dye leakage; (ii) changes in the redox state of the hepatocytes which interfered with the fluorescence produced by the dye at various excitation wavelengths; (iii) compartmentalization of the dye producing high local intracellular concentrations; and, of particular importance for this study, (iv) enhanced photobleaching of the dye in the presence of halothane. To aid in the interpretation of the Fura-2 data, the Ca2(+)-sensitive photoprotein aequorin was also used to monitor changes in [Ca2+]i. The aequorin and Fura-2 techniques qualitatively yielded the same result, that the volatile anesthetic agents halothane, enflurane, and isoflurane induce an immediate and transient increase of [Ca2+]i. The durations of these transients were approximately between 5 and 10 min and were not related to any evident acute cell toxicity. The [Ca2+]i increases induced by the volatile anesthetic agents were dose-dependent, with halothane the most potent. The exact mechanism governing these increases in [Ca2+]i induced by these anesthetics in rat hepatocytes is unknown, but is likely to involve effects on both the cell surface membrane and endoplasmic reticulum components of the signal transducing system.

Surface enhanced resonance Raman study of phenobarbital-induced rabbit liver cytochrome P-450 LM2

Surface enhanced resonance Raman (SERR) spectroscopy has been used to study the vibrational spectra of the heme of purified rabbit liver cytochrome P‐450 LM2 which was adsorbed on colloidal silver suspensions or on a silver electrode. Based on a comparison with the resonance Raman (RR) spectra of the ‘solute’ species the high sensitivity of the SERR technique is demonstrated. Two different features were chosen in order to determine the structural and functional integrity of the adsorbed P‐450. Both, substrate‐induced spin state changes on the oxidized P‐450 and the effect of the thiolate ligand on the oxidation state marker band ν4 in the reduced P‐450 could be observed in the SERR spectra of the adsorbed as well as in the RR spectra of the dissolved enzyme. These findings indicate that the protein structure near the substrate binding site and the coordination by thiolate are not affected by the interaction with the metal surface. Both structural elements are crucial for the function of P‐450. Thus the elementary processes of the enzymatic action of P‐450 can be investigated by this highly sensitive version of RR spectroscopy.

A perfusion chamber for high-resolution light microscopy of cultured cells*

A perfusion chamber suitable for general microscopic work on upright and inverted microscopes with cultured cells is described. The device features a minimal internal volume and large field of view within small outer dimensions. In particular the chamber body is very flat to accommodate large‐aperture condenser and objective lenses. It is constructed of innocuous, sterilizable materials, incorporates permanent seals for the windows, is robust and simple to use.

Resonance Raman study of the cytochrome P-450 LM2-halothane intermediate complex

Resonance Raman (RR) and absorption spectroscopic studies of purified rabbit liver cytochromes P‐450 show that the form 2 isomer (LM2) but not the form 4 isomer (LM4) forms a long‐lived complex with halothane after dithionite reduction, absorbing light at 470 nm, in which ferric 6‐coordinated heme iron in the low‐spin configuration is liganded to 2‐chloro‐1,1‐difluoroethylene. The RR data exclude the possibility that the CF3CHCl− carbanion is a ligand and are consistent with the involvement of an active‐site pocket in the cytochrome P‐450 polypeptide.