Benno Hess

Cooperation of glycolytic enzymes.

Abstract 1. 1.|With an injection technique, glycolytic oscillation can be induced in yeast extract with hexoses, glucose-6-phosphate, fructose-6-phosphate, but not fructose-1,6-diphosphate, with an average rate corresponding to a QNglucose of 70 (per intact cell). 2. 2.|The enzyme and metabolic concentration pattern of glycolysis of a yeast extract is presented. Enzyme molarities per cytosol in the order of 10−6 to 10−4 are found. The enzyme activity pattern for optimal and oscillating conditions reveals large amplitude oscillations (∼ 60-fold) of the activity changes of phosphofructokinase during the oscillation and a mean activity of all glycolytic enzymes in the range of 10–40% of the optimal activity. 3. 3.|Glycolytic self-excitation is induced by substrate addition and generated by the allosteric oscillophor phosphofructokinase. The pulsed generation of products by phosphofructokinase is propagated with a dependent phase shift by means of the coupling variable adenosine phosphate via the kinases, phosphoglycerate kinase and pyruvate kinase. The function of fructose-diphosphate during the oscillations is masked because oscillations only occur above the stresshold level of fructose-diphosphate being critical for the activation of pyruvate kinase. The function of ATP and ADP is amplified by the strong activation of phosphofructokinase by AMP. 4. 4.|Oscillations are a general property of metabolic systems and an implicit function of their feedback structure, which involves cross-coupling and self-coupling with opposite sign in a two variable structure and might produce kinetic instability involving more than one singularity of the trajectories in a phase plane. The study of oscillations reveals the dynamics of a pathway over a large range of states. The physiological significance of controlled oscillations can not yet be evaluated.

The Structure of the Core of the Spiral Wave in the Belousov-Zhabotinskii Reaction

The quantitative structure of the core of the spiral-shaped traveling wave of chemical activity appearing in a thin excitable layer of the Belousov-Zhabotinskii reaction, in which the oxidation and decarboxylation of malonic acid by bromate ions is catalyzed by ferroin, was analyzed experimentally. Light absorption by ferroin as the reduced reaction catalyst and indicator was measured by means of a video-and computer-based two-dimensional spectrophotometer with 10-micrometer spatial, 2-second temporal, and 256-digital units intensity resolution. The spiral core is a singular site (diameter, 30 micrometers or less) at which intensity modulations due to ferroin-ferriin distributions are at least ten times smaller than in the surrounding area of spiral propagation. Archimedian spirals were fitted to isoconcentration lines.

On the control of metabolism in ascites tumor cell suspensions.

In spectroscopic studies of isolated rat liver mitochondria, it has been pussible to identify different oxidation-reduction levels of the respiratory enzymes, especially cytochrome b and dihydrode phosphopyridine riucleotide (DPNH), depending upon the presence or absence of adenosine diphosphate (ADP), and to correlate these spectroscopically defined states with the acceleration or inhibition of respiration caused by the addition or withdrawal of ADP from such mitochondria.* * Based on the idea that the glucose inhibition of respiration in ascites tumor cells3’ 4 , may be related to the intracellular ADP level, we have applied thc above spectroscopic test to such cells in the transition from respiration with endogenous substrate to the glucose-inhibited state. Such spectroscopic methods have already been used with ascites cells to record their cytochrome6 and pyridine nucleotide7 content and to show by measurements of the rapid kinetics of the components of their respiratory chain that capabilities for rapid electron transport exist, but are not utilized.* 111 the current study, spectroscopic measurements of cytoclirome b show definite changes in the intracellular ,4DP level caused by glucose addition. Other aspects of the glucose effect are revealed under our experimental conditions. The effect of glucose on respiration is found to include an initial transitory phase of stimulation in which the ,4DP level is high. This phase is followed by a severely inhibitory phase in which the ADP (or phosphate) level is too low to support appreciable respiration. Measurements of glucose uptake show that i t follows a similar course of brief stimulation and then inhibition. These observationsg allow us io evaluate various theories for the Pasteur reaction and to suggest that i t may he a special case of extensive interactions of glycolytic and respiratory pathways in which exchange of adenosine polyphosphates between these two enzyme systems may he an important control mechanism*.’O The Ehrlich ascites tumor cells were prepared as before’ but, in this case, special precautions were taken to avoid appreciable changes of pH due to fermentative activity of the cells. Thc Ringer solution was buffered with from 10 to 40 mM “tris” instead of phosphate. At the higher concentrations, the pH changed niucli less than 0.1 unit (0.04) during the cycle of respirat.ory changes. In view of such x small change and of the fact that addition of alkali did not reverse the inhibition, we may conclude that the effects recorded were not caused by a pH change. It: was found thal cells suspendcd in higher concentrations of “tris” buffer Cell preparations and reaction media.

Allosteric properties of yeast pyruvate decarboxylase

Pyruvate decarboxylase (EC 4.1.1.1) of yeast was discovered by Neuberg and Karczag [l] and subsequently isolated and partially purified by several groups of investigators [2,3]. The authors reported, that the overall kinetics of the enzyme follow the Michaelis-Menten equation. However, as pointed out earlier [4] in studies on the control mechanism of glycolysis we found that the enzyme has allosteric properties. We here report the results of kinetic measurements carried out with cell-free extract and highly purified yeast enzyme.

Kinetic interaction between aromatic residues and the retinal chromophore of bacteriorhodopsin during the photocycle

In an earlier communication [ I] a reaction of the protein fluorescence following 570 nm light activation of bacteriorhodopsin (BR) was described, indicating the participation of the aromatic amino acid residues in the mechanism of its photoand protoncycle and a conformational change. Recently confirmation and extension of these data were presented suggesting a deprotonation and reprotonation of tyrosine and possibly tryptophane during the photocycle [2,3]. This notion is supported by the elucidation of the amino acid analysis of BR [4], the pH-sensitivity of its aromatic spectrum [S] as well as the results of chemical substitution experiments of tyrosine residues [6]. Here we report on the transient and steady state difference spectra of the aromatic amino acid residues, their reaction velocity constants, their approximate stoichiometry during a photocycle as well as on their interactions and time correlation with photocycle intermediates.

Curvature and Propagation Velocity of Chemical Waves

The collision of circular chemical waves in an excitable medium, the Belousov-Zhabotinskii reaction, leads to characteristic cusplike structures. The high curvatures of these structures are especially suitable for experimentally verifying the predicted proportionality between the velocity and the shape of traveling waves. A computerized spectrophotometric video technique with microscopic resolution was used to determine the proportionality factor (2 x 10-5 square centimeter per second), which in this case is the diffusion coefficient of the autocatalytic species of the reaction system. A numerical calculation of the spatiotemporal evolution of the cusp structure is in good agreement with the experimental observations.

Two-dimensional spectrophotometry of spiral wave propagation in the Belousov—Zhabotinskii reaction. I. experiments and digital data representation

Abstract The spiral-shaped wave of chemical activity propagating in a 1 mm layer of an excitable solution of the Belousov-Zhabotinskii reaction is analysed quantitatively for a 5 min time interval in a 4.5 × 4.5 mm area. The spatial distribution of the reaction catalyst and indicator ferroin is measured by light absorption at 490 nm with a video- and computer-based two-dimensional spectrophotometer. Digital images with 10 μm spatial and 256 digital units intensity resolution are acquired at a frequency of 20 per minute. Initially, the spiral has a pitch of 1.18 mm, a revolution time of 17.2 s, and the wavefronts propagate outwards with a velocity of 69 μm/s. After 5 min these quantities have changed by up to 5%. Three-dimensional graphic procedures and image analysis reveal that the wave profiles are symmetric close to the spiral center and become highly asymmetric during their outward displacement. The core of the spiral has a radius of 0.35 mm. Its center is a singular site (diameter

Chaotic dynamics in yeast glycolysis under periodic substrate input flux

The numerical analysis for a glycolytic model containing the enzymes phosphofructokinase and pyruvate kinase reveals different types of entrainment, as well as chaotic response under sinusoidal substrate input. Entrainment with response periods 1, 2, 3, 5 and 7‐times the input flux period and aperiodic behaviour is verified by measurements of NADH fluorescence in extracts of Saccharomyces cerevisiae in the theoretically predicted range. The stroboscopic transfer function obtained from the aperiodic signal admits period 3, implying chaos according to the Li‐Yorke theorem.

Hydration effects on cis--trans isomerization of bacteriorhodopsin.

Cis-tram isomerization is the primary photochemical event in the photochemistry of natural and artificial visual pigments [ 1,2]. Although the retinal-opsin interaction is the underlying constraint for the photochemistry of vision, not much information is available about the role of the opsin in these processes. A possible approach to such a question would be the study of the effects of protein conformational changes on the primary photochemical event. We have adapted such an approach in the study of the cistrdns isomerization of bacteriorhodopsin. We present here the reversible effects of hydration changes on the cis-trans isomerization of bacteriorhodopsin and demonstrate that the isomerization process is stopped at low hydration states. Furthermore the quantum yield for 13&s + all-trans isomerization, is shown to be wavelength independent. It is suggested that 13cis + all-trans photoisomerization takes place after complete thermal relaxation in the excited state. Bacteriorhodopsin can be found in two interconvertible forms [3]: light adapted and dark adapted. Bacteriorhodopsin when kept in dark at 25°C has its longest wavelength absorption maximum at 560 nm, and is defined as dark-adapted bacteriorhopdopsin (BR

Allosteric influence of anions on mitochondrial ATPase of yeast

&). Irradiation of BRDA causes a red shift 6f the absorption maximum to 570 nm together with an increase of the molar extinction coefficient. This form which is defined as lightadapted bacteriorhodopsin (BRLA), reverts thermally to BRDA. Chemical analyses of the retinal configuration in BRLA have shown [3-61 that it consists of a single isomer, all-trans retinal. Thin layer chromatog-