Guy C. Brown

Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase

Nitric oxide (NO) reversibly inhibited oxygen consumption of brain synaptosomes. Inhibition was reversible, occurred at the level of cytochrome oxidase, and was apparently competitive with oxygen, with half‐inhibition by 270 nM NO at oxygen concentrations around 145 μM and by 60 nM NO at around 30μM O2. Isolated cytochrome oxidase was inhibited by similar levels of NO. These levels of NO are within the measured physiological and pathological range for a number of tissues and conditions, suggesting that NO inhibition of cytochrome oxidase and the competion with oxygen may occur in vivo.

Increased apoptosis in the cingulate sulcus of newborn piglets following transient hypoxia-ischaemia is related to the degree of high energy phosphate depletion during the insult

An increase in the number of cells undergoing apoptosis was observed in the cingulate sulcus of newborn piglets 48 h after a global hypoxic-ischaemic insult. Apoptotic death was identified morphologically (by light and electron microscopy) and by DNA fragmentation, detected by in situ end labelling. The number of apoptotic cells was directly related to the degree of high-energy phosphate depletion during hypoxia-ischaemia, measured using continuous 31P magnetic resonance spectroscopy. These results may have implications for the understanding and treatment of perinatal hypoxic-ischaemic brain injury.

Total cell protein concentration as an evolutionary constraint on the metabolic control distribution in cells

Cell protein occupies 15-35% of cell volume. This level is argued to be the maximum compatible with cell function. Because of this constraint, selection pressure during evolution is likely to have maximized pathway fluxes for minimum total protein level. Pathways optimized in this way are shown to have the following characteristics: (1) the simple flux control coefficients of all enzymes are equal, (2) the normal flux control coefficients depend on the relative kinetic constants of the enzymes, such that enzymes with low specific activity are present at relatively high levels and have high flux control, (3) the normal flux control coefficients are proportional to enzyme levels. A single rate limiting step located at the first step in a pathway is likely to be inefficient in terms of protein levels, and the major metabolic pathways are therefore expected to have control distributed throughout the pathway. This has important implications for metabolic control.

The leaks and slips of bioenergetic membranes.

Bioenergetic membranes use proton gradients to transduce energy. However, when the proton gradient is large 1) there is a passive leak of protons (and other ions) across these membranes, and 2) some proton pumps may fail to pump protons (a phenomenon known as slip). Leaks and slips may be an unavoidable consequence of membrane structure and cause significant wastage of free energy, but they may also have functional roles beneficial to the organism.— Brown, G. C. The leaks and slips of bioenergetic membranes. FASEB J. 6: 2961‐2965; 1992.

Brain-metabolite transverse relaxation times in magnetic resonance spectroscopy increase as adenosine triphosphate depletes during secondary energy failure following acute hypoxia-ischaemia in the newborn piglet.

The adenosine triphosphate (ATP)-dependent sodium/potassium pump extrudes intracellular sodium in exchange for extracellular potassium. Low ATP causes pump dysfunction increasing both intracellular sodium and water thereby enhancing metabolite mobility. This should be detectable by proton magnetic resonance spectroscopy (MRS) as increased metabolite transverse relaxation times (T2s). During secondary cerebral energy failure in the newborn piglet, proton and phosphorus MRS showed large increases in the T2s of choline, creatine, N-acetylaspartate, and lactate that correlated with ATP depletion. These results provide insight into factors affecting metabolite T2s and show that T2s may be useful for studying cellular oedema.

Cytochrome oxidase content of rat brain during development.

The cytochrome oxidase concentration and content of rat brain during development was measured using a simple new assay for cytochrome a. The cytochrome oxidase concentration increased from 1.2 nmol/g wet wt. of brain at birth to about 5.5 nmol/g in the adult, most of the change occurring between 5 and 25 days after birth.

Rate limitation within a single enzyme is directly related to enzyme intermediate levels.

The extents to which different rate constants limit the steady‐state rate of an isolated enzyme can be quantified as the control coefficients of those constants and elemental steps. We have found that the sum of the control coefficients of rate constants characterising unidirectional rates depleting a particular enzyme intermediate is equal to the concentration of that enzyme intermediate as a fraction of the total enzyme concentration. Together with simple measurements this powerful relation may be used (i) to estimate certain enzyme intermediate levels, in particular the free enzyme concentration, and (ii) to estimate the control coefficients of rate constants and steps.

Rate control within the Na+/glucose cotransporter

The intestinal Na+/glucose cotransporter has recently been cloned and expressed in Xenopus oocytes, and kinetically characterised to produce a 6-state model of the transporter. We have analyzed this model using control analysis to determine the extent to which each rate constant and step in the mechanism limits the steady-state rates under various conditions. We show that control (rate limitation) is distributed among a number of rate constants and changes with the membrane potential and the concentrations of external and internal sodium and sugar. There is no single rate-limiting step but, with saturating concentrations of external sugar and sodium and negligible internal concentrations, the sugar and sodium fluxes are limited by sodium dissociation on the inside, translocation of the unloaded carrier to the outside and translocation of the fully loaded carrier to the inside. With more physiological external and internal concentrations there is a significant leak flux, which causes the cotransporter to act as a sugar uniporter, and entails that control over the sugar flux differs from control over the sodium flux. In these conditions control is widely distributed among many of the rate constants. The analysis shows that the concept of a rate-limiting step is not generally applicable to transporters.

Phenomenological Kinetics and the Top-Down Approach to Metabolic Control Analysis

In investigating the regulation of metabolic pathways one is often faced with a problem of the following type: some agent or change (e.g. hormone, drug, disease) has caused a change in a pathway flux, and you wish to know whether the agent has caused this change by altering the kinetics of one end of the pathway, or the other, or both. One approach to this problem is an analysis related to Cross-over analysis, which I shall call Phenomenological Kinetic Analysis. The pathway is conceptually divided into two parts around some intermediate metabolite (B in the scheme below) which can be measured in the system. The top end of the pathway (1 in the scheme) produces the intermediate (B), and the bottom end of the pathway (2 in the scheme) consumes the intermediate.

Severity of delayed (“secondary”) cerebral energy failure after acute hypoxia-ischemia is related to the time integral of acute ATP depletion

The aim of this study was to reproduce the delayed (“secondary”) cerebral energy failure previously described in birth-asphyxiated newborn infants and to investigate relationships between primary insult severity and the extent of the delayed energy failure. Phosphorus (31P) magnetic resonance spectroscopy (MRS) at 7 T was used to study the brains of 12 newborn piglets during an acute, reversible, cerebral hypoxic-ischemic episode which continued until nucleotide triphosphates (NTP) were depleted. After reperfusion and reoxygenation, spectroscopy was continued for 48 h. High-energy metabolite concentrations returned to near normal levels after the insult, but later they fell as delayed energy failure developed. The time integral of NTP depletion in the primary insult correlated strongly with the minimum [phosphocreatine (PCr)]/[inorganic orthophosphate (Pi)] observed 24–48 h after the insult. (Linear regression analysis gave slope −8.04 h−1; ordinate intercept=1.23;r=0.92;P<0.0001.) This model is currently being used to investigate the therapeutic potential of various cerebroprotective strategies including hypothermia.