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PubMed2005

Inducing coproporphyria in rat hepatocyte cultures using cyclic AMP and cyclic AMP-releasing agents.

Cyclic AMP (c-AMP), added on its own to rat hepatocyte cultures, caused a marked accumulation of coproporphyrin III. The results obtained by comparing the effect of c-AMP to that of exogenous 5-aminolevulinate (ALA), and from adding c-AMP and ALA together, indicated that the coproporphyrinogen III metabolism was blocked, even though no inhibition of the relevant enzyme, coproporphyrinogen oxidase, could be demonstrated. Preferential accumulation of coproporphyrin could also be produced in cultures of rat hepatocytes by agents that raise the cellular levels of cyclic AMP, such as glucagon. The effect of supplementing the culture medium with triiodothyronine (T3) on the response of rat hepatocytes to c-AMP was also investigated. T3, which is known to stimulate mitochondrial respiration, uncoupling O2 consumption from ATP synthesis, produced a c-AMP-like effect when given on its own and potentiated the effect of c-AMP, with an apparent increase in the severity of the metabolic block. It is suggested that an oxidative mechanism may be activated in c-AMP and T3-induced coproporphyria, preferentially involving the mitochondrial compartment, leading to oxidation of porphyrinogen intermediates of haem biosynthesis, especially coproporphyrinogen. Coproporphyin, the fully oxidized aromatic derivative produced, cannot be metabolized and will therefore accumulate.

Aminolevulinic Acid↗
PubMed1980

Improved renal cortical tubule suspension: spectrophotometric study of O2 delivery.

An improved cortical tubule suspension from the rabbit kidney is described that contains almost entirely proximal convoluted tubules with little contamination by cellular debris or glomeruli. These tubules appear to be capable of active transepithelial transport, since their tubular lumina are open and ouabain inhibits 70% of the QO2. In previous preparations with closed tubular lumina, ouabain inhibited QO2 only 40% and the base-line QO2 was one-half to one-quarter that of the present preparation. The delivery of oxygen to this tubule suspension was compared to that of slices by three different means: oxygen consumption kinetics, ATP content, and, more directly by spectrophotometric monitoring of the redox state of cytochrome oxidase. Results demonstrate that the cortical slice is evidently oxygen deficient even when the bath PO2 is in excess of 570 mmHg. In contrast, the tubule suspension is shown to be adequately oxygenated even at a bath PO2 of 10 mmHg. This tubule suspension will be highly applicable for the analysis of aerobic metabolism in a functionally intact renal preparation by optical, electrode, and biochemical assay technologies.

Adenosine Triphosphate↗
PubMed1990

Bulk isolation of renal PCT and PST. I. Glucose-dependent metabolic differences.

A new procedure for separately isolating milligram quantities of rabbit renal proximal straight (PST) or convoluted (PCT) tubules is described, and the differential abilities of these segments to utilize glucose as a metabolic substrate are investigated. Separate dissection of the cortical cortices and the outer medullary stripe, followed by collagenase digestion and discontinuous Percoll centrifugation, provide enriched populations (greater than 98% pure) of PCT (37 mg) and PST (14 mg), respectively, per rabbit. The purity of PCT and PST fractions was quantitated morphologically and by comparing the enriched activity of the proximal tubular marker leucine aminopeptidase and deenriched activity of the distal marker hexokinase to previously published values reported from microdissection studies. To investigate glucose-dependent metabolic differences, PCT and PST suspensions (1 mg/ml) were preincubated in Dulbecco's modified Eagle's-Ham's F-12 medium for 1 h before being incubated for 30 min in buffer with or without glucose as the only available metabolic substrate. In glucose-containing buffer, PST segments maintained their oxygen consumption and ATP contents at levels significantly higher than PCT segments. These differential responses between PST and PCT were glucose-dependent because they were abolished when segments were incubated under glucose-free conditions. Because responses in PCT were glucose-independent, these results suggest that PCT cannot utilize glucose to support oxidative metabolism, whereas PST segments can oxidatively metabolize this substrate. These differences in glucose utilization do not correlate with the distribution of glycolytic enzyme activities, suggesting that differential metabolic regulation of these enzymes may determine the ability of each segment to utilize glucose.

Adenine Nucleotides↗
PubMed1982

Stimulatory action of verapamil on transport of organic acids in rat kidney cortical slices.

Effect of verapamil on the organic acid transport was examined with rat kidney cortical slices. Verapamil increased the initial rate of p-aminohippurate (PAH) uptake, markedly enhanced its maximal accumulation under steady-state conditions and depressed the efflux of PAH. The accumulation of urate was also stimulated by verapamil. D600, a derivative of verapamil, showed the same effect as verapamil with regard to the stimulation of PAH accumulation. Kinetic studies revealed that verapamil resulted in an increase in the Vmax of the transport of PAH. The apparent Km remained essentially constant. The PAH accumulation was enhanced by aerobic preincubation of the slices with verapamil at 37 degrees C. On the other hand, the preincubation of the slices with verapamil at 0 degrees C did not alter the PAH accumulation. Oxygen consumption and ATP content in the slices and microsomal (Na+ + K+)adenosine triphosphatase activity were not affected by verapamil. Verapamil enhanced a Na+ gradient to some degree. however, the PAH accumulation in the presence of verapamil and ouabain was increased approximately the same amount as in the absence of these drugs regardless of the dissipation of the Na+ gradient by ouabain. These results suggest that verapamil accumulated by the slices stimulates the PAH uptake and its stimulatory action cannot be explained by the increase in the Na+ gradient and stimulation of (Na+ + K+)adenosine triphosphatase activity.

Adenosine Triphosphatases↗
PubMed1994

Inhibition of flagellar motility of fowl spermatozoa by L-carnitine: its relationship with respiration and phosphorylation of axonemal proteins.

The action of carnitine in regulating fowl sperm motility was investigated. As the concentration of L-carnitine was increased (0-20 mM), the motility of intact and demembranated fowl spermatozoa was reduced at 30 degrees C. Even the presence of 1 mM CaCl2 before the addition of 10 mM carnitine could not prevent the inhibition of motility at 30 degrees C and 40 degrees C. However, motility was restored by reducing the concentrations of carnitine. Carnitine also inhibited the oxygen consumption and ATP concentrations of intact spermatozoa, and caused a reduction in intracellular free Ca2+ concentrations. Phosphorylation of a 50 kDa protein and dephosphorylation of 24 kDa and 30 kDa proteins of demembranated spermatozoa were observed after the addition of carnitine. In contrast, the flagellar ATPase activity of crude dynein extract was not affected by the addition of carnitine. These results suggest that inhibitory effect of carnitine for motility may be directly on the axonemal phosphoproteins, but not directly on the dynein ATPase activity. The physiological role of carnitine for fowl spermatozoa in the ductus deferens is discussed.

Adenosine Triphosphate↗
PubMed1996

Lack of correlation between para-aminophenol toxicity in vivo and in vitro in female Sprague-Dawley rats.

The present study was designed to test the hypothesis that para-aminophenol (PAP) nephrotoxicity is due to autooxidation. We compared renal functional responses following PAP administration to female Sprague-Dawley rats and following incubation of renal proximal tubules with PAP. The concentrations of PAP selected for in vitro incubations produced cytotoxicity (for example, a decrease in oxygen consumption or adenine nucleotide concentration) in rat renal epithelial cells or rabbit proximal tubule suspensions. In rats, PAP (300 mg/kg i.p.) caused proximal tubular necrosis within 24 hr. Changes in renal function 24 hr following PAP administration included increased kidney weight and blood urea nitrogen concentration and decreased renal glutathione (GSH) content and adenine nucleotide concentrations. PAP did not cause hepatic damage. Within 2-4 hr following PAP administration, renal GSH content and adenine nucleotide concentrations were significantly decreased. In renal cortical slices prepared from PAP-treated rats, oxygen consumption and accumulation of organic ions (para-aminohippurate and tetraethylammonium) were significantly decreased compared with renal cortical slices prepared from control rats. In liver, GSH content was significantly decreased from 1 to 4 hr following PAP administration. In contrast to the effects of PAP in vivo, renal proximal tubules showed little evidence of injury when incubated with 0.1 or 0.5 mM PAP for up to 4 hr in the presence or absence of amino acids in the incubation medium. When tubules were incubated with 1 mM PAP for 4 hr in the presence of amino acids, GSH content, AMP concentration, and TEA uptake were significantly decreased. When amino acids were removed from the incubation medium, 1 mM PAP caused decreases in oxygen consumption and ATP concentration after 4 hr of incubation. Functional changes observed during incubation with PAP in vitro were not consistent with functional changes observed in vivo. The discrepancy between PAP toxicity in vivo and in vitro suggests that autooxidation is unlikely to be responsible for PAP nephrotoxicity and that nephrotoxicity in vivo is primarily mediated by extrarenal bioactivation. Further, depletion of hepatic GSH content prior to changes in renal function suggests that PAP or a PAP metabolite may conjugate with hepatic GSH. These observations suggest that PAP nephrotoxicity may be mediated by PAP-GSH conjugates rather than autooxidation of PAP in the kidney.

Adenosine Triphosphate↗
PubMed2002

Potassium channel openers protect cardiac mitochondria by attenuating oxidant stress at reoxygenation.

K(+) channel openers have been recently recognized for their ability to protect mitochondria from anoxic injury. Yet the mechanism responsible for mitochondrial preservation under oxidative stress is not fully understood. Here, mitochondria were isolated from rat hearts and subjected to 20-min anoxia, followed by reoxygenation. At reoxygenation, increased generation of reactive oxygen species (ROS) was associated with reduced ADP-stimulated oxygen consumption, blunted ATP production, and disrupted mitochondrial structural integrity coupled with cytochrome c release. The prototype K(+) channel opener diazoxide markedly reduced mitochondrial ROS production at reoxygenation with a half-maximal effect of 29 microM. Diazoxide also preserved oxidative phosphorylation and mitochondrial membrane integrity, as indicated by electron microscopy and reduced cytochrome c release. The protective effect of diazoxide was reproduced by the structurally distinct K(+) channel opener nicorandil and antagonized by 5-hydroxydecanoic acid, a short-chain fatty acid derivative and presumed blocker of mitochondrial ATP-sensitive K(+) channels. Opener-mediated mitochondrial protection was simulated by the free radical scavenger system composed of superoxide dismutase and catalase. However, the effect of openers on ROS production was maintained in nominally K(+)-free medium in the presence or absence of the K(+) ionophore valinomycin and was mimicked by malonate, a modulator of the mitochondrial redox state. This suggests the existence of a K(+) conductance-independent pathway for mitochondrial protection targeted by K(+) channel openers. Thus the cardioprotecive mechanism of K(+) channel openers includes direct attenuation of mitochondrial oxidant stress at reoxygenation.

Adenosine Diphosphate↗
PubMed2007

Role of nitric oxide on mitochondrial biogenesis during the ovarian cycle.

The mitochondrial changes in the ovary during the ovarian cycle are adapted to a cyclically increased-decreased energy demand. In the proliferative phase, the increased energy needs are sustained by the recruitment of mitochondria in active state 3, by an increased tissue O2 consumption and ATP production and by an increase in the number of mitochondria. In the phase of decreased energy needs, mitochondria-dependent apoptosis reduces tissue and mitochondrial O2 uptake. The ovary morphological changes during the cycle describe a process in which the follicles undergo a clear cycle with two sequential phases: proliferation and apoptosis. The follicular growth stimulated by FSH characterizes a tissue that shows a quick cell proliferation. During the ovarian cycle, tissue and mitochondrial O2 uptakes, mitochondrial mass, mitochondrial NO production and cytochrome oxidase activity exhibit a biphasic pattern, with marked increases in the ovary proliferative phases. Relatively low levels of NO seem to drive the cell signaling for follicle proliferation, whereas relatively high NO levels trigger mitochondria-dependent follicle apoptosis.

Animals↗
PubMed2004

Hypoxia-induced decrease of UCP3 gene expression in rat heart parallels metabolic gene switching but fails to affect mitochondrial respiratory coupling.

Mitochondrial uncoupling proteins 2 and 3 (UCP2 and UCP3) are postulated to contribute to antioxidant defense, nutrient partitioning, and energy efficiency in the heart. To distinguish isotype function in response to metabolic stress we measured cardiac mitochondrial function and cardiac UCP gene expression following chronic hypobaric hypoxia. Isolated mitochondrial O(2) consumption and ATP synthesis rate were reduced but respiratory coupling was unchanged compared to normoxic groups. Concurrently, left ventricular UCP3 mRNA levels were significantly decreased with hypoxia (p<0.05) while UCP2 levels remained unchanged versus controls. Diminished UCP3 expression was associated with coordinate regulation of counter-regulatory metabolic genes. From these data, we propose a role for UCP3 in the regulation of fatty acid oxidation in the heart as opposed to uncoupling of mitochondria. Moreover, the divergent hypoxia-induced regulation of UCP2 and UCP3 supports distinct mitochondrial regulatory functions of these inner mitochondrial membrane proteins in the heart in response to metabolic stress.

Animals↗
PubMed2006

Early post-mortem AMP-activated protein kinase (AMPK) activation leads to phosphofructokinase-2 and -1 (PFK-2 and PFK-1) phosphorylation and the development of pale, soft, and exudative (PSE) conditions in porcine longissimus muscle.

Pale, soft, and exudative (PSE) meat has been recognized for decades. Fast glycolysis during early post-mortem stage while the muscle temperature is still high is the cause of PSE meat. To elucidate the molecular mechanism underlying this fast glycolysis in muscle to become PSE meat, post-mortem ATP metabolism, fructose-2,6-diphosphate content, and the activities of AMPK, glycogen phosphorylase, and pyruvate kinase were examined in post-mortem muscle. Earlier and faster post-mortem AMPK activation was responsible for the significantly lower pH and higher lactic acid accumulation (p<0.05) seen in PSE muscle, which resulted in the occurrence of PSE meat. In muscle that became PSE meat, AMPK was activated at 0 h post-mortem and reached maximal activation at 0.5 h post-mortem, whereas AMPK reached maximal activation at 1 h post-mortem in the normal pork loin. Higher fructose-2,6-diphosphate content (p<0.05) was detected in PSE muscle compared to normal muscle at early post-mortem stage. However, no difference in the activities of glycogen phosphorylase and pyruvate kinase, rate-controlling enzymes in glycogenolysis and glycolysis, respectively, was detected between PSE and normal pork loins. Because fructose-2,6-diphosphate is a product of phosphofructokinase-2 (PFK-2), these data suggest that AMPK regulates post-mortem glycolysis through its phosphorylation and activation of PFK-2, which then up-regulates the activity of phosphofructokinase-1 (PFK-1), a key rate-controlling enzyme in glycolysis. Early AMPK activation in PSE muscle is associated with early consumption of ATP, because higher AMP and IMP contents and lower ATP content were detected in PSE meat compared to normal meat. Other mechanisms causing early AMPK activation in PSE meat may exist, which warrants further investigation.

AMP-Activated Protein Kinases↗
PubMed2003

Characterization and function of mitochondrial nitric-oxide synthase.

The mitochondrial production of nitric oxide is catalyzed by a nitric-oxide synthase. This enzyme has the same cofactor and substrate requirements as other constitutive nitric-oxide synthases. Its occurrence was demonstrated in various mitochondrial preparations (intact, purified mitochondria, permeabilized mitochondria, mitoplasts, submitochondrial particles) from different organs (liver, heart) and species (rat, pig). Endogenous nitric oxide reversibly inhibits oxygen consumption and ATP synthesis by competitive inhibition of cytochrome oxidase. The increased K(m) of cytochrome oxidase for oxygen and the steady-state reduction of the electron chain carriers provided experimental evidence for the direct interaction of this oxidase with endogenous nitric oxide. The increase in hydrogen peroxide production by nitric oxide-producing mitochondria not accompanied by the full reduction of the respiratory chain components indicated that cytochrome c oxidase utilizes nitric oxide as an alternative substrate. Finally, effectors or modulators of cytochrome oxidase (the irreversible step in oxidative phosphorylation) had been proposed during the last 40 years. Nitric oxide is the first molecule that fulfills this role (it is a competitive inhibitor, produced at a fair rate near the target site) extending the oxygen gradient to tissues.

Adenosine Triphosphate↗
PubMed1987

The use of hyperosmolar, intracellular-like solutions for the isolation of epithelial cells from guinea-pig small intestine.

Isolated small intestine epithelial cells were prepared by using either (a) hyperosmolar, low sodium, high potassium containing (intracellular-like) solutions, or (b) isosmolar, high sodium, low potassium containing (extracellular-like) solutions. Both (a) and (b) cells show high viability as estimated by Trypan blue exclusion, oxygen consumption, cellular ATP content, lactate-dehydrogenase liberation, intracellular ion concentrations and significant Na+-dependent alanine and uridine uptakes. Although (a) and (b) cells show in the cold similar ion concentration, after reincubation at 37 degrees C for 30 min (a) cells show intracellular ion concentrations of 31 mM Na, 129 mM K and 88 mM Cl, whilst (b) cells have 71 mM Na, 93 mM K and 102 mM Cl. Cells prepared with (a) concentrate much more alanine and uridine than cells prepared with (b), probably because the latter have a lower Na+ gradient across the plasma membrane. Cells prepared with intracellular-like solutions would be an ideal system to study Na+-dependent transport mechanisms and the regulatory systems of intracellular ion concentrations.

Adenosine Triphosphate↗
PubMed1997

Induction of mitochondrial dysfunction and apoptosis in HeLa cells by bis-pyridinium oximes, a newly synthesized family of lipophilic biscations.

When tested on HeLa cells, bis-pyridinium oximes (BPO), a family of newly synthesized molecules whose charged pyridinium moieties are linked by a linear polymethylene chain of variable length (N = 3 to 12) have been shown to possess an inhibitory effect on cell growth and finally to provoke cell death. BPO-affected cells displayed reduced mitochondrial oxygen consumption and ATP stores and were blocked in the G1 phase of the cell cycle. Mitochondrial membrane potential, as assayed with the dye 3,3'-diexyloxacarbocyanine iodide [DiOC6(3)], increased in BPO-treated cells with time of exposure. Cell growth inhibition as well mitochondrial dysfunction were observed only with derivatives having a long polymethylene linking chain (N > or = 6). Furthermore, the concentration of the compound eliciting such effects was inversely related to the number of methylene groups in the linking chain. None of the BPO with N = 6 to 12 modified the mitochondrial DNA content, relative to the nuclear DNA content. In BPO (N = 8 and N = 12)-treated cells, chromatin fragmentation and internucleosomal DNA cleavage occurred massively, indicating that the death mode induced by these compounds is apoptosis. The possible pathway of action and the potential pharmacological interest of these compounds are discussed.

Adenosine Triphosphate↗
PubMed2000

Cytotoxicity of nickel-chromium alloys: bulk alloys compared to multiple ion salt solutions.

OBJECTIVE: Nickel-based alloys have been in use since the 1930s; however, there are concerns regarding the biocompatibility of the metallic ions released from these alloys to surrounding tissues. The objective of this study was to better understand nickel-based alloy cytotoxicity as well as determine if multiple ion salt solutions can be used to model the cytotoxic effects of bulk implant alloys. METHODS: This study evaluated cellular morphology, viability, membrane integrity, and alterations in metabolic activity, including DNA synthesis, RNA synthesis, protein synthesis, oxygen consumption, intracellular ATP levels, and glucose-6-phosphate dehydrogenase in response to bulk alloys and multiple ion salt solutions. RESULTS: Over a 24- or 72-h exposure time, the nickel-based alloys released a total ion concentration in the parts per billion range and caused alterations in DNA, RNA, and protein synthesis, intracellular ATP levels, and glucose-6-phosphate dehydrogenase activity. Interestingly, cellular responses to the salt solutions representing the ions released from the alloys were not consistently significantly similar to those elicited from the alloys. SIGNIFICANCE: From these studies, it was shown that a number of cellular functions are altered in response to ions released from these implant alloys. However, cellular functions were not similarly altered in response to salt solutions representing the ions released from the alloys. These results demonstrated salt solutions cannot be easily used to represent alloy cytotoxicity, and ionic release from alloys is a complex process dependent on variables including ion chemistry, ion valence, and dose-time dependence. This study provides a better understanding of the metabolic response of fibroblasts to ions released from dental alloys; and is a good first step towards developing a more reliable cell culture model of cytotoxicity.

Adenosine Triphosphate↗
PubMed2000

Growth characteristics and metabolic flux analysis of Candida milleri.

The growth characteristics of the sourdough yeast Candida milleri was studied in a carbon-limited aerobic chemostat culture on defined medium. The effect of glucose, xylose, and glucose-xylose mixture on metabolite production and on key enzyme activities was evaluated. Xylose as a sole carbon source was not metabolized by C. milleri. Glucose as a sole carbon source produced only biomass and carbon dioxide. When a glucose-xylose mixture (125:125 C-mM) was used as a carbon source, a small amount of xylose was consumed and a low concentration of xylitol was produced (7.20 C-mM). Enzymatic assays indicated that C. milleri does not possess xylitol dehydrogenase activity and its xylose reductase is exclusively NADPH-dependent. In glucose medium both NAD(+)- and NADP(+)-dependent aldehyde dehydrogenase activities were found, whereas in a glucose-xylose medium only NADP(+)-dependent aldehyde dehydrogenase activity was detected. The developed metabolic flux analysis corresponded well with the experimentally measured values of metabolite production, oxygen consumption (OUR), and carbon dioxide production (CER). Turnover number in generation and consumption of ATP, mitochondrial and cytosolic NADH, and cytosolic NADPH could be calculated and redox balance was achieved. Constraints were imposed on the flux estimates such that the directionality of irreversible reactions is not violated, and cofactor dependence of the measured enzyme activities were taken into account in constructing the metabolic flux network.

Candida↗
PubMed1998

Specific interaction of methionine adenosyltransferase with free radicals.

Although free radicals have been traditionally implicated in cell injury, and associated to pathophysiological processes, recent data implicate them in cell signaling events. Free radicals are naturally occurring oxygen-,nitrogen-and sulfur-derived species with an unpaired electron, such as superoxide, hydroxyl radical or nitric oxide. In order to assess the role of free radicals in cell signaling, we have studies the modulator effect of oxygen and nitrogen active species on liver methionine adenosyltransferase (MAT), a key metabolic enzyme. The presence of 10 cysteine residues per subunit, makes liver MAT a sensitive target for oxidation/nitrosylation. Here we show that purified MAT from rat liver is nitrosylated and oxidized in vitro. Incubation with H202 or the NO donor S-nitrosylated GSH (GSNO), diminish MAT activity in a dose-and time-dependent manner. Furthermore, the inactivation derived from both oxidation and nitrosylation, was reverted by GSH. MAT inactivation originates on the specific and covalent modification of the sulphydryl group of cysteine residue 121. We also studied how free radicals modulate MAT activity in vivo. It was previously shown that MAT activity is strongly dependent on cellular GSH levels. Generation of oxygen and nitrogen active species in rats by injection of LPS, induced a decrease of liver MAT activity. This effect might derive from nitrosylation and/or oxidation of the enzyme. Modulation of liver MAT by NO is further supported by the inactivation of this enzyme observed in experimental models in which NO is produced; such as the administration of NO donors to rats and in hepatocytes cultured in hypoxia, a condition that induces the expression of the inducible nitric oxide synthase (iNOS). Oxidation also controls liver MAT activity in a cell environment as shown in CHO cells stably transfected with rat liver MAT cDNA upon addition of H2O2 to the culture medium. This effect depends upon the generation of the hydroxyl radical. On the basis of the metabolic implications of liver MAT, together with the structural features accounting for the sensitivity of this enzyme to active oxygen and nitrogen species, we propose that modulation of MAT by these agents could be a mechanism to regulate the consumption of ATP in the liver, and thus preserve cellular viability under different stress conditions.

Animals↗
PubMed2005

Proteomics of ischemia/reperfusion injury in rabbit myocardium reveals alterations to proteins of essential functional systems.

Brief periods of myocardial ischemia prior to timely reperfusion result in prolonged, yet reversible, contractile dysfunction of the myocardium, or "myocardial stunning". It has been hypothesized that the delayed recovery of contractile function in stunned myocardium reflects damage to one or a few key sarcomeric proteins. However, damage to such proteins does not explain observed physiological alterations to myocardial oxygen consumption and ATP requirements observed following myocardial stunning, and therefore the impact of alterations to additional functional groups is unresolved. We utilized two-dimensional gel electrophoresis and mass spectrometry to identify changes to the protein profiles in whole cell, cytosolic- and myofilament-enriched subcellular fractions from isolated, perfused rabbit hearts following 15 min or 60 min low-flow (1 mL/min) ischemia. Comparative gel analysis revealed 53 protein spot differences (> 1.5-fold difference in visible abundance) in reperfused myocardium. The majority of changes were observed to proteins from four functional groups: (i) the sarcomere and cytoskeleton, notably myosin light chain-2 and troponin C; (ii) redox regulation, in particular several components of the NADH ubiquinone oxidoreductase complex; (iii) energy metabolism, encompassing creatine kinase; and (iv) the stress response. Protein differences appeared to be the result of isoelectric point shifts most probably resulting from chemical modifications, and molecular mass shifts resulting from proteolytic or physical fragmentation. This is consistent with our hypothesis that the time course for the onset of injury associated with myocardial stunning is too brief to be mediated by large changes to gene/protein expression, but rather that more subtle, rapid and potentially transient changes are occurring to the proteome. The physical manifestation of stunned myocardium is therefore the likely result of the summed functional impairment resulting from these multiple changes, rather than a result of damage to a single key protein.

Amino Acid Sequence↗
PubMed2001

Ratcheting in post-translational protein translocation: a mathematical model.

We have developed a non-steady-state mathematical model describing post-translational protein translocation across the endoplasmic reticulum membrane. Movement of the polypeptide chain through the channel in the endoplasmic reticulum membrane is considered to be a stochastic process which is biased at the lumenal side of the channel by the binding of BiP (Kar2p), a member of the Hsp70 family of ATPases (ratcheting model). Assuming that movement of the chain through the channel is caused by passive diffusion (Brownian ratchet), the model describes all available experimental data. The optimum set of model parameters indicates that the ratcheting mechanism functions at near-maximum rate, being relatively insensitive to variations of the association or dissociation rate constants of BiP or its concentration. The estimated rate constant for diffusion of a polypeptide inside the channel indicates that the chain makes contact with the walls of the channel. Since fitting of the model to the data required that the backward rate constant be larger than the forward constant during early diffusion steps, translocation must occur against a force. The latter may arise, for example, from the unfolding of the polypeptide chain in the cytosol. Our results indicate that the ratchet can transport polypeptides against a free energy of about 25 kJ/mol without significant retardation of translocation. The modeling also suggests that the BiP ratchet is optimized, allowing fast translocation to be coupled with minimum consumption of ATP and rapid dissociation of BiP in the lumen of the ER. Finally, we have estimated the maximum hydrophobicity of a polypeptide segment up to which lateral partitioning from the channel into the lipid phase does not result in significant retardation of translocation.

Adenosine Triphosphate↗