Amino acids and fatty acids are oxidized in which of the following organelles?

8.9.2.2 Amino Acid Metabolism

Mitochondria are also important for the synthesis, breakdown, and interconversion of amino acids (Figure 4). Amino acids are also used for the synthesis of proteins that are encoded by the mitochondrial DNA. Glutamate and aspartate are transported by the aspartate/glutamate carriers,41 which are involved in the aspartate-malate shuttle for the oxidation of cytosolic NADH. Glutamate is also transported by the glutamate carriers.42 Inside mitochondria, amine groups of amino acids are removed and fixed to carbamoyl phosphate, which reacts with ornithine to form citrulline. Citrulline is transported out of mitochondria by the citrulline/ornithine carrier43 and enters the urea cycle, which leads to the production of urine. Lysine and tryptophan are broken down to oxoadipate, which is transported to the mitochondrial matrix in exchange for oxoglutarate by the oxodicarboxylate carriers and oxidized.44,45 Alanine, serine, and cysteine are degraded in the cytosol to pyruvate, which enters the mitochondrion via the functionally but not molecularly identified pyruvate transporter,19 whereas phenylalanine and tyrosine are broken down to malate, which is transported by the dicarboxylate carrier.31,32 Alanine and serine are also interconverted in the mitochondrial matrix, which requires folate.46

6.7.3.4 Endogenous Generation of Ceramide

Isolated mitochondria are able to generate ceramide through reverse ceramidase activity, dihydroceramide desaturase activity, and sphingomyelinase activity. The importance of these pathways in vivo is not clear. Ceramidase is found in mitochondria,37 and the addition of sphingosine generates ceramide in a way that is inhibitable by ceramidase inhibitors28 but not by inhibitors of ceramide synthase (also reported in mitochondria38).

In the de novo pathway for the formation of ceramide, dihydroceramide is formed first by the action of ceramide synthase on sphinganine and acyl-CoA. Dihydroceramide is converted to ceramide by a desaturase that uses NADH and oxygen to form the trans double bond at the 4,5 position. Isolated mitochondria are associated with special endoplasmic reticulum vesicles. These are known as MAM. These have a dihydroceramide desaturase capable of converting dihydroceramide to ceramide.39 The ceramide produced can move rapidly to the mitochondria by an unknown mechanism, resulting in transient permeabilization of the outer membrane to cytochrome c (Figure 23). Control experiments showed that this permeabilization was not observed with the short-chain dihydroceramide because this is not a substrate of the desaturase. Also, no permeabilization was observed if NAD was added instead of NADH.39

1. MITOCHONDRIA

Mitochondria are organelles typically ranging in size from 0.5 micrometer to 1 micrometer in length, found in the cytoplasm of eukaryotic cells. Mitochondria contain the inner and outer membranes, separated by a space. Both the inner and outer membranes are constructed with tail-to-tail bilayers of phospholipids into which mainly hydrophobic proteins are embedded. One portion of the lipid molecule is hydrophilic (water-attracting) and the other portion is hydrophobic (lipid-attracting). As a result of this unique character, lipids spontaneously form a bimolecular lipid bilayer in aqueous solution. The self-assembled lipid bilayer is in a dynamic and liquid-crystalline state. The outer membrane contains proteins and lipids. The smooth outer membrane holds numerous transport proteins, which shuttle materials in and out of the mitochondrion The outer membrane is 60–70 Å thick and permeable to small molecules including salts, adenine and nicotinamide nucleotides, sugars and coenzyme. The inner membrane contains all the enzymes and less lipid than the outer membrane. These membranes produce two separate compartments creating the intermembrane space (C-side) and the space enclosed by the inner membrane called matrix (M-side) (Fig. 1). The intermembrane space is usually 60–80 Å in width and contains some enzymes. The matrix however is very viscous and rich in protein, enzymes and fatty acids. A membrane component exhibits allotropy and changes its property when separated. Experimental evidence shows that the mitochondria exhibit anisotropy.

The inner membrane houses the respiratory chain and ATP synthesis, and is permeable to small neutral molecules such as water, oxygen, and carbon dioxide. Its permeability to charged molecules such as proton and ions is limited. The inner membrane has numerous folds called cristae, which have folded structure greatly increasing the surface area where ATP synthesis occurs. Transport proteins, molecules called electron transport chains, and enzymes that synthesize ATP are among the molecules embedded in the cristae (Figs. 1 and 2). The cristae have the major coupling factors F1, (a hydrophilic protein) and Fo (a hydrophobic lipoprotein complex), which together comprise the ATPase complex activated by Mg+2. ATPase catalyses hydrolysis of ATP to adenosine diphosphate (ADP) and phosphate, while ATPsynthase produces ATP using the energy released by the redox reactions of the respiratory chain. Both reactions are inhibited by the antibiotics such as oligomycin.

Mitochondria contain deoxyribonucleic acid (DNA) and ribosomes, protein-producing organelles in the cytoplasm. Within the mitochondria, the DNA directs the ribosomes to produce proteins as enzymes, or biological catalysts, in ATP production. Mitochondria are responsible for converting nutrients into the energy-yielding ATP to power the cell's activities. The number of mitochondria in a cell depends on the cell's function. Cells with particularly heavy energy demands, such as muscle cells, have more mitochondria than other cells.

The main function of the mitochondria is to provide energy for cellular activity by the process of aerobic respiration. In this process, glucose is broken down in the cell's cytoplasm to form pyruvic acid, which is transported into the mitochondrion. In a series of reactions, part of which is called the citric acid cycle or Krebs cycle, the pyruvic acid reacts with water to produce carbon dioxide and hydrogen atoms. These hydrogen atoms are transported with special carrier molecules called coenzymes to the cristae, and some eventually combine with oxygen to form water.

The electrons flow from the coenzymes down to the oxygen atoms, and protons are pumped from the matrix to the intermembrane space. When the protons flow back into the matrix, a phosphate group is added to ADP to form ATP, which is transported to the cytoplasm of the cell and hydrolyzed into ADP for virtually every energy-requiring reaction and process. ADP is returned to the mitochondrion to be reutilized.

Food containing sugars or carbohydrates is converted to basic chemicals that the cell can use. Sugars are broken down by enzymes into the glucose, which is broken down further to make ATP in two pathways. The first pathway is called glycolysis, which occurs in the cytoplasm outside the mitochondria, and requires no oxygen. During glycolysis, glucose is broken down into pyruvate, which is a 3-carbon molecule. After it enters the mitochondria, it is broken down to a 2-carbon molecule by a special enzyme, and carbon dioxide is released. The 2-carbon molecule is called Acetyl CoA and it enters the Kreb's cycle by joining to a 4-carbon molecule called oxaloacetate. Once the two molecules are joined, they produce citric acid. This is the first reaction that makes citric acid, and the citric acid cycle gets its name from that. Only 4-ATP molecules can be produced by one molecule of glucose.

There are two types of carrier molecules for the electrons: one is called the nicotinamide adenine dinucleotide (NAD+) and the other is called the flavin adenine dinucleotide (FAD+). The third molecule, of course, is oxygen. Eventually, the process produces the 4 carbon oxaloacetate again, and is called a cycle, because it ends up always where it started with oxaloacetate available to combine with more acetyl CoA. Oxidation of NADH and the flow of electrons through the electron transport system leads the transfer of protons from the matrix into the intermembrane space. This creates the vital proton electrochemical gradient to power the synthesis of ATP.

Membrane proteins transfer material and information between the cells and their environment and between the compartments housing the organelles. Some of these proteins selectively transport specific molecules and ions, and some others are receptors for chemical signals from outside the cell. They can support the transport of ion and electron, and the energy conversion and conservation. They act as transducers capable of gathering information, processing it, and delivering a response. This indicates the electronic and molecular character of their functions. Their electrical activities are measurable as an electric potential difference across the membrane. Changes in the membrane permeability would yield a change in the potential difference. In the cotransport system the movement of one permeant is dependent on the simultaneous movement of a different permeant either in the same direction called the symport or in the opposite direction called the antiport. The best-known antiport system is the Na+/K+-ATPase pump that is present in the plasma membrane of all animal cells. This pump transports sodium ions out of the cell and potassium ions into the cell through the lipid bilayer against their electrochemical potential gradients, and operates as an antiport This antiport, like every active transport, needs to couple to a dissipative process in the metabolic activity, like ATP hydrolysis. Transmembrane activities are thermodynamically driven by the gradients of chemical and electrochemical potentials. The metabolic processes are generally able to maintain steady nonequilibrium conditions across the cell membranes by generating the flows of ions or electrons. Therefore transport and rate equations occur in the formulation of metabolic activities.

Cytoplasm houses many metabolic cycles and synthetic pathways, as well as the protein synthesis. Beside the matter and information transfer across the cell membrane, there is the essential interaction of living bodies with the external surroundings. A coordinated body action requires integration of respiratory, nerve, sensing, muscle, etc. At the cellular level, communications via the membrane are called the signal transduction, and facilitated with the ligands or messengers, such as proteins, peptide hormones. These ligands facilitate the communication by directly entering the cell, or interacting with a specific receptor situated on/in the lipid bilayer of the membrane.

Electron transfer and associated reactions leading to the ATP synthesis are completely membrane-bound. Photosynthetic energy conservation occurs in the thylakoid membrane of plant chloroplasts; oxidative phosphorylation takes place in the mitochondrial inner membrane. These membranes facilitate the interactions between the redox system and the synthesis of ATP, and are referred to as coupling membranes. The coupling mechanisms of oxidative phosphorylation may change during development. Firstly, the membrane is an efficient and regulated energy-transducing unit as it organizes the redox systems and associated enzymes. Secondly, the membrane is a permeability barrier of the cell, controls the transport of certain solutes and the effects of osmotic imbalance.

5.4.1 Historical Background

Mitochondria are tubular organelles found in every eukaryotic cell and consist of two specialized membranes embedded with many transport proteins. While the outer membrane contains many channels formed by the protein porin to filter out large molecules, the inner membrane contains a group of transport proteins to allow the passage of different ions and metabolites from cytosol to the mitochondrial matrix. The mitochondrion possesses an intricate transport system for facilitating the transport of Ca2+ ions across its inner membrane to regulate cytosolic Ca2+, to serve as a buffer during excess Ca2+ overload, and to modulate mitochondrial matrix Ca2+, thereby controlling the activities of Ca2+-sensitive enzymes (e.g., the dehydrogenases of the TCA cycle), for its bioenergetic function.

Ca2+ uniporter is a membrane protein located in the inner mitochondrial membrane (IMM) and serves as the primary pathway for Ca2+ transport into the energized mitochondria [14,15]. The mechanism of mitochondrial Ca2+ transport has been a subject of investigation from various standpoints for over five decades. Originally, mitochondrial Ca2+ uptake was described as an active transport mechanism [16]. However, with the evolution of chemiosmotic theory and measurement of a large negative IMM potential (ΔΨ) [17] led to our current understanding, which is that Ca2+ is transported into the energized mitochondria via the Ca2+ uniporter down the electrochemical gradient maintained across the IMM without utilizing any metabolic energy or directly coupling to other ion transport. Therefore, any kinetic study on mitochondrial Ca2+ uptake should concern both the effects of concentration gradient of Ca2+andΔΨ across the IMM.

The earliest measurements of mitochondrial Ca2+ uptake in response to variations in extra-matrix [Ca2+] were performed in isolated respiring mitochondria from both rat livers and rat hearts [18–24]. These studies showed higher-order kinetics that are characteristic of cooperative binding, and a saturation mechanism associated with carrier-mediated transport. Most studies reported sigmoidicity in the plots of mitochondrial Ca2+ uptake vs. extra-matrix [Ca2+] with positive cooperativity and considerable variations in the Ca2+ binding affinity (Km) and maximum uptake velocity (Vmax) . The Hill coefficient for the Ca2+ uptake has usually been reported to be around 2, indicating two Ca2+ ions bound in cooperative transport sites or one at a transport site and one at a separate activation site, such that binding of one Ca2+ ion at the activation site increases the affinity for the binding of another Ca2+ ion at the transport site. In addition, a large variation was reported in the apparent Km value (Km ranges from ∼1 to 90 μM; describing the extra-matrix [Ca2+] at which the transporter shows the half-maximal activity) under different experimental conditions with other divalent metal ions (e.g., Mg2+).

The IMM ΔΨ dependency of mitochondrial Ca2+ uptake has also been studied extensively using isolated mitochondrial preparations [15,22]. These studies suggest a non-linear Goldman-Hodgkin-Katz (GHK) type of dependency of Ca2+ uptake on ΔΨ [25]. Until recently, the uniporter-mediated mitochondrial Ca 2+ uptake has been described to be consistent with carrier, gated pore and with channel modes of transport [15]. Nevertheless, the mechanism of trans-acceleration has not been shown in any of the Ca2+ uniporter kinetic studies. In this regard, by comparing the turnover rates of Ca2+ per site, a large turnover rate for the uniporter was suggested, indicating the uniporter might work more like a gated pore [15]. Later, a patch clamp study in mitoplasts isolated from COS-7 cells demonstrated that the uniporter is a highly selective ion channel [26]. More recently, the structural identity of the uniporter was reported, which suggests that it is a 40 kDa protein that forms oligomers in the IMM, and resides within a high molecular weight complex. It is also thought to consist of two predicted trans-membrane helices [27,28]. An EF-hand-containing protein MICU1 has been suggested to interact with the uniporter and may serve as a putative site for binding of extra-matrix Ca2+ for uniporter operation [27,28]. However, identification of the actual structure and composition of the uniporter continues to be an active research topic in the field.

The kinetics of mitochondrial Ca2+ uptake is primarily determined by the catalytic properties of the Ca2+ uniporter, the electrochemical gradient of Ca 2+ across the IMM and other regulatory factors (e.g., Mg2+ inhibition of the uniporter function, effects of cytosolic Pi and pH on the uniporter activity). Over the past five decades, the biophysical and catalytic properties of the Ca2+ uniporter in respiring mitochondria have been extensively studied using many initial velocity measurements [18–23] and mathematical models [25,29–32]. Mathematical modeling along with the experimental data has been shown to provide deeper insights into the kinetics and regulation of many membrane transport systems, including the Ca2+ uniporter. In the study of mitochondrial metabolism, the Ca2+ uniporter has been extensively modeled by many investigators, in attempts to understand its integrated functions in mitochondrial Ca2+ homeostasis and energy metabolism [33–35].

The initial mitochondrial Ca2+ uniporter model was developed by Magnus-Keizer[32], based on a simple binding scheme of Ca2+ for the uniporter (4-state model), which has been used by many researchers for integrated modeling studies of mitochondrial Ca2+ dynamics [33–35]. In this uniporter model, the ΔΨ dependency of Ca2+ transport was described based on a linear GHK constant field approximation for electrodiffusion, with an offset potential ΔΨ∗=91mV to describe the ΔΨ dependent data. However, this model fails to provide a unique, consistent explanation for the experimentally observed kinetics of uniporter-mediated Ca2+ transport, and hence was considered to have limited applications for studying integrated mitochondrial functions [30]. The major drawback of the Magnus-Keizer uniporter model is that it collapses for membrane potentials ΔΨ≤ΔΨ∗=91mV, and is not thermodynamically balanced. This model also fails to explain the extra-matrix [Ca2+] dependent data on Ca2+ uptake [20,21] measured in isolated rat liver and rat heart mitochondria. Furthermore, the Magnus-Keizer integrated model of mitochondrial Ca2+ handling [32] predicts a high steady state mitochondrial [ Ca2+] (∼15 μM) in response to a low cytoplasmic [Ca2+] (∼1 μM) which is unusual for most cardiac cells.

Recently, our group has systematically developed a series of kinetic models [25,30,31] of the mitochondrial Ca2+ uniporter which mechanistically characterizes various driving forces that govern the uniporter function, based on a large body of experimental data [18–23] concerning the kinetics of mitochondrial Ca2+ uptake. Figure 5.3 shows the general schematics and proposed mechanisms (a 5 state carrier model and a reduced 3 state carrier model) for Ca 2+ transport via the uniporter from the cytoplasmic side to the matrix side, driven by the electrochemical gradient of Ca2+ across the IMM, in the absence of any other effector interactions (e.g., Mg2+ and Pi) [30]. This preliminary Ca2+ uniporter model was developed on the basis of Michaelis-Menten kinetics for multi-state catalytic binding and an interconversion mechanism associated with carrier-mediated facilitated transport, combined with Eyring’s free energy barrier theory for absolute reaction rates associated with interconversion or electrodiffusion (Ca2+ translocation). The model provides a biophysical basis for the catalytic cycle associated with Ca2+ transport via the uniporter and also depicts the mechanisms of cooperative binding of Ca2+ to the uniporter which are observed experimentally.

In the next subsections, we focus on details to illustrate how a step by step approach is applied to characterize mechanistically the kinetics of the mitochondrial Ca2+ uniporter and the regulation of its transport function by other cytosolic factors (e.g., Mg2+ and Pi).

8.1. THE STRUCTURE OF MITOCHONDRIA

Similar to chloroplasts, mitochondria are semiautonomous organelles in the cells of animals, plants, and fungi. Their shape is similar to that of chloroplasts. They contain cyclic DNA and ribosomes. Like chloroplasts, mitochondria are believed to be endosymbionts that entered eukaryotic cells in an early stage of evolution and lost their genetic independence during the succeeding development. Some of the enzymes that catalyze reactions in mitochondria are synthesized in the mitochondrial matrix, others are imported from the cytosol of the cell. Mitochondria are sometimes also called cellular power plants, because most of the energy in eukaryotic cells is produced in mitochondria. This energy is produced in the form of ATP that can be directly used as energy source by many biochemical reactions. The number of mitochondria per cell may vary between one and several thousand.

Mitochondria consist of an inner and an outer membrane (see Fig. 14). The inner membrane is highly folded to enlarge the surface and forms the so-called cristae. All membrane proteins that are involved in the oxidative phosphorylation are located at the inner membrane. These proteins pump protons into the intermembrane space driven by an electron transfer. The proton gradient is used by the membrane enzyme ATP synthase to form ATP. The inner membrane consists of approximately 70% protein and 30% lipid. Most of these proteins are directly involved in respiration. The majority of the rest are transport proteins and ion channels. Because of the high degree of folding between the inner membrane, more proteins can be placed into the membrane to allow a higher effectiveness of the reactions. The inner membrane encloses a plasmatic mitochondrial matrix. The matrix accommodates the enzymes of the Krebs Cycle and fatty acid degradation (β-oxidation) and a whole machinery for protein and nucleic acid synthesis. The outer membrane has an enzyme composition that is very different from that of the inner membrane. It binds mainly membrane pore proteins, transports, and enzymes that are involved in amino acid oxidation, phospholipid synthesis, and other reactions.

8.04.3.6.3 Cartilage

Cartilage is a distinctive skeletal tissue that also mineralizes with calcium phosphate. There are multiple locations in the vertebrate skeleton that are predominantly cartilage but only some sites biomineralize (e.g., in utero), where an aggregation of cells, chondroblasts, proliferate into chondrocytes, and create a preformed model of a bone. The cartilage will eventually be replaced as part of the normal gestation and maturity sequence of the organ by membranous bone (Ogden and Grogan, 1987; Ogden et al., 1987). This precursor bone formation system, known as endochondral ossification, is typical of vertebrate long bones where elongation of the organ takes place at the growth plate and the cells as they are produced assemble in columns (Figure 29). The chondroblasts produce a water-rich (up to 80%) gel-like aggregate (anlage) of two organic molecular species: proteoglycans, or protein–polysaccharides, and type II collagen, which becomes mineralized with apatite (Lowenstam and Weiner, 1989, pp. 167–175). Sharks and some fish keep such cartilaginous “hard” tissues to maturity (McLean and Urist, 1968; Moss and Moss-Salentijn, 1983), whereas in humans this “cancellous bone” is replaced by “membranous bone.”

The other sites where cartilage occurs are not meant to mineralize. Articular cartilage is the shiny slippery textured material found at the ends of many bones, a tissue that facilitates the motion between two bones at the joints. The deposition of mineral in articular cartilage and joints is pathological, and briefly discussed in Skinner (2000). Cartilagenous tissues that occur in the ear, epiglottis, and intervertebral disks are normally not mineralized, and the organic components at these sites are distinct chemically and histologically from the mineralized cartilage sites. Miller (1985) compares the collagen compositions and the associated proteoglycan molecules that have molecular weights upwards of 200 kDa. These molecules have a core of hyaluronic acid (a polymer of glucuronic acid and N-acetylglucosamine), ∼1,500 nm in length. This aliphatic protein base has upward of 100 protein-linked monomers with noncovalently linked “side chains.” Each of the monomers, formed intracellularly by chondroblasts, also has a protein backbone ∼300 nm long with tens of negatively charged glycosaminoglycan chains covalently attached through serine and threonine residues. The assembly of such very large molecules, some of which contain sulfated species (e.g., chondroitin sulfate) results in extracellular, negatively charged species that, like bone, provides sites where mineralization commences and continues.

II.A Group I Introns

Group I introns are widely distributed in fungal mitochondria, chloroplasts, rRNA genes of protists, T-even phages, and the genomes of eubacteria. Group I intron self-splicing (in vitro) in the absence of proteins was first observed for the intervening sequence (IVS, intron) of the nuclear 26S rRNA gene in Tetrahymena thermophila.

Group I splicing proceeds by two consecutive trans-esterification reactions. These reactions are initiated by a nucleophilic attack by the 3′-hydroxyl of a guanosine (or a phosphorylated derivative: GMP, GDP, or GTP) at the phosphodiester bond between the 5′-exon and the intron (5′-splice site). The new 3′-hydroxyl group of the 5′-exon then initiates a second nucleophilic attack, this time on the phosphodiester bond between the 3′-exon and the intron (the 3′-splice site). This results in ligation of the exons and excision of the intron.

Despite all the evidence for self-splicing in vitro, it is clear that splicing in vivo requires protein factors. Even the Tetrahymena IVS, which at low levels of Mg2+ splices efficiently in vitro, is splicing at a rate of about 50-fold less than the level estimated for splicing in vivo. Proteins therefore aid in the folding of these complex RNAs to allow the self-splicing reaction to occur.

Self-splicing is, by definition, an intramolecular event, and the intron is therefore not acting as a true enzyme. However, the catalytic activity found within the conserved core, with a small deletion, can be dissociated into distinct active enzyme and substrate molecules. Cleavage at the 5′- and 3′-splice sites of group I introns can also occur slowly in the absence of a guanosine cofactor, due to the sensitivity of these sites to base hydrolysis which generates cleaved products consistent with the splicing reaction (3′-OH and 5′-P) but unusual for the hydrolysis reaction. The rate of this type of hydrolysis at the splice sites is much greater than expected (10-fold higher), implying that the folded RNA structure influences the susceptibility of certain phosphodiester bonds to alkaline hydrolysis.

Shortened versions of the Tetrahymena IVS (L-19 IVS and L-21 Scal IVS) have been shown to be true enzymes in vitro, for example, as a restriction endoribonuclease and as a template-dependent polymerase.

2.5.7.1 Induced Changes in Organelle and Plasma-Membrane Morphology

The study of the molecular basis of crista junction formation in mitochondria is an example of CET being used in conjunction with other, more common microscopic and biochemical methods.161 Changes in the morphology of crista junctions and cristae membranes and in the localization of the mitochondrial ATP-synthase upon genetic ablation of Fcj1, a molecule identified in this study that is present in oligomeric complexes and specifically enriched in crista junctions, contributed to the conclusion that the formation of cristae depends on Fcj1, as well as on some of the subunits of ATP-synthase.

In a comparative approach, structural features such as the morphology of the cytoplasmic membrane, the outer membrane and the associated complexes were investigated in three related species of Borrelia, the bacteria that cause Lyme disease. The partial removal of the outer membrane of Borrelia induced by an antibody directed against an outer surface protein led to the conclusion that the peptidoglycan layer localized between the two membranes is strongly linked to the cytoplasmic membrane.162

Those previous studies depended on a faithful detection of cell membranes and their morphological changes. Other cellular structures that possess strong intrinsic contrast can also be readily identified. For example, one study compared the location and spatial arrangement of magnetosomes in wild-type and mutant cells lacking proteins that were expected to be relevant for magnetosome organization and function.122,123 In both studies, the wild-type organization of magnetosomes along partially resolved filaments that presumably consisted of actin homologs was restored following the reintroduction of the deleted proteins in mutant cells.

III.E General Features of Eukaryotic DNA Replication

Unlike the genomes in bacteria and plasmids (as well as in mitochondria and chloroplasts) which consist of a circular duplex DNA, with a single ori sequence, the genomes of eukaryotes are not only much larger and linear, but also contain multiple ori sequences for DNA replication and thus multiple replicons. Thousands of replicons are simultaneously fired in mammalian genomes, as is needed to complete replication of the genome in a few hours. Mammalian genomes are three orders of magnitudes larger than the E. coli genome for which one round of replication requires about 40 min at 37 °C. Replication of a mammalian genome, initiated at a single ori, would thus take more than 1 week with the same rate of synthesis. In fact, it would be even longer because the rate of DNA chain elongation is slower in eukaryotes than in E. coli, possibly because of the increased complexity of eukaryotic chromatin.

As mentioned earlier, DNA replication in eukaryotes occurs only during the S phase, which can last for several hours but whose duration varies with the organism, the cell type, and also the developmental stage. For example, in a rapidly growing early embryo of the fruitfly D. melanogaster, cellular multiplication with duplication of the complete genome occurs in less than 15 min. The details of temporal regulation of firing of different replicons are not known. However, euchromatin regions are replicated earlier than the heterochromatin regions.

The details of initiation of replication at individual replicons have not been elucidated in eukaryotes. Some ori sequences of the yeast genome, known as autonomous replication sequences (ARS), have been determined. Although such sequences in the mammalian genomes have not been isolated, the ori regions of certain genes which could be selectively amplified have been localized by two-dimensional electrophoretic separation. Nevertheless, a significant amount of information has been gathered regarding regulation of DNA replication at the global level.

II.C ATP Synthesis

ATP synthesis in chloroplasts is called photophosphorylation and is similar to oxidative phosphorylation in mitochondria. The light-driven transport of electrons from water to NADP+ is coupled to the translocation of protons from the stroma across the thylakoid membrane (the green, energy-converting membrane) into the lumen. Electron transport from Q− to P700+ is exergonic. Part of the energy released by electron transport is conserved by the formation of an electrochemical proton gradient. The cytochrome b6f complex of chloroplasts functions not only in electron transport, but also in proton translocation.

The active site of the oxygen-evolving enzyme is arranged so that the protons formed during water oxidation are released into the thylakoid lumen. These protons contribute to the electrochemical proton potential. The thylakoid membrane contains a protein that functions to transport Cl− across the membrane. Proton accumulation in the thylakoid lumen is electrically balanced in large part by Cl− uptake. As a result, thylakoids accumulate HCl and the membrane potential across the membrane is low. The pH inside the lumen during steady-state photosynthesis is about 5.0.

One of the earliest experiments that supported the hypothesis that ATP synthesis and electron transport were linked by the electrochemical proton potential was carried out with isolated thylakoid membranes. Thylakoid membranes were placed in a buffer at pH 4.0 and after a few seconds the pH was rapidly increased to 8.0, which resulted in the formation of a proton activity gradient. This artificially formed gradient was shown to drive the synthesis of ATP from ADP and Pi. The experiments were carried out in the dark so that the possibility that electron transport contributed to the ATP synthesis was excluded. Thus, a proton activity gradient was proven capable of driving ATP synthesis.

The thylakoid membrane enzyme that couples ATP synthesis to the flow of protons down their electrochemical gradient is called the chloroplast ATP synthase (see Fig. 10). This enzyme has remarkable similarities to ATP synthases in mitochondria and certain bacteria. For example, the β subunits of the chloroplast ATP synthase have 76% amino acid sequence identity with the β subunits of the ATP synthase of the bacterium E. coli.

The reaction catalyzed by ATP synthases is

(11)nHa++ADP+Pi+H+→nHb++ATP+ H2O,

where n is the number of protons translocated per ATP synthesized, probably three or four, and a and b refer to the opposite sides of the coupling membrane. Provided the electrochemical proton potential is high, the reaction is poised in the direction of ATP synthesis. In principle, when the proton potential is low, ATP synthases should hydrolyze ATP and cause the pumping of protons across the membrane in the direction opposite that which occurs during ATP synthesis. ATP-dependent proton transport by the ATP synthase is of physiological significance in E. coli under anaerobic conditions in that it generates the electrochemical proton potential across the plasma membrane of the bacterium. This potential is used for the active uptake of some carbohydrates and amino acids.

In contrast, ATP hydrolysis by the chloroplast ATP synthase in the dark has no physiological role and would be wasteful. In fact, the rate of ATP hydrolysis by the ATP synthase in thylakoids in the dark is less than 1% of the rate of ATP synthesis in the light. Remarkably, within 10–20 msec after the initiation of illumination, ATP synthesis reaches its steady-state rate. Thus, the activity of the chloroplast ATP synthase is switched on in the light and off in the dark. In addition to being the driving force for ATP synthesis, the electrochemical proton potential is involved in switching the enzyme on. Structural perturbations of the enzyme induced by the proton potential overcome inhibitory interactions with bound ADP as well as with a polypeptide subunit of the synthase. An additional regulatory mechanism that is unique to the chloroplast ATP synthase is reductive activation. Reduction of a disulfide bond in a subunit of the chloroplast ATP synthase to a dithiol enhances the rate of ATP synthesis, especially at physiological values of the proton potential. The electrons for this reduction are derived from the chloroplast electron transport chain.