Chapter 3.

Molecular Basis of Chemical Organization of the Cell

3.1 Chemical Elements in Living Organisms Living matter is made up of more than 60 chemical elements, mainly light ones. Heavy elements are not proper for life because they are chemically inert and unsolvable in water. The chemical elements can be grouped into: macroelements, microelements and oligoelements. 1) Macroelements (major chemical elements in amount of 2-60% each) are core of the cell structures. They are: C, H, O, N. The main element of organic compounds, carbon has unique features. It is a tetravalent element whose valence lines are pointed towards the tips of a regular tetrahedron. Carbon can be bound by simple double or triple bonds with other atoms (H, N, O), to make long linear, ramified or cyclic chains. This results in an enormous variety of macromolecules. The ability of carbon to make double or triple bonds with other elements also leads to unsaturated, highly reactive compounds of vital importance in metabolic processes of the organisms. 2) Microelements (less abundant elements, about 0.02-0.1 % each) are: P, S, Cl (metaloid elements), and Na, K, Ca, Mg (metals) respectively. Among these, Na is more abundant outside the cell, while K is more abundant inside the cell. The first 2 groups of elements constitute the so-called plastic elements (of which all biological structures are made up). 3) Oligoelements (trace elements, less than 0.02% each) are very important for life and medicine. There are dozens of such elements. Some are part of protein structures, as is the case of Fe in hemoglobin and myoglobin, with role to bind and transport oxygen. Iodine (I) is part of thyroid hormones. Co, Zn, Lead (Pb) or Cd are enzymatic activators/inhibitors, influence cardiovascular system, formation of gametes, intra-uterine evolution of embryo and foetus, or induce neuropsychical conditions in children. Thus, in areas polluted with Pb or Cd compounds there is a higher incidence of neuropsichically retarded children. Absence of oligoelements from water or soil can induce endemic diseases in those geographical areas (biochemical endemics): lack of iodine in the water and soil results in increased volume of thyroid in adults and a type of cretinism in children; Fe insufficiency produces feriprive anemia; lack (or insufficiency) of Se is associated with a higher risk of cancer (i.e. in Northern Europe); lack of F in water leads to dental caries (compensated by toothpaste usage); Mg deficiency in water is associated with a higher incidence of cardiovascular diseases. 1

3.2 Chemical substances Relative ratio of chemical substances is constant in all living beings. For example, both human body, in all its complexity, and an Escherichia coli (E. coli) cell contain about 70% water, 15% proteins, 7% nucleic acids, 3% glucides and their metabolites, 2% lipids and their metabolites, 1% inorganic ions and several other compounds (under 1%). This is an illustrative aspect of material unity of biosphere. However, at cellular level, differences between cells are found, even in the same body (consider neurons as compared to red blood cells, muscle or epithelial cells).

3.2.1 Inorganic substances 3.2.1.1 Water Water is the essence of life, a primordial and most abundant molecule in all living cells. In a human body of 70 kg, water weights about 40 kg! In organism, water is present in 2 compartments: intracellular water (about 55%) and extracellular water (about 45%), found in plasma, lymph, interstitial liquids (that fill the empty spaces between the cells), digestive secretions, cephalorachidian liquid or liquids in serous cavities (pleura, peritoneum). Within the cell, water is the main constituent of each and every cell. Younger cells (metabolically more active) have a higher content of water (up to 95%) compared with older cells (about 60%). Sometimes, the water molecules represent up to 99% of the total number of molecules in a cell! Water is the only solvent of the living matter, in which all other compounds are diluted or suspended. Chemistry of life is given by chemical reactions occurring in aquatic solutions. Physico-chemical properties of water explain why life cannot exist without water: 1) Arrangement of oxygen and hydrogen atoms produces electric dipole of the water molecule, with the negative (anionic) pole at oxygen and the positive (cationic) one at hydrogen atoms. In fact, the electrical charges are oriented towards the tips of a regular tetrahedron whose centre is occupied by the oxygen atom, while the hydrogen atoms occupy two of the apexes. The polar nature of water confers it a high dielectric constant (80 times higher than of vacuum). This leads to a marked attenuation effect of electrical interactions in water and consequently to defence of living structures against intense electrical fields (so-called electrical 2

shield). The same polar nature makes the water a very good solvent for majority of substances with ionic bonds (which dissociate in water whose molecules provide a hydration cover of ions). Water is also a good solvent for polar substances with covalent bonds. 2) Water molecules disassociate in protons and hydroxyl ions; a proton can attach to another water molecule resulting in a hydronium ion: H2O H+ + OH-; H+ + H2O H3O+ As a result: 2H2O H3O+ + OH-

In this form water participates in chemical reactions within living organisms. 3. Hydrogen bonding: one water molecule can bind 1 to 4 water molecules by hydrogen bonds. Depending on the aggregation state these bonds are stable (in the crystalline structure of ice) or labile (in liquid water dynamic molecular packages are continuously formed and broken). These bonds confer water a high caloric capacity, being an excellent cooler. Water absorbs a lot of energy for the breaking of hydrogen bonds, thus protecting the living structures from caloric energy (heat) released in different biochemical processes (is a thermal shield). On another hand, the hydrogen bonds confer water a high value of vaporization heat, important for cooling of the living organisms by water evaporation (thermoregulatory property). The aquatic phase consists of the so-called free water (95%), representing a solvent or dispersion medium for organic and inorganic substances, and the bound water (5%), which consists of water molecules bound by hydrogen bonds, water bound to other structures (mainly proteins), as well as the water trapped inside elongated fibrous molecules. Besides the aquatic phase, a non-aquatic phase is found in the living cells. Such areas where water is excluded are the inside space of macromolecules (proteins or nucleic acids) or inside cell membranes. Transport of water through membranes in all cells, from bacteria to humans, is accomplished by specialised proteins called water-channel proteins or aquaporins (from latin aqua=water, porus=passage). The first water-channel protein, later called aquaporin 1 (AQ1), was discovered in membranes of the human red blood cells by a team led by Professor Gheorghe Benga in Department of Cellular Biology, Cluj-Napoca University of Medicine and Pharmacy, in 1985. For the discovery of the water-channel proteins the US scientist Peter Agre, who redescovered AQ1 several years later, was awarded the 2003 Nobel Prize for Chemistry. Aquaporins were found in bacteria, in plants (over 200 various types), as well as in animals. In humans 11 types have already been described, among those AQ1 in the red blood cell membrane, in epithelial cells of the proximal contorted tubes (role in the concentration of urine), in endothelium of the capillary vessels and in choroid plexi. AQ2 is found in the renal collecting 3

tubes, having role in the concentration of urine, together with the antidiuretic hormone (ADH). AQ3 is located in the renal collecting tubes, in lungs and brain, and AQP0 in the crystalline (keeps its transparency). Thus, aquaporins are responsible for regulating all processes of quick water transport in organisms. Numerous implications of the aquaporins in pathology (diabetes insipidus, edematous cardiac insufficiency, diseases of the nervous system etc.) were described.

3.2.1.2 Mineral salts

Mineral salts can be found as ions, or combinations bound to proteins, nucleic acids, etc. Among the cations, Na+ (the main extracellular cation), K+ (the main intracellular cation), Ca2+, Mg2+ could be mentioned, while among anions are phosphates (PO43-, HPO42-, H2PO4-), sulphate (SO42-), carbonates (HCO3-, CO32-) and nitrate (NO3-). The presence of ions in organisms is of great importance, as they influence enzymes activity and various other cellular processes such as permeability, excitability, conductibility, contractility, citoplasmic viscosity or cellular divisibility. Also, the ions participate to the osmotic pressure and the acido-basic equilibrum (intra- and extra-cellular pH), being vital for living organisms. Small variations of certain ions concentration in plasma or at cellular level can sometimes result in major functional alterations (such as cardiac arythmias) or even sudden death. The ionic concentration is relatively constant in all the living bodies (about 1% of total weight). This is another proof of the material unity of the biosphere.

3.2.2 Organic substances 3.2.2.1 Glucids

Glucids (sugars) can have either a plastic or an energetic role in the cells. Certain monosaccharides, such as ribose or deoxyribose, have a plastic role as they are present in the structure of nucleic acids. On the other hand, glucose is the main monosaccharide used as cellular fuel; it has mainly an energetic role. The glucose molecule is particularly suited for such a role. Being highly soluble in water, it is easily absorbed from the digestive tube and transported by the blood flow into the whole organism. Also, as the glucose molecule is extremely stable (it is a hexose, more stable than a pentose or a heptose), when the covalent bonds are broken an extremely high energy is released that provides important energetic resources. Finally, glucose is easily metabolised (via glycolysis and other metabolic pathways), thus producing the energy used 4

for the synthesis of ATP (adenosine triphosphate). A representative of polysaccharides is glycogen as the main storage form of glucose in human and animal organisms (in plants, this role is accomplished by starch). Glycogen is made of glucose residues bonded via !1-4 glicosidic bonds that form long chains and via !1-6 glicosidic bonds that form ramifications. Several aspects of the glycogen structure-function relationship are noteworthy: a glycogen molecule stores several thousands of glucose molecules, thus osmotic pressure in the cell being reduced to a minimal level (the osmotic pressure depends on the number of particles, not on their size); enzymes that attach or separate glucose molecules to/from glycogen may work simultaneously on many ramifications, thus the glycogenesis (the formation of glycogen from glucose molecules), and the glycogenolysis (the release of glucose molecules form glycogen) depending on the momentary needs of the organism, are very quick metabolic processes. In the human body, glycogen is present in high amounts in liver and muscles cells. After meals (especially if rich in sugars), as the glucose is absorbed in blood, hyperglycemia occurs; however, the glucose is quickly stored in liver and muscles, glycemia thus falling back to normal ranges. On the other hand, between meals and particularly during periods of intense physical efforts, the organism comes to a state of hypoglycemia; this is rapidly counterbalanced by the release of glucose molecules from the hepatic glycogen for energetic purposes. If the rapidly restored hepatic glycogen is currently used for glucose release, the muscular glycogen (which, once consumed, is restored over a longer period) is used only after the hepatic glycogen is consumed: in inanition, extreme physical efforts (such as marathon running, mountain climbing or professional boxing). Although the nervous system doesnt have a glycogen storage system, it is a big consumer of glucose (the main fuel for neurons), being highly sensitive to hypoglycemia. The liver glycogen can be studied with the light microscope using special colorations (i.e. with carmine Best), red glycogen granules being visible, while at the electronic microscope one can observe it as black granules in the cytoplasm. Mucopolysaccharides are polysaccharides in which the monomer units contain amino derivatives of monosaccharides (glucoseamine, galactoseamine etc.), forming long chains and resulting in very big fibrous molecules. Mucopolysaccharides can attach to lower-size polypeptidic chains, forming the so called proteoglicans. These are important components 5

especially for the extracellular matrix, the fundamental substance of the conjunctive tissue. Examples of mucopolysaccharides are hyalouric acid, codroitin-sulphuric acid, keratan-sulphate, dermatan-sulphate or heparine. Observation of mucopolysaccharides is possible after their staining in specific histochemical reactions. Because they are highly hydrated (gels with high viscosity), the mucopolysaccharides accomplish mainly mechanical functions in the conjunctive tissue (support, shock absorption, lubrication), but they also actively participate in the tissue metabolism.

3.2.2.2 Lipids

Lipids (fats) have various roles in the cell: plastic role: they participate to the structure of cell membranes; energetic role: they constitute the fuel with the highest energetic value (lipids provide more energy than equivalent quantities of glucides or proteins); regulatory role: accomplished by several hormones (e.g. steroid ones) and vitamins of lipidic nature, as well as prostaglandins. a. Simple lipids, used for energy gain via oxidative processes or for the synthesis of other types of lipids, are called free fatty acids. The free fatty acids with chains of 16 and 18 carbon atoms are used by the cells. Those with less than 14 C12 atoms are detergents that destroy the cellular membranes, while those with more than 20 C12 atoms are highly insoluble and thus useless in metabolic reactions. b. Triglycerides (also called neutral fats) are the storage form of lipids in specialised cells with depositing function (i.e. adipose cells or adipocytes). In such cells the nucleus is pushed towards periphery and the cytoplasm is occupied by a large lipidic vacuole. In pathological conditions, triglycerides can also be found in the cytosol of other cells, for example in cases of hepatic steatosis in obese and alcoholic patients, or in patients with hepatic diseases or presenting intoxications due to organic solvents. Lipids present in cytosol are observed using specific staining methods: with Sudan Black they turn to black, while with Sudan III they are stained in orange. c. Complex lipids are present in the cellular membranes as phospholipids or glycolipids.

Some phospholipids (glycerin-phosphatides) are based on the glycerol molecule, where two carbon atoms have esters of 16-18 C12 fatty acids (with up to 6 double covalent bonds in case of the unsaturated ones), while to the third carbon atom a polar group with a phosphoric acid radical and various other substances are attached. Pending on the polar group, they may be phosphatidylcholine (lecitin), phosphatidylethanolamine, phosphatidylserine or

phosphatidylinositol. Another important phospholipid is sphingomyelin, in which sphingosine (an amino-alcohol) is esterified with a fatty acid, while the polar group is phosphorylcholine (the same as in phosphatidylcholine). Glycolipids are also based on sphingosine, on which a fatty acid is bound, but the polar groups consist of one or several glucidic radicals (in cerebrosides or gangliosides respectively). Phospholipids and glycolipids rearrange in water forming the so called micelles" or lipidic bilayers. This is possible due to the hydrophobic property of the fatty acid chains (with the hydrophobic groups orientated towards the inside of the micelle or bilayer) and the hydrophylic property of the polar groups. Such molecules are called amphiphiles. The phospholipids bilayer represents the basis of biological membranes structure.

3.2.2.3 Proteins

Proteins have numerous and various important roles at cellular level: plastic role: they are part of all cellular structures, including membranes; participate in chromatin structure; role in transport and storage of various substances (Fe in the liver, O2 to the cells etc.) role in muscle contractions and all other cell movements; resistance and elasticity of different tissues; regulatory roles (in cell growth, development and multiplication control of cellular division); catalytic role: almost all enzymes (with clearly defined roles in all metabolic processes) are proteins; role in immune defensive reactions of the organisms; receptor role (synaptic receptor etc.) role in nutrition (e.g. ovalbumine) role in maintaining osmotic pressure and pH at constant levels. 7

Accomplishing such various roles is explainable if we consider the structural diversity of proteins on one hand, their specificity on the other. Proteins are macromolecules formed by polycondensation of amino-acids by peptide bonds (-CO-NH-), resulting in chains of a great variety of length, from very short to very long (thousands of amino-acids). Chemically, proteins display a great diversity of structures and functions as well. 20 different amino-acids compose the proteins. Their heterogeneity in size (from small to highly voluminous amino-acids) and polarity (hydrophilic polar, or neutral) is reflected upon the heterogeneity of proteins. Amino-acid availability for a large variety of combinations results in an enormous number of possible sequences. The amino-acid sequence in the proteins (primary structure) largely determines the spatial arrangement of the polypeptide chain (secondary = bidimensional and tertiary= spatial structures) so that the most thermodynamically stable conformation is always adopted. The tri-dimensional (spatial) structure is essential for the biological activity. Various sequences correspond to extremely various spatial structures and, consequently, functions. Each family of proteins has a different spatial structure. Moreover, some proteins have in their structures lipidic radicals (lipoproteins), glucidic radicals (glycoproteins), phosphoric groups (phosphoproteins), sulphur (sulphur proteins), metals (metaloproteins) etc. Protein heterogeneity also resides in the association of several polypeptidic chains in a single functional protein (i.e. the hemoglobin consists of 4 polypeptide chains). Protein specificity implies their propriety of specifically combining with particular substances, even if those substances are present in very small concentrations in the environment. Specific bondage is a consequence of the structure-function relationship underlying several types of molecular interactions in the living world: antigen-antibody reactions, enzyme-substrate or receptor-ligand reactions. Proteins can be studied with the light and electronic microscopes in various ways: a) amino- or sulphidryl- groups in the polypeptide chains can be observed after cytochemical reactions resulting in coloured precipitates (visible in the light microscope) or electron-dense precipitates (visible in electron microscope); b) enzymatic reactions (for enzymes) on tissue sections lead to stained precipitates; c) specific antibodies targeted against the respective protein can be used: antibodies marked with fluorescent groups can be seen with light microscopes (in UV), while the electron microscope allows identification of electron-dense metals such as gold or iron, via antibodies marked with colloidal gold or ferritin (a protein rich in Fe atoms). 8

3.2.2.4 Nucleic acids Nucleic acids are macromolecules formed by nucleotide polycondensation. A nucleotide consists of the following components: a nitrogenous base, purine (adenine, guanine) or pyrimidine (thimine, cytosine, uracil); a pentose, either ribose (in RNA) or deoxyribose (in DNA); a phosphoric ester (radical) In the molecule of any nucleic acid four nitrogenous bases (two purines and two pyrimidines) can be found that constitute four different types of nucleotides: ribonucleotides (in RNA) and deoxyribonucleotides (in DNA). The sequences generated by the nitrogenous bases or the nucleotides in the nucleic acids are very diversified: not as the sequences generated by the 20 amino-acids, but diversified enough to secure biological role of the nucleic acids. The nucleic acids are heredity molecules; they represent genetic material, storage system of genetic information. Heredity is the particular feature of the living organisms to give offspring similar to the parents, in certain (environmental) conditions. It involves transferring hereditary characteristics. The genetic material is contained in genes, formed out of hundreds to thousands of nucleotides that determine all biochemical, morphological, behavioural and other characteristics of an organism. Heredity implies stability. The DNA macromolecule has a double-helix (duplex) universal structure in which two polynucleotide chains are whirled (forming a huge spiral). In the case of RNA, universal types of spatial structures are seen in the tRNA (transfer RNA), mRNA (messenger RNA) or rRNA (ribosomal RNA). Due to their structure, the nucleic acids are fit for their biological role: to confer interspecific and intraspecific (individual) variability, each individual being unique genetically speaking (with the exception of monozygotic twins, where in similar material changes are generated by environmental factors). Location of the nucleic acids within the cell: DNA is located in nucleus, mainly in chromosomes (chromosome DNA constitutes major part of the genetic material in the eukaryote cell), in nucleolus (small amount of nucleoloassociated DNA), and in cytoplasm (in mitochondria, forming mitochondrial genes). In plants, DNA is located in cytoplasm, mitochondria and chloroplasts. RNA is located within the nucleus (in nucleolus) and in cytoplasm: rRNA is located in ribosomes, while tRNA and mRNA are found in the soluble phase of the cytoplasm.

Nucleic acids within the cell can be observed by specific cytochemical reactions with methyl green and pyronine (combined): methyl green gives DNA a green-blue colour, while pyronine turns RNA to pink. Generally speaking, the cells are visualised with a green-blue nucleus (DNA excess), pink nucleolus and cytoplasm (due to the large amount of rRNA).

3.3 Biochemical organisational unity of the (living) cell

There are several unitary principles concerning the chemical and biological organisation of the living cell: a) Cells are composed of identical chemical elements and substances, in similar percentages; b) Ionic composition of the body fluids is constant in various animal species; c) Biochemical processes in biosphere follow a unitary scheme: the main metabolic processes in the cell (such as glycolysis, citric cycle, fatty acids oxydation, biosynthesis of fatty acids and phospholipids) follow the same stages, catalyzed by similar enzymes. d) Some molecules are universally distributed within living cells: D-glucose, fatty acids with 16 to 18 C12, the 20 amino-acids, sterols, purine and pyrimidine basis are found in every cell. Also, co-factors such as NAD, NADP, CoA, coenzyme Q or vitamins are omnipresent. e) ATP is the prototype of the macroergic molecules, the universal currency in energetic exchanges at cellular level. There are three basic systems for ATP generation identical in all cells: glycolysis, oxidative phosphorylation in mitochondria (oxidative process coupled with ATP synthesis) and photosynthesis. Evidently, the latter is specific for plants. f) DNA replication and protein biosynthesis also follow the same mechanisms in all living cells. g) Control mechanisms for all cellular processes are also common for the whole biosphere.

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