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THE LIFE SCIENCES disulfides might have recombined, only one of which would have assured catalytic activity. Accordingly, the manner in which the sulfhydryl groups "find each other" and are reoxidized is an inherent property of the primary . . , amino acid sequence. Yet another illustration of this principle is the manner in which whole viruses are constituted. For example, a bacteriophage virus consists of a core of nucleic acid surrounded by a coat protein, a base-plate protein, at least one enzyme, and tail fibers. Assembly of the virus normally occurs by an orderly, stepwise process. But mutants are known that are unable to conduct one or another of the syntheses required and, as shown in Figure 13, accumulate incomplete parts of the virus. When such pieces are simply mixed, the complete virus forms without any guiding agent, enzyme, or energy source much as simpler molecules assemble to form orderly crystals. ~ - , ~ , , mu. ~ ~ . . . . . ~ . . ~ . ~ .. The final structure of the virus is the obligatory consequence of the three- dimensional structures of its various parts. By extension of this concept, the variety of structures common to a cell membranes, ribosomes, chloro- plasts, mitochondria, etc. are all considered to come into being by spon- taneous self-assembly in consequence of the existence in the cell of the requisite subunits, the system attaining in every instance the state of lowest free energy. This concept can be extended to the manner in which cells "recognize each other" and form the cell aggregates that are tissues and organs. It is an intriguing stretch of the imagination to project this still further to the fact that biological individuals recognize each other and dwell communally as colonies of bacteria, schools of fish, herds of mice, or clans of human beings. THE :~IF E AND TIMES OF A CELL A living cell is a remarkable and rather unlikely object. Within itself it fabricates, from a few low-molecular-weight materials received from the environment, a great variety of additional molecular species, some of which are then utilized for the further synthesis of various high-molecular-weight polymeric materials, including nucleic acids, proteins, and polysaccharides. And these continue to exist within the cell despite the presence therein of enzymes capable of degrading them back to their original monomeric forms. Numerous low-molecular-weight compounds and mineral ions are maintained in concentrations decidedly greater than those that prevail in the cell's environment. In general, the medium surrounding the cell is rich in sodium ions and low in potassium ions, whereas the interior of the cell is rich in potassium and low in sodium, despite the fact that the outer mem

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FRONTIERS OF BIOLOGY 53 Head ; 20, 21, 22 ,,23,24,31,40,66 3 Tail 5,6,7,8, 10,25,26 ",`27,28,29,51,53 <11,12 FIGURE 13 Sequential construction of the T-4 bacteriophage. At bottom of figure is a complete virus particle. The viral genome itself conveys instructions for manufacture of its parts. Specific individual mutants of the virus denoted by the numbers in the figures that represent the position of the defective gene in the chromosomal map of the virus, are unable to perform the specific operation shown on the arrow. (From W. B. Wood, R. S. Edgar, I. King, I. Lielausis, and M. Henninger, "Path- way of T-4 Morphogenesis," Federation Proc., 27: 1 163, 1968. Copyright (I) 1968 Federation of American Societies for Experimental Biology.) W? 54, 48, 19 ~8 ' 16, 17, 49 t: ; ,2,4 t50, 64, 65 :15 113, 14 ~ (Spontaneous) 1: /~ brane of the cell permits passage of both substances. This remarkable dynamic steady state, so remote from equilibrium with the environment, is maintained by the cell through the continual expenditure of energy. Still more remarkably, such a structure is capable of accepting additional materials from the environment and proceeding through a complex set of operations that results in the formation of two daughter cells identical in \\~ ~/35 Tail fiber ' 37 j38 ~/36

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54 THE LIFE SCIENCES structure and potential to the original parent cell. Conceivably, the earliest "cell" was rather like some currently existing bacteria, although it may be assumed that it did not possess an outer coating or cell wall. Presumably, no such coating was required because the osmotic pressure of the environ- ment was much the same as that of the cell interior. With the passage of eons, such single-celled objects found themselves in environments inimical to their existence, where extracellular concentrations differed from those of the original primordial soup. Under these circumstances, mutations that resulted in greater capability for meeting the environmental challenge im- parted survival value. Among these was acquisition of the ability to sur- round the cell with a tough outer wall, which is evident in all current plant cells and in bacteria. The latter show a great diversity of such structures, all of which share one aspect: The entire wall surrounding a single cell is a single "bag-shaped" molecule made of several different types of repeating units. In higher plants, the wall is fashioned of cellulose that imparts not only stability against changes in the salt concentration of the environment, but also vertical rigidity to the growing plant. The Energy for Cell Work From all available evidence, at the time these events occurred the atmos- phere of the planet was rather different from what it is today and contained such gases as methane, hydrogen, and carbon monoxide, as well as some carbon dioxide, but little if any oxygen. One can only assume that the earliest cells "learned" to utilize as their energy source the energy available in anhydrides of phosphoric acid, particularly that of adenosine triphosphate (ATP) (Figure 14), since it is the energy available from hydrolysis of the bonds between the phosphoric acid components of this compound that, in all currently living cells, is utilized to drive all synthetic chemical processes and all other events requiring a source of energy, e.g., secretion, contraction, conduction of electrical impulses, or emission of light (Figure 141. As the supply of this compound, available from the primordial soup, disappeared, an advantage accrued to those cells that had "learned" to synthesize ATP for themselves by utilizing the energy potentially available in the chemical structure of glucose. Such processes have persisted in all living cells to the present time. When performed by bacteria, they are called fermentations; in animal cells the related process is called glycolysis. In human muscle, the process can be summarized by the following equation, which requires the operation of 12 distinct enzymes: 1 glucose + 2ADP + 2Pi 2 lactic acid + 2ATP.

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FRONTIERS OF BIOLOGY 55 NH2 No ARC-NO ON/ \N 1 O O O ||A |IB ll -O PRO PRO P-O CH2 am)\ -O -O O ~ H H ~ 1 OH OH H ~ By H /CH FIGURE 14 Structure of adenosine triphosphate (ATP). The wavy lines represent bonds whose hydrolytic rupture is accompanied by a relatively large change in free energy. Hydrolysis of bond A yields adenosine diphosphate (ADP); hydrolysis of bond B yields adenosine monophosphate (AMP). Pi in these equations denotes inorganic phosphate. The process may be more familiar in the manufacture of beer, in which yeast conducts an essen tially similar process: glucose + 2ADP + 2Pi ~ 2 ethanol + 2CO, + 2ATP. PHOTOSYNTHESIS As long as the supply of glucose in the medium sufficed, this would have permitted generation of ATP to meet the requirement of the cell for an energy source. But the time came when the sugar in the cellular environ- ment was consumed; at that time an advantage accrued to any cell that "learned" to utilize some other means of trapping energy e.g., solar energy- in such a way as to generate a supply of ATP. This may be pre- sumed to have happened in the early progenitors of current forms of algae or purple bacteria. The particular novelty required was a pigment that could absorb the energy of light and in some manner take advantage of that circumstance. That pigment was, and still is, chlorophyll. In some- what loosely organized minute membranous bodies, called "chromato- phores," the chlorophyll can accept the energy of sunlight. (It looks green because it is absorbing the red light of sunlight and reflecting green light back to the observer.) From the light-activated chlorophyll molecule is

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THE LIFE SCIENCES ejected an electron that is transferred to some acceptor and then to an iron-protein called ferredoxin, the chlorophyll being left as a free radical. In the most primitive instance, this electron is transferred in turn from one carrier to another and ultimately returns to the original chlorophyll; in the course of this electron passage along the consecutive carriers, one or two molecules of ATP can be synthesized. In a cell to which there was still available a reasonable abundance of diverse organic chemicals, this process would suffice as a source of energy because the cell still could utilize substances taken from its environment for building materials. But, in time, the organic chemical supply of that environment must have dwindled; then those cells gained an advantage that could not only make ATP by the photosynthetic process but also manage to utilize photosynthetic energy for the reductive formation of carbohydrate from CO. in the environment. Even now, such processes are evident in photosynthetic bacteria and algae. The actual reactions by which CO. is fixed into carbohydrate are not, strictly speaking, photosynthetic; they readily occur in the dark in a sequence of reactions made possible by an assortment of 12 different enzymes. The overall process may be summarized as: SCOW + 18ATP + 12TPNH + 12H+ glucose + 1 8ADP + 1 8P + 1 2TPN+. ~ l ~ TPN is a small molecule (triphosphopyridine nucleotide) that can accept a pair of electrons and is thereby reduced to TPNH. But Equation 1 thus defines the role of the true photosynthetic process, which is to provide the requisite ATP and TPNH. The overall photosynthetic process may be summarized as: h, MH, + TPN+ + xADP + xPi ~ M + TPNH + H+ + xATP. (2) Some details of this mechanism are shown in Figure 15. The reaction expressed in Equation 2 would be thermodynamically impossible were it not for the energy of sunlight coupled into the system through the action of chlorophyll. As shown in Figure 15, the ultimate source of reducing power, the source of the electrons to reduce the TPN to TPNH, is an organic compound, like succinic acid. Electrons flow over a series of carriers to the chloro- phyll free radical that is formed when photoactivated chlorophyll releases its electrons to the iron-protein, ferredoxin, and thence to TPN. But again we may imagine that with the passage of time the supply of compounds like succinic acid was seriously depleted. Ultimate advantage then accrued

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FRONTIERS OF BIOLOGY 57 LIG HT E'o volts +0.008 -0 32 -043 +034 Cytochrome c / Cytochrome b:A ~ P Quinones ADP Flavoprotein Succinic Acid FIGURE 15 Path of electron flow in bacterial photosynthesis. CH LOROPHYLL r ~ C! 3LOROPHYLL+ _ . Flavoprotei :\ 1 1 TPN Ferredoxi n ~ Ferredoxin Reducing Substance to those plant cells that acquired the capability to replace the succinic acid of Equation 2 (MH,) with water (H,O). This remarkable capability ap- peared in the higher plants. h, 2H.,0 + 2TPN~ + xADP + xPi -a O. + 2TPNH + 2H-- + xATP. (3) This process is even less likely, thermodynamically, than that summarized in Equation 2. The overall reaction, glucose + oxygen to yield water and CO,, is by far the more likely event, familiar as the combustion of wood. The reverse process, ~ 3 + 1 ), is made possible by the extraordinary organi- zation of molecules in the chloroplast, summarized in Figure 16. Two distinct chlorophyll centers collaborate in the process. The electrons ejected from the first chlorophyll (on the left side of the figure, as drawn) are replaced from water molecules with the evolution of oxygen gas. Elec- trons ejected from that activated chlorophyll traverse a set of carriers until they reach a second chlorophyll center (P,OO). This must be irradiated in

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58 THE LIFE SCIENCES Eo +1.0 +0.8 +0.6 +0.4 +0.2 -0.0 -0.2 -0.4 -0.6 --- TEN --- Ferredoxin System 11 Chl + ---OH /O2L~ l O2 1 --P700 --- Plastocyanin ~~ Cytochrome f Light Chl Acceptor System I _-~ Light - 1- Wo Plastocyanin Cytochrome f ---Cytochromeb6 | | Cytochromeb6 | _ Rubimedin --- Quinones Plastoquinone A , Plastoquinone C I TEN 1: / HI Flavoprotein I \ | Ferredoxin | FIGURE 16 Path of electron flow in photosynthesis by higher plants. (From priM ciples of Biocllemis~ry, 4th ea., A. White, P. Handler, and E. L. Smith. Copyright (if) 1968 McGraw-Hill, Inc. Used with permission of McGraw-Hill Book Company.)

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FRONTIERS OF BIOLOGY turn; at the second center an electron is ejected and accepted by ferredoxin as in simpler systems, and the electron ejected from this second chlorophyll is replaced by the one that was ejected from the first chlorophyll. About 15 different kinds of molecules are required in this overall process, all of which must be located quite specifically with respect to each other in the working machine. Given a supply of TPNH and ATP made by this marvel- ous machinery, these can be utilized elsewhere in the cell to "fix" carbon dioxide into glucose. This is the process upon which all life on this planet ultimately depends. This is the primary source of organic materials gen- erated by the chemistry of higher plants and made available to animals by ingestion, as well as the source of the oxygen that animals require in order to metabolize the organic compounds they have eaten. It was this process in times past that generated the organic compounds left to us as the carbon and hydrocarbons of our fossil fuels. These concepts of the evolution of the photosynthetic machinery, based entirely upon living forms now available, were entirely unknown a decade ago. Not only the overall outlines, but all the intimate details of these processes, have been revealed in the years since World War II, utilizing a great variety of techniques and instruments developed during this period. The appearance of animal cells necessarily had to wait until plant-photo- synthetic activities were well advanced and had enriched the atmosphere with oxygen while generating a supply of organic compounds. Animal cells developed their own miniature power plants, termed "mitochondria," in which electrons taken from ingested foodstuffs similarly pass, hand to hand as it were, over a series of intermediary carriers and are delivered to oxygen with the formation of water. As in chloroplasts, this passage of electrons is coupled to the operation of a molecular machinery that can generate ATP, so that, in animals, the overall process is described by the following reaction: glucose + 60 + 38ADP + 38P Metabolism METABOLIC PATHWAYS -a 6CO + 3 8ATP + 6H,O. In animal cells, as in plant cells, it is the energy intrinsically available in the structure of ATP that is utilized to drive all other processes associated with the life of the cell. In both series of cells, function is intimately asso- ciated with the architecture of the minute bodies-chloroplasts or mito

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60 THE LIFE SCIENCES chondria in which these events occur. The "life" of the cell then consists of the utilization of ATP and other raw materials to synthesize the variety of compounds required for cell function and for cell growth and division, as well as to maintain the constancy of the interior of the cell in the face of the challenges of its environment. Each cell, therefore, is a miniature chemical factory: Plant, algal, and bacterial cells have wider capacities for synthetic activity than do animal cells, which rely on the previous chemical activity of the plant material that they ingest as the source of many of their raw materials. The incredible complexity and diversity of this chemical activity is shown in Figure 3, pages 38 and 39, in outline form only. Starting with a trivial number of precursors in the environment inorganic elements and carbon dioxide~ells synthesize the huge number of compounds shown in this "metabolic map." As the 1940's opened, only a handful of those compounds had been identified. By the mid-1960's, the major features of the metabolic map were well-ni~ complete and, where gaps existed, intel- ligent guesses could be made with respect to the missing compounds. The sequential events from a common starting material such as glucose to an end product e.g., an amino acid required for protein synthesis is termed a metabolic pathway. A few generalities in this regard are noteworthy. METABOLIC CONTROLS A pathway commences with a reasonably readily available material, e.g., glucose, made by the photosynthetic apparatus m glucose >a >basic defend product n Each step thereafter is made possible by the catalytic activity of a specific protein called an enzyme, which serves no other function in the life of the cell. Early in the pathway, there may be branches in which intermediates can be utilized for entry into other pathways, but one step, in our example the reaction basic, is called the "committed step" in the sense that, thereafter, the intermediates serve no purpose in the life of the cell but as stages in the ultimate formation of the desired end product. Most importantly, the rate at which the overall process proceeds, in most cells, is dictated in turn by the requirements of the cell for the end product. This is achieved in a variety of ways. In mitochondrial oxidation, the for- mation of ATP is tightly coupled, as if by a clutch, to the passage of elec- trons. If no ADP is available for synthesis into ATP, electron transfer

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FRONTIERS OF BIOLOGY comes to a halt, viz., the cell oxidizes glucose only when the energy so released can be utilized for the formation of ATP, a process strikingly evi- dent in muscle cells, in which the requirement for ATP can rise spectacu- larly when the muscle begins to contract. In a synthetic pathway, the end product itself, when present in sufficient amount, can "turn off" the pathway, so Mat no more is manufactured until it is needed. This is brought about in two quite different ways, both of which are examples of "negative feedback." The most thoroughly studied example of negative feedback is the formation of cytidylic acid (CTP) (related to the "C" of the genetic code) in a reaction sequence that begins with aspartic acid, ax amino acid. This is schematically represented in Figure 17. The committed step consists of the carbamylation of aspartic acid to form N-carbamyl aspartic acid, catalyzed by aspartyl transcarbamy- lase. The rate of this enzymic reaction is markedly reduced by the presence of CTP. This enzyme can be dissected into two types of subunits. The larger variety, even in the absence of the smaller, can catalyze the reaction in question but is not affected by CTP. The smaller subunits tightly bind CTP but have no catalytic properties. When both types of units are recom- bined, catalytic property is retained, but CTP binding to the smaller units so alters the three-dimensional structure of the enzyme complex that its A ~Y'C0 .30 B FIGURE 17 Schematic representation of allosteric inhibition. A shows the enzyme, constructed of two enzymic subunits with active sites at the angular clefts, which exactly fit the substrate (triangles). The smaller units have no substrate binding sites but can bind the final product (circles) of the reaction sequence in which the enzyme participates. When the latter binding occurs (B), the structure of the reacting sub- unit is deformed, in turn deforming the enzymic units so that they no longer snugly fit the substrate. Hence, enzymic function is inhibited by the end product of its catalysis. (From Biology and the Future of Man, P. Handler, ed. Copyright (I) 1970 by Oxford University Press, Inc.)

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62 THE LIFE SCIENCES catalytic properties are markedly inhibited, a process termed "allosteric inhibition." Numerous examples of such allosteric inhibition have been observed in the last decade; all conform to this general pattern. An additional foe of feedback control is evident in bacteria. It has long been known that bacteria such as Escherichia cold are capable of syn- thesizing for themselves all 20 of the amino acids that they require. How- ever, when placed in a medium rich in those amino acids, they rapidly cease to manufacture any and grow exclusively by use of the amino acids from the medium. In part, this is accounted for by the kind of feedback control described above, viz., the amino acids inhibit the enzymes respon- sible for the committed steps in their own syntheses. In addition, however, with time, as the cells multiply, the actual amounts of those enzymes dwindle. Such cells actually cease to make the enzymes responsible for the synthesis of the amino acids already present in the medium, a phenomenon called "repression." Understanding of this process came from studies of the converse of this process, which has also been observed. When E. cold are placed in a medium that contains milk sugar (lactose) instead of glucose, they very rapidly begin to produce enzymes that permit them to metabolize the milk sugar; this process is called "induction." In both repression and induction, control is exercised by regulation of the expression of the genetic apparatus. When E. cold is placed in a medium containing lactose, rapid accumulation of three enzymes is observed. One, -galactosidase, makes possible the hydrolysis of lactose, a double sugar of glucose linked to galac- tose, into its two components; a second is a protein, galactose permease, which serves in the membrane of the cell to facilitate entry of galactose into the cell interior; and the third catalyzes the acetylation of galactose, a process whose metabolic meaning is obscure. Detailed analysis of these events has led to the following formulation as summarized in Figure 18. Within the circular chromosome of E. coli, there is a region that can be mapped as five consecutive genes. The terminal three are the genes that when expressed, give rise to the synthesis of the three proteins cited above. The first gene, however, directs synthesis of a protein, the repressor, which, when formed, attaches itself to the second gene. When it is so attached (the manner of attachment is unknown at this writing), the subsequent three genes cannot be transcribed; no mRNA is made on their surfaces, and accordingly the cell is unable to metabolize lactose. If, however, a small amount of lactose enters the cell, it prefer- entially binds to the repressor protein made by the activity of the regulatory gene, removing it from its attachment to the operator gene, thus freeing the rest of this locus for its expression, and the cell rapidly gains ability to metabolize milk sugar. That this is the case has been demonstrated by the actual isolation of the repressor protein, by the finding that it does indeed

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FRONTIERS OF BIOLOGY 63 bind to some portion of the chromosome, but not as tightly as it does to lactose, thereby giving direct experimental confirmation to postulates de- duced from the general behavior of the system. Thus, "induction" is more properly regarded as "derepression." The process of repression typical of the cell growing in a medium rich in amino acids is visualized as a modification of the process described above. $-Galactosidase operon 3-Galactosidase operator [o] Regulatorygene (i) 3-Galactosidase Galactoside permease gene (z) gene (y) ~ ~or 7 1 1 ~1 1 Start Stop ~ ~ signal signal / \ No synthesis of &3 53 43 j3 / $-galactosidase mRNA ~/ ..... , .. ~ Repressors (i) (a) (Z) (Y) 1 1 11 1 1 ,, ~ it 24~.~:~.~.~= I nactive .e ~[repressors . Repressors . 3-Galactosidase Galactoside permease 3-Galactosides FIGURE 18 Structural relationships and function of the lactose operon. In the top figure, repressor protein, made by action of regulatory gene, binds to operator gene, and the other genes of the operon are not functional. Newly arrived substrate, below, binds to repressor protein, which departs from regulator gene, and the structural genes of the operon go to work, as indicated by the growing protein chains on the ribosomes. (From J. D. Watson, Molecular Biology of the Gene, W. A. Benjamin, Inc., New York, 1965. Copyright (0 1965 W. A. Benjamin, Inc.)

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THE LIFE SCIENCES The repressor gene is thought to make a protein that can lock onto a regulator gene, but the repressor protein is successful in repressing only when it is in the proper three-dimensional conformation that results from binding to it of the amino acid-purine, pyrimidine, etc.-which is the repressing agent. By a combination of such modifications of the genetic apparatus and allosteric inhibition of enzymes, the cell's chemical activities are brought into a harmonious whole, all activities proceeding at rates commensurate with the cell's requirements at any time. ACTIVE TRANSPORT Every cell is in contact with its environment at its interface, the plasma membrane. But movement across this membrane is not the mere conse- quence of passage through little holes. Although a few components e.g., water and lipid-soluble gases can move freely, most materials viz., amino acids, sugars, and charged ions cross the membrane only in con- sequence of a process termed "active transport." As noted earlier, galactose permease takes its place in the bacterial cell membrane when the cell has been derepressed by the presence of milk sugar. Available information indicates that on the surface of this protein there is a site that tightly binds the sugar galactose. Thereafter, the protein rotates within the membrane so that the galactose so bound now faces inward. A second site on the protein then binds a molecule of ATP. When the latter attaches, the three- dimensional structure of the protein is so altered that the galactose falls off and becomes available for the metabolic activities of the cell interior. Hydrolysis (rupture of a covalent bond by addition of the elements of water), by that same protein, of the ATP into ADP and Pi discharges these from its surface, and the lactose permease molecule becomes free to rotate in the membrane to repeat the cycle. Only presumptive evidence suggests that similar events account for the active transport of the potassium ions into cells and sodium ions of cells, of amino acids, of other sugars, and so on. But for the moment, this appears to be an acceptable working model. ENZYMES The working machinery of the cell is its complement of enzymes. Enzymes have been studied for more than 50 years, but the pace of this endeavor has accelerated enormously in recent times. The primary task has always been to relate the structure of an enzyme to its catalytic function. Many types of enzymes have been under continuous study; the best understood of all enzymes is ribonuclease, made in the pancreas and secreted into the intestine, where it serves for digestive purposes by hydrolyzing long-chain

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FRONTIERS OF BIOLOGY 65 ribonucleic acids into the smaller nucleotide units of which they are com- posed. The initial approach to this problem was to establish the linear sequence of amino acids along the chain, an accomplishment that was completed by the early 1960's (Figure 19~. A great body of evidence was then assembled that indicated that the catalytic properties of this enzyme are dependent upon the coordinated, concerted activity of the histidine residues in positions 119 and 12 of the chain. It was then assumed that the poly- peptide chain must be so coiled as to offer a crevice into which the substrate, ribonucleic acid, would fit, such that these two histidine residues would then sandwich the bond to be subjected to hydrolysis. Convincing proof that this was so awaited the advent of x-ray crystallography, which pro- vided a total three-dimensional representation of the ribonuclease model (Figure 20~. In complete accord with expectations, in this as in every other hydrolytic enzyme examined to date, there is indeed a crevice into which the substrate molecule can fit, and, as predicted, the two working histidine residues are so located as to sandwich the portion of the substrate molecule to be attached. This description also accounts for the catalytic activities of enzymes generally. Each must be so arranged that the substrate is attracted to its surface and tied down by appropriate groups on that surface so as to permit 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 5' / Ala_ Alo_ Lys ~ Phe _ Glu ~ Arg _ Gln ~ His _ Met_ Asp ~ Serf Set _ Thr ~ Ser _Ala-Ala _. 4 3 1 Ala t Thr Glu AH, 55 75 80 ~i Ser Sir Asn Tyr Cys A;n Gln Met Met (Gin 4~ Cys -Tyr_ Gln~ Or ~ lyre Her_ Or ~ Mer_oer~ I) ~ ) /; Gln- Ala _Val ~ Cysuser-Gln~Lys~ ~ s~ /v^',~15 116 117 118 119 120 121 122 123 124 53 52 1 51 50 Asp Ala Lou 5;d~ r /~'Val-Ala ~ Cys-Glu ~Giy~Asn~Pro-Tyr-Val~Pro_Val~ His ~ Phe_Asp_Ala~Ser-val | \ His ~ :`Lys ~ Asn ~ Ala ~ Gln ~ Thr ~Thr ~Lys ~Tyr ~ Ala. Cys ~ Asn ~ Pro~Tyr ~Lys Server 100 lieu l Thr Asp Cys Arg Glu Thr 22 23 24 25 26 27 28 29 30 31 49 V1U~t5 ~ Vol ~Phe ~Thr ~ Asn ~ Val ~ Pro ~Lys ~ Cys ~Arg ~ Asp ~ Lys ~Ihr~Leu ~ Asn ~ Erg ~ ~ 48 47 46 45 44 43 42 4 1 4C 39 38 37 36 35 34 33 FIGURE 19 The amino acid sequence of bovine pancreatic ribonuclease. (From D. G. Smyth, W. H. Stein, and S. Moore, "Sequence of Amino Acid Residues in Bovine Pancreatic Ribonuclease: Revisions and Confirmations," J. Biol. Chem., 238: 227, 1963. Copyright (A 1963 The American Society of Biological Chemists, Inc. )

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66 THE LIFE SCIENCES FIGURE 20 Three-dimensional model of ribonuclease. The continuous strand of tubing follows the peptide chain. c`-Helix constitutes about 15 percent of the molecule: residues 2 to 13 (partly visible), residues 26 to 33, and residues 50 to 58 (in the back of the molecule). There is a section of antiparallel ,x-structures com- prising residues 71 to 92 and 94 to 110. The pairs of balls represent sulfur atoms in disulfide bridges, e.g., between 40 and 95 at the upper right. The single balls repre- sent sulfur atoms of methionine residues. The imidazole rings of histidines 12 and 119 are, as predicted from the chemical data, near one another in a groove. The competitive inhibitor, 5-iodouridine-2'(3')-phosphate, is bound in this groove. (From H. W. Wyckoff, K. D. Hardman, N. M. Allewell, T. Inagami, L. N. Johnson, and F. M. Richards, "The Structure of Ribonuclease-S at 3.5 A Resolution," J. Biol. Chem., 242: 3984, 1967. Copyright (I) 1967 The American Society of Biological Chemists, Inc.) attack by specific "working" residues on the enzyme surface. Such residues include phenolic groups from tyrosine, carboxyl groups from glutamic acid, and sulfhydryl groups from cysteine, as well as the imidazole group of histi- dines. It is the combination of specific binding and "concerted attack" that

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FRONTIERS OF BIOLOGY gives enzymes both their high degree of specificity for substrates and their remarkable catalytic rates. The final proof in the present instance was the total synthesis of ribo- nuclease from component amino acids by completely chemical procedures. When a chain of amino acids was synthesized with the sequence that had been ascertained by analytical procedures, it spontaneously assumed the three-dimensional conformation that results in normal catalytic activity of this enzyme, the ultimate triumph capping five decades of research. At the same time, this new capability offers a bold vista for the future, the prospect of synthetic proteins that will serve a variety of purposes. Perhaps the most important immediate prospect is the availability of the polypeptide hor- mones of the anterior and posterior pituitary glands, lack of which results in a variety of human disorders. In several cases-e.g., growth hormone specificity for man is absolute in that material obtained from bovine or porcine sources is ineffective; patently, the supply of human hormones is limited. SUBCELLULAR ORGANELLES The advent of electron microscopy ushered in a new era of understanding of the structure and functioning of living cells; the seemingly simple little box with an enclosed nucleus is, in fact, a highly detailed, organized struc- ture (Figure 211. The nucleus itself was found to be enclosed in a double membrane. Occasionally, that membrane is interrupted and becomes con- tinuous with membranous sheets that continue through the cell, some of which find their way to the plasma membrane at the cell surface and thence to the cell exterior. These sheets of membranes through the cell are termed the "endoplasmic reticulum." In cells engaged in protein synthesis, these membranes are stippled with ribosomes, the complex little machines that achieve protein synthesis. It is noteworthy that ribosomes also come into being by self-assembly. By appropriate means, separated ribosomes can be made to fall apart into a collection of several different kinds of nucleic acids and approximately 18 different proteins, the roles of which are not yet evident. When the medium is changed, all these components reaggre- gate and assemble spontaneously into working ribosomes. Thus the genetic machinery does not have to give additional instructions to assure construc- tion of these little chemical factories. Nor is there reason to think that any additional instructions are needed for the synthesis of the membranes themselves. These are constructed of lipids (fatty materials) and proteins; some are enzymes or participate in the active transport process and some may be purely structural. As the cell grows, these membranes continue to increase by the incorporation of additional proteins and lipids, and no

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68 THE LIFE SCIENCES Cell membrane ~ 5~.: ~ ~ ~ t~ ~ .:, ~ .... Ribosomes~5~ I: :\ Golg' complex . . . : Nucleolus: Nuclear membrane M itochond rion EndoPlasmic reticulum Lysosome FIGURE 21 Modern diagram of a typical animal cell, based on what is seen in electron micrographs. The mitochondria are the sites of the oxidative reactions that provide the cell with energy. The dots that line the endoplasmic reticulum are ribo- somes: the sites of protein synthesis. In cell division the pair of centrosomes, shown in longitudinal section as rods, part to form poles of the apparatus that separate two duplicate sets of chromosomes. (Reproduced from Biological Science by William T. Keeton, with the permission of the publisher, W. W. Norton & Company. Eric. Copy- right (if) 1967 by W. W. Norton & Company, Inc.) instructions need be given because this process is a consequence of the structure of the lipids and proteins themselves. Like the membranous sheets through the cell and the one surrounding the nucleus, the outer

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FRONTIERS OF BIOLOGY 69 membrane of the cell consists of two layers, one facing the environment and the other facing inward toward the cell. A similar double-membrane structure is evident in mitochondria, the small powerhouses to which reference was made earlier. Their outer membranes are exposed to the cell cytoplasm; the inner membrane consists of the working parts of the electron-transfer and ATP-generating machinery. Chloroplasts in plant cells are rather similarly constructed. Withal, the great advantage to the cell of this double-membranous structure remains to be understood, although it is a primary characteristic of life itself. Some portions of the endoplasmic reticulum are particularly concentrated with respect to enzymes that engage in the metabolism of lipid-soluble ma- terials e.g., the synthesis of cholesterol and steroid hormones presumably because these water-insoluble materials can be managed only when dis- solved in an appropriate medium, the lipid of the membranous structure. Unexpectedly, both mitochondria and chloroplasts have been found to contain DNA. The latter is insufficient to specify all the proteins of these structures, so they are not fully autonomous bodies. But these DNA's do find expression; in the living cell, mRNA is made on their surfaces and is translated into proteins on ribosomes in the usual manner. But the special virtues of this process are not apparent. Cell Division The great marvel of the living cell is the cycle of events whereby one parent cell gives rise to two identical daughter offspring. Indeed, for unicellular organisms and for the early stages of the embryonic growth of more complex organisms, cell division is itself the very essence of life. The life cycle of most cells can be viewed as a sequence of events shown as follows: S- BIG, G1 ~ D Stage S is the period of DNA synthesis, during which the total DNA com- plement of the cells is doubled. Stage D is the process of mitosis or gene segregation, in which the total DNA is separated into two identical sets of genetic material, after which cell division occurs. Stage G1, the gap in time between cell division and the next onset of DNA synthesis, varies from only a few seconds in some cells to many hours in others. During this period the cell engages in all the synthetic activities required for its own growth and metabolism, reaching its mature size, form, and function. If the cell is not to divide again, it remains in this stage.

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70 THE LIFE SCIENCES Nothing is known of the trigger that initiates the next burst of DNA synthesis, nor is it clear how this actually occurs in animal cells with com- plex chromosomes. The DNA content of a human cell is thousands of times greater than that of a simple bacterium. It is organized in chromo- somes, structures which, at greatest magnifications in some cells, somewhat resemble a bottle brush. Whereas a bacterial chromosome consists of a single circular DNA molecule, it is unknown whether mammalian chromo- somal DNA is circular, whether each chromosome is a single molecule, or whether each of the protruding filaments from the main axis is a separate discrete molecule of DNA. During the period of DNA synthesis, the chromosomes are not recognizable morphologically, losing their charac- teristic structure. When the total DNA content has doubled, a second resting stage, G., is apparent. During this period the marvelous mitotic apparatus is generated. Centrioles appear at either side of the cell, and spindle fibers, made of an unknown protein, are organized. Phase D, mitosis itself, then commences, and the spindle fibers attach to the paired chromosomes that are aligned in parallel at mid-cell. The fibers somehow shorten and contract, drawing one of each chromosome pair to the opposite sides of the cell. A membrane begins to grow at the mid-line; when this is completed, there are two cells. The chromosomes in each daughter cell compact themselves into a dense nucleus within which it is almost impossible to recognize specific chromo somes. Each daughter cell then enters Stage Go. It will be evident that some chemical event must initiate each of the four phases of cell life and serve as a signal inaugurating the processes char- acteristic of that phase. But in no case is the nature of the signal known. The mitotic apparatus requires a signal for fiber production, some mode for fiber attachment to the chromosomes, a mechanism for spindle short- ening, a signal that occasions formation of new membrane, and the dis- appearance of the spindle fibers after two cells are fully formed, as well as the subsequent compacting of chromosomes into nuclei. These triggers and many other details governing the process are unknown. A decade of careful electron microscopy and study of cell division has served only some- what to fill in details of this remarkable machinery. But the cardinal aspects remain elusive and warrant intensive investigation because the potential of control of these events by external manipulation would provide a means for an approach both to cancer therapy and to the alleviation of a multitude of other disorders. A powerful tool for the study of these events was made available by the discovery of techniques for the cultivation of mammalian cells in disasso- ciated suspended culture, much like the growth of bacteria or algae. By such techniques it has been possible to learn the nutritional requirements