高等生物化学.pdf

高等生物化學: Life 簡介 生命體的基本組成:細胞 (cell) Prokaryote cell 的組成及各別功能 Eukaryote cell 的組成及各別功能 組成生命體的基本化學分子 生化域的有關期刊 Biochemistry-Chpater1: Life 生物化學及其研究範疇 麼是生物化學? 它和化學有麼同? Biochemistry is the study of the chemical reactions on the molecular level that occur in living organism. 生物化學研究的目的? to interpret the biological functions of living organisms in the language of chemistry. 生物化學研究對象-生命體的定義為何? 生命體具有下特質:由環境中吸取營養，因應環境變化，成長及分化，繁衍。 Norman Horowitz提出: All living things grow, reproduce (replication) Obtain energy from their surroundings, build new molecules from basic organic molecules (catalysis) Repair themselves, and respond to external stimuli (mutability) .tw國立中興大學生化所 李天雄 生命體的基本組成單位:細胞 All living things are composed of one or more cells and that cells are more or less alike and carry out similar functions to keep themselves alive. Matthias Schleiden transport of solutes; energy generation; location of numerous enzyme systems Plasma membrane Protein against phagotrophic engulfment Attachment to surfaces; protection Common pili or fimbriae Protein Mediates DNA transfer during conjugation Sex pilus Pili Protein Swimming movement Flagella Predominant chemical composition Function(s) Structure Prokaryotes Prokaryotes Inclusions Inclusions are distinct granules that may occupy a substantial part of the cytoplasm. Inclusion granules are usually reserve materials of some sort. For example, carbon and energy reserves may be stored as glycogen (a polymer of glucose) or as polybetahydroxybutyric acid (a type of fat) granules. Polyphosphate inclusions are reserves of PO4and possibly energy; elemental sulfur (sulfur globules) are stored by some phototrophic and some lithotrophic procaryotes as reserves of energy or electrons. Some inclusion bodies are actually membranous vesicles or intrusions into the cytoplasm which contain photosynthetic pigments or enzymes. Mesosomes The plasma membrane of prokaryotes may invaginate into the cytoplasm or form stacks or vesicles attached to the inner membrane surface. These structures are sometimes referred to as mesosomes Such internal membrane systems may be analogous to the cristae of mitochondria or the thylakoids of chloroplasts which increase the surface area of membranes to which enzymes are bound for specific enzymatic functions. The photosynthetic apparatus (light harvesting pigments and ATPase) of photosynthetic prokaryotes is contained in these types of membranous structures. Mesosomes may also represent specialized membrane regions involved in DNA replication and segregation, cell wall synthesis, or increased enzymatic activity. .tw國立中興大學生化所 李天雄 Energy Sources of Prokaryotes Organisms that use radiant energy (light) are called phototrophs. Organisms that use (oxidize) an organic form of carbon are called heterotrophs or chemo(hetero)trophs. Organisms that oxidize inorganic compounds are called lithotrophs. The carbon requirements of organisms must be met by organic carbon (a chemical compound with a carbon-hydrogen bond) or by CO2. Organisms that use organic carbon are heterotrophs and organisms that use CO2as a sole source of carbon for growth are called autotrophs. Most Bacteria, some Archaeaorganic compoundsOrganic compounds Chemoheterotrophs or Heterotrophs A few Bacteria and many Archaea CO2 Inorganic compounds, e.g. H2, NH3, NO2, H2S Chemoautotrophs or Lithotrophs (Lithoautotrophs) Some Purple and Green Bacteria organic compounds Light Photoheterotrophs Cyanobacteria, some Purple and Green Bacteria CO2 Light Photoautotrophs ExamplesCarbon SourceEnergy SourceNutritional Type Environmental Requirements for Microbial Growth : Oxygen Oxygen is a universal component of cells and is always provided in large amounts by H2O. However, procaryotes display a wide range of responses to molecular oxygen O2. Obligate aerobes require O2for growth; they use O2as a final electron acceptor in aerobic respiration. Obligate anaerobes (occasionally called aerophobes) do not need or use O2as a nutrient. In fact, O2is a toxic substance, which either kills or inhibits their growth. Obligate anaerobic procaryotes may live by fermentation, anaerobic respiration, bacterial photosynthesis, or the novel process of methanogenesis. Facultative anaerobes (or facultative aerobes) are organisms that can switch between aerobic and anaerobic types of metabolism. Under anaerobic conditions (no O2) they grow by fermentation or anaerobic respiration, but in the presence of O2they switch to aerobic respiration. Aerotolerant anaerobes are bacteria with an exclusively anaerobic (fermentative) type of metabolism but they are insensitive to the presence of O2. They live by fermentation alone whether or not O2is present in their environment. .tw國立中興大學生化所 李天雄 Effect of Oxygen on the growth of prokaryotes Growth Growth Growth Toxic No growth No growth Anaerobic Not required and not utilized Growth Aerotolerant Anaerobe Not required for growth but utilized when available Growth Facultative Anaerobe (Facultative Aerobe) No growth Obligate Anaerobe Required but at levels below 0.2 atm Growth if level not too high Microaerophile Required (utilized for aerobic respiration) Growth Obligate Aerobe O2EffectAerobicGroup Other environmental factors are pH, temperature, water, etc. Prokaryotic classification In Determinative Microbiology, some of the features that have been used to classify prokaryotes include: Gram stain (cell wall structure), Mole percent G+C in the genome, Growth temperature, Ability to form heat stable spores, Electron acceptors for respiration (if any), Photosynthetic ability, Motility, Cell shape, Ability to use various carbon and nitrogen sources, Special nutritional requirements (e.g., vitamins) 優缺點: (1) From the standpoint of many areas of microbiology: A determinative classification is sufficient. For example, in clinical microbiology the identification of organisms permits the physician to assess pathogenicity, and to select a treatment. In this context, the purely determinative nature of a classification was not crucial. If the organism has previously been described, and hence is already in the classification, then it can be identified and treated. (2) From a biological point of view: The lack of a natural system was an unsatisfactory state of affairs. It does not permit the projection of properties of previously described organisms onto new ones that might be closely related, but not identical, to those known before. In addition, it does not help us understand an organism that we have been unable to cultivate in the laboratory. Finally, it does not permit studies of the origin and evolution of cellular functions (e.g., drug resistance, aerobiosis or photosynthesis), because there is no evolutionary (historical) framework. .tw國立中興大學生化所 李天雄 MOLECULAR PHYLOGENY分法(1) Method 1: DNA-DNA Hybridization One important technique for comparing prokaryotes at the molecular level is DNA-DNA hybridization. In this test, the genomic DNA from one species is mixed with the DNA from a second species and the similarity of the DNAs is reflected in the extent to which strands of DNA from one organism anneal with strands of DNA from the other organism. The sensitivity of DNA DNA hybridization declines rapidly as the organisms become more diverged, limiting the method to characterization of closely related strains, species and genera. *使用DNA-DNA Hybridization方法時首要條件: (1) The gene must be present in all organisms of interest. Thus, to infer relationships that span the diversity of prokaryotes (or life), we must look at the central (universal) cellular functions. Examples include genes whose products function in replication, transcription, or translationthe processes constituting the Central Dogma of molecular biology. (2) The gene cannot be subject to transfer between species (lateral transfer). (3) The gene must display an appropriate level of sequence conservation for the divergences of interest. (4) The gene must be sufficiently large to contain a record of the historical information. Thus, although transfer RNA (tRNA) genes are present in all species, they are too small (about 75 nucleotides) to provide an accurate sample of evolutionary history. Although the morphologies and physiologies of prokaryotes are much simpler than those of eukaryotes, there is a large amount of information in the molecular sequences of their DNA, RNAs and proteins. Thus, it is possible to use molecular similarities to infer the relationships of genes, and, by extension, to learn the relationships of the organisms themselves. MOLECULAR PHYLOGENY分法(2) Method 2: Ribosomal RNA Genes and Their Sequences Most prokaryotes have three rRNAs, called the 5S, 16S and 23S rRNA. If relationships were analyzed by comparing sequence data, rather than hybridizing the molecules, one could infer relationships without having all of the molecules in hand (only the sequence data from previous studies are necessary). This was already being done with protein sequences. Carl Woese recognized the full potential of rRNA sequences as a measure of phylogenetic relatedness. He initially used an RNA sequencing method that determined about 1/4 of the nucleotides in the 16S rRNA (the best technology available at the time). This amount of data greatly exceeded anything else then available. Using newer methods, it is now routine to determine the sequence of the entire 16S rRNA molecule. Today, the accumulated 16S rRNA sequences (about 10,000) constitute the largest body of data available for inferring relationships among organisms. The name is based on the rate that the molecule sediments (sinks) in water. Bigger molecules sediment faster than small ones. Large subunit of ribosome290423S Small subunit of ribosome154216S Large subunit of ribosome1205S LocationSize (nucleotides)Name Ribosomal RNAs in Prokaryotes The 5S has been extensively studied, but it is usually too small for reliable phylogenetic inference. The 16S and 23S rRNAs are sufficiently large to be quite useful. .tw國立中興大學生化所 李天雄 Examples Comparison of small subunit rRNA gene sequences from organisms spanning the known diversity of life .GTGTCAGCCGCCGCGGTAATACCAGCTCCGCGAGTGGTCGGGGTGATTACTGGGCCTAAAGCG. Sulfolobus sulfotaricus .GTGGCAGCCGCCGCGGTAATACCGGCGGCCCGAGTGGTGGCCGCTATTATTGGGCCTAAAGCG. Thermococcus celer .GTGCCAGCAGCCGCGGTAATACCGACGGCCCGAGTGGTAGCCACTCTTATTGGGCCTAAAGCG. Methanococcus vannielii .GTGCCAGCAGCCGCGGTAATACGTAGGGGGCAAGCGTTACCCGGATTTACTGGGCGTAAAGGG. Thermotoga maratima .GTGCCAGCAGCCGCGGTAATACGGGAGAGGCAAGCGTTATCCGGAATTATTGGGCGTAAAGCG. Anacystis nidulans .GTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCG. Escherichia coli .GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTTAAGTTGTTGCAGTTAAAAAG. corn .GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGTTGCAGTTAAAAAG. yeast .GTGCCAGCAGCCGCGGTAATTCCAGCTCCAATAGCGTATATTAAAGTTGCTGCAGTTAAAAAG. human Prokaryote descent An analysis of 16S rRNA- based phylogenetic tree showing the three (identified) Domains of lifeBacteria, Archaea and Eucarya. Archaea consists f three different kinds of organisms: (1) methanogens (2) halobacteria (3) thermoacidophiles .tw國立中興大學生化所 李天雄 Eukaryotes 通常Eukaryotic cells在10-100 m直徑，其大小為 prokaryote的千至百萬倍。 Eukaryotic cells的特徵 在體積的大小，而在內含各佐式膜包覆，及具有特定 功能的organelle。 Cells are surrounded by a bordering membrane called the cytolemma, plasmalemma, cell membrane, or plasma membrane. The nucleus is the central region of the cell containing the cells DNA (genetic code, or genome). The nuclear membrane is biochemically similar to, and often anatomically continuous with, the cell membrane.The prominent structure in the nucleus is the nucleolus. The nucleolus produces ribosomes, which move out of the nucleus to positions on the rough endoplasmic reticulum where they are critical in protein synthesis. Eukaryotes architecture-核酸, ribosome, ER Nucleic Acids (DNA, RNA) are the genetic code molecules of cells. All cells use DNA as the genetic code molecule, and in eukaryotes the DNA is packaged in a nuclear region inside of a spherical nuclear membrane that contains pores. A gene is a length of DNA coding for a cell protein (enzyme or structural protein). The photo shows a piece of cellular DNA. It is highly coiled and is called a chromosome. Ribosomes are small snowmen-shaped structures floating free in the cytoplasm or attached to endoplasmic reticulum. They are made of proteins and also of ribonucleic acid (rRNA). Ribosomes are the site where amino acids are assembled into proteins, under the guidance of the genetic code molecule messenger RNA (mRNA). mRNA is a mirror image copy of a DNA gene segment. Transfer RNA (tRNA) is a special type of RNA that carries specific amino acids to the ribosome. Endoplasmic Reticulum, or ER for short, is a membranous tubule system throughout a cell that serves several functions: Smooth ER (SER), devoid of ribosomes, detoxifies cell poisons and also is the site of steroid synthesis; Rough ER (RER), studded with ribosomes, modifies proteins synthesized on ribosomes, sometimes glycosylating (adding sugars to) proteins to create glycoproteins (such as the ABO proteins of infamous blood type notoriety). Study the portion of a cell shown below. It has a large amount of rough endoplasmic reticumulum (RER), appearing as stacked flattened membranes. The RER is covered with ribosomes. The nucleus is seen occupying the majority of the cell (it is large and round). .tw國立中興大學生化所 李天雄 Eukaryotes architecture-Golgi, centrioles, mitochondria Golgi material looks like a stack of flattened pancakes with vesicles at the edges. It functions for packaging material into vesicles for transport to other parts of the cell or for cell export (for example packaging of synthesized antibody proteins for export in the case of an immune system plasma cell). Mitochondria are submarine-shaped structures with membranous infoldings called cristae. The mitochondrion is the powerhouse of the cell, where most of a cells ATP energy is made during cell metabolism using oxygen (aerobic metabolism). Mitochondria contain DNA (called m-DNA) and ribosomes. The bacteria-like functions of mitochondria leads to the hypothesis that mitochondria evolved from originally aerobic bacteria, which formd a symbiotic relationship with a primordial anaerobic eukaryote.(the origin of endosymbiotic). Centrioles are short arrays of microtubules, appearing like a piece of cut licorice candy, located near a cells nucleus. Centrioles function analogous to fishing rod and reels with hooks. During cell division, centrioles elongate, seeking out cell chromosomes (supercoiled DNA and histone protein molecules) to attach to and reel (move) to ends of the cell for orderly cell division following DNA replication. Eukaryotes architecture Inclusion bodies are chunks of miscellaneous material inside of cells, such as starch or fat granules, and so on. Lysosomes are membranous sacs that store powerful destructive enzymes, used to destroy other cell organelles, to destroy the cell at some point, or to destroy engulfed microbes or debris in the case of certain immune system cells. Cell such as certain white blood cells have numerous lysosomes to help destroy foreign engulfed particles or bacteria. Lysosomes are produced by the Golgi apparatus. Peroxisomes(microbodies) are membrane-enclosed organelles which contain oxidative enzymes. Some peroxisomal reactions generate hydrogen peroxide (H2O2), a reactive substance used in the enzymatic oxidation of other substances or degraded through a disproportionation reaction by enzyme catalyse. The proper functioning of the cell requires careful control of the levels of important structural proteins, enzymes, and regulatory proteins. The only way that cells can reduce the steady state level of a particular protein is by proteolytic degradation. Thus, complex and highly-regulated mechanisms have been evolved to accomplish this degradation. An additional role of intracellular proteolysis is in the stress-response. Cells which are subject to stress such as starvation, heat-shock, chemical insult or mutation respond by increasing the rates of proteolysis. One function of this enhanced proteolysis is to salvage amino acids from non-essential proteins. These amino acids can then be reutilized in the synthesis of essential proteins or metabolized directly to provide energy. Another function is in the repair of damage caused by the stress. For example, oxidative stress has been shown to damage a variety of proteins and cause them to be rapidly degraded. .tw國立中興大學生化所 李天雄 Eukaryotes architecture Microtubules and Microfilaments are linear protein arrays that function in the cell for transport of cell materials within the cell, for movement of cell DNA during cell division, for creation of the cell cytoskeleton (cell skeleton), and for creation of cilia and flagella. The cytoskeleton maintains the cells shape, anchors organelles in place, and moves parts of the cell in processes of growth and motility. Mitosis is the process by which cells divide. The parent cell has already duplicated its chromosomes , providing both daughter cells with a complete copy of genetic information. Eukaryotic animal cell This is a transmission electron micrograph of tissue removed from the small intestine of a mouse and magnified approximately 26,000 times. 由上述課程所的資 ，請你猜一猜圖中 的各個號碼所對應的 organelle名稱，請問 圖片中只有一個cell嗎? 試試看是否可找出有 幾個cells? .tw國立中興大學生化所 李天雄 Eukaryotes (植物) architecture Vacuolesare membranous sacs that store nutrients, wastes, or water. They are more prominent in plant cells (90% cell volume). Chloroplast are the organe