Cell Biology - Class Notes for Exam #2

• Energy
• Enzymes
• Cell Surface Molecules
• Cell-Cell Communication
• Cell Adhesion
• Extracellular Matrix

LSc 202 Energy Notes Winter 1999

All organisms need a constant supply of energy to live

Cells contain molecules, molecules undergo 1000’s of chemical reactions.
Essential for:

• Growth
• Reproduction
• Development
• Homeostasis

Why do chemical reactions occur?

Energy: agent of change, the capacity to do work

• Potential energy (stored energy): energy stored in chemical bonds between atoms of molecules
  Ex: wood -- has potential energy stored in chemical bonds, when burned wood releases that energy as heat and light energy
• Kinetic energy: energy of a body in motion
  Ex: molecules moving, and when they collide the energy may be enough that they react

Thermodynamics: the study of energy conversions

First Law of Thermodynamics – Law of conservation of energy, energy can not be created or destroyed, it can only be converted from one form to another

Ex: gas in car—burned – gives mechanical energy and heat energy (some of the heat energy is lost)

Second Law of Thermodynamics – when energy is converted from one form to another, the conversion is never 100% efficient, some energy is lost as heat

(never is the amount of potential energy in a system fully converted to work)

the disorder of the system and its surroundings always increases

Metabolism: all chemical reactions occurring in an organism taken together

• Endothermic (Endergonic) or Anabolic reactions: synthetic rxns, smaller molecules combine to build larger molecules; require energy; the potential energy of the products is more than that of the reactants
• Energy Profile diagram

  Ex: anabolic steroids build muscle protein, protein synthesis reactions are an anabolic reactions

  • Exothermic (Exergonic) or Catabolic reactions: breakdown rxns, molecules are split apart & dissociated; release energy; the potential energy of the reactants is more than that of the products
    Energy Profile diagram
    The potential energy stored in covalent bonds is released by cells as they catabolize carbohydrates & other molecules (like triglycerides), a series of reactions breaks covalent bonds & energy is released
    Ex: "carbo-loading" starch is readily broken down into glucose. When needed the glucose molecules in muscle are broken down in a series of chemical reactions releasing large amounts of energy.
• Net Energy Yield = potential energy of the reactants - potential energy of the products
• Activation Energy (E_a) – the energy invested to make a reaction go; even exergonic (spontaneous) reactions require a small investment of energy to get things started, basically to overcome the repulsion forces between the electron clouds of atoms
  Ex: combustion of gasoline is an exergonic reaction, must be ignited to proceed (small investment of heat energy)
• Coupled reactions: the energy released by catabolic reactions is used to drive anabolic reactions; the transfer of energy is never 100%, some energy is lost as heat ("pulling reactions uphill")

Ex: glucose breakdown & ATP formation à ATP hydrolysis & protein synthesis

• ATP serves as a carrier molecule linking exergonic and endergonic reactions

(third phosphate group bond requires a lot of energy to create, but is fairly easily broken; houses a lot of potential energy)

• ATP produced in the catabolic breakdown of glucose, ATP transports this energy where needed in the body (shuttle, short-term energy storage)

Ex: E.coli (bacteria in human digestive tract)

2.5 million ATP molecules broken down/second

(only has 10,000 - 100,000 total ATP molecules, so ATP is recycled 25-250X / second !)

• Stable molecules (glucose, starch, glycogen) for long-term storage of energy
• Chemical reactions are reversible.

Ex: in blood

CO₂ + H₂O D H₂CO₃

Chemical equilibrium: when the rate of the forward reaction equals the rate of the reverse reaction, once equilibrium is achieved the concentrations of the products and reactants remain constant (this does NOT mean that [reactants] = [products]).

LSc 202 Enzymes Notes Winter 1999

ENZYMES

• Most chemical reactions require the presence of a specific enzyme.
  Enzyme: protein molecule that speeds up the rate of an anabolic or catabolic reaction
  • Enzymes catalyze reactions by lowering the activation energy
  • Each enzyme is specific for one or few reactions
    • helps regulate chemical reactions
    • conserves energy & cytoplasmic supplies
  • Enzymes are typically large proteins with small active sites (pocket in the enzyme where the chemical reaction occurs)
  • Substrate (molecule undergoing chemical reaction) binds the pocket of an enzyme
    • "lock and key" fit
    • induced fit ("hand in glove")
  • Enzymes provide a place for reactions to occur.
    • do not need to rely on collisions
    • overcomes repulsion forces between the electron clouds of atoms
    • orient molecules to increase chance of reaction
    • stabilize a transition state (intermediate)
    • placing proton donors and acceptors in position to promote acid/base reactions
  • Each cell has about 2000 different enzymes (human body has about 10,000 different enzymes)
  • Enzyme catalyzed reaction are 100 million – 10,000 million times faster than uncatalyzed reactions (10⁸-10¹⁰)
  • Some enzymes catalyze reactions in sequential steps of a metabolic pathway, a missing or defective enzyme in the pathway at any step can cause breakdown in the whole process
  • many enzymes have non-protein group called cofactors
  • enzymes are saturated in high substrate concentrations
  • Enzymes are a subgroup of catalysts (remain unchanged), can be reused (a single enzyme molecule can catalyze 1000 reactions/sec).
  • Enzymes can be regulated.
    • negative feedback (feedback inhibition): end products of chemical reactions often block further enzyme activity
      • competitive inhibitor -- inhibitor molecule binds to the active site of the enzyme and competes with the substrate for binding to the enzyme
      • non-competitive inhibitor -- inhibitor molecule binds to an allosteric site of the enzyme (not the active site) and alters the shape of the active site preventing the substrate from binding the enzyme
    • positive feedback: when the presence of a molecule enhances or turns on enzyme activity
    • temperature
    • pH
• Enzymes also catalyze catabolic reactions
  cleavage: requires enzymes, the active site provides a place where bonds can break with less energy

LSc 202 Cell Surface Molecules and Receptors Notes Winter 1999

Cell Surface Molecules

Glycocalyx (the "sugar coating" of a plasma membrane), molecules are involved in recognition , receptors, signaling, cell adhesion ……

 

Carbohydrate groups of glycoproteins and glycolipids can form in many diverse straight and branched chain patterns

• form linkages between 1 à 1, 1 à 2, 1 à 3, 1 à 4, 1 à 6, 2 à 3, 2 à 6 carbons of sugars in either the a (-OH down) or b (-OH up) form
• these combinations can create many different carbohydrate chains from even a small number of carbohydrate molecules (Ex: 4 monosaccharides can link to form over 35,000 distinct tetrasaccharides!), ever more diverse than proteins
• glycoproteins and glycolipids on the cell surface can have single or multiple carbohydrate chains from a single monosaccharide to chains of over 70 units

Cell Recognition glycoproteins and glycolipids can serve as sites of cell recognition

• identifying a cell as foreign or self (MHC molecules)
• identifying a cell as a particular tissue type (tissue specific antigens)

MHC (Major Histocompatibiility Complex) molecules: primary molecules responsible for recognition of self from non-self

• MHC molecules are also known as transplantation antigens: individuals mount an extremely strong immune response against foreign MHC molecules (thus, MHC important in transplant rejection)
• class I molecules (found on essentially all cells in the body):
  • heterodimer of a heavy chain (3 immunoglobulin domains, 2 of which are variable, highly polymorphic) and a constant lighter chain of b ₂ microglobulin
  • primarily function in the presentation of antigens to CD8⁺ cytotoxic or killer T cells, which can destroy target cells, typically class I molecules present viral antigens which are endogenously produced during a viral infection
• class II molecules (normally found on a subset of cells in the immune system including B cells, macrophages, and dendritic cells)
  • heterodimer of an a and b chain (each having 2 immunoglobulin domains, 1 constant and one variable)
  • primarily function in the presentation of antigens to CD4⁺ helper T cells, which can secrete mediators of the immune response and help with humoral immunity, typically class II molecules present antigens which are exogenous and have been engulfed or endocytosed by the antigen presenting class II positive cell

Blood Group Antigens:

• MN Antigens -- glycoprotein (glyocophorin) on erythrocytes
• ABO blood groups – glycolipid carbohydrate groups
  • Type A N-acetylgalactosamine at one position
  • Type B galactose at the same position
  • Type AB have glycolipids with both
  • Type O corresponding position is empty
  • Caused by a difference in the glucosyl transferase enzyme (encoded in a single gene that has several single base substitutions causing the differences between A and B, and a deletion in O), which add sugar groups to protein in the Golgi
• Cell surfaces may contain many receptors
• Distinct sets of receptors

Receptors bind ligands:

• free in extracellular fluid
• part of the extracellular matrix
• linked to the surface of other cellsBinding at the cell surface -- complete cellular response

Receptor Groups

1. receptors which bind first messengers (Table 7-2)
   • triggers a complex biochemical response
     • rate of transport
     • secretion
     • oxidative metabolism
     • initiation of cell division
     • cell movement
   • small number of bound ligands is sufficient for full cellular response
   • conformational change in receptor activates enzymatic site on cytoplasmic side
   • activates and series of reactions (cascade)
2. receptors which bind and internalize the ligand (receptor-mediated endocytosis Ch.20)
3. receptors which connect cells to other cells and the extracellular matrix (Ch.8)
   • hold cells in place in tissues and organs
   • supportive function NOT biochemical

Tools for investigating receptors:

• enzymes: identify receptors as glycoproteins
  • carbohydrate-digesting enzymes: galactosidase (attacks galactose units) neuraminidase (attacks sialic acid residues)
  • proteases
• covalent linkage of tagged ligands to receptors
  • gel electrophoresis
  • affinity chromatography
• molecular genetics: families of receptor genes

Common aspects of receptor signaling pathways:

• protein kinase activity (add a phosphate group derived from ATP to target proteins)

  Phosphorylations control:

  • enzymes in metabolic pathways
  • transport proteins (ion channels)
  • ribosomal proteins
  • gene activity regulator proteins
  • receptors
• protein phosphatase activity (remove phosphate groups from target proteins)
  • reverse or inhibit effects of kinases

LSc 202 Cell Communication Notes Winter 1999

first messengers: external molecules bind to cell surface and initiate a cellular response

second messengers: small rapidly diffusing intracellular molecules which can directly or indirectly activate protein kinases involved in a cellular pathway

(Ex: cyclic AMP (cAMP), InsP₃, DAG)

Nobel Prize 1971 E.W. Sutherland

Two Major Pathways generating second messengers:

• cyclic AMP (cAMP)
• diverse array of cellular responses in animals
• generated from ATP by the effector adenylate cyclase (Fig 7-5)
• cAMP is quickly degraded by cyclic nucleotide phosphodiesterase
• cAMP activates protein kinase A family
• cAMP binds to regulatory subunits of the kinases -- conformational change in the enzymes
• subsequent phosphorylations by kinases activate or inhibit target proteins
• regulation of cAMP by cGMP : guanylate cyclase converts cytoplasmic GTP into cGMP, cGMP activates a phosphodiesterase which breaks down cAMP
• inositol triphosphate (InsP₃) and diacylglycerol (DAG)
• involved in all eukaryotes (Ex: secretion of hormones, cell division, fertilization early events, sugar transport, contraction of smooth muscle)
• effector phospholipase C breaksdown membrane phosphoglyceride: phosphatidyl inositol (Fig. 7-6)
• Ca²⁺ critical for function
• activates protein kinase C family, Ca²⁺ - activated

G proteins

• peripheral membrane proteins on the cytoplasmic side of membrane
• consisting of 3 polypeptides (a - b - and g - chains)
• bind nucleotides GTP and GDP
• functional links between receptors and effectors which activate second messengers (Fig. 7-7)

Regulation

• amplification: many molecules in both cAMP and InsP₃/DAG pathways are enzymes which can repeatedly catalyze steps in the pathway à amplifying the signal, combined effect of a single receptor, G protein, effector, and kinase can amplify 10⁸ times!
• need for mechanism the detects when the surface receptor is disengaged: rapid "off switch"
• rapid degradation of second messengers
• serine/threonine or tyrosine phosphatases
• "crosstalk" regulation
• regulatory molecules outside of the pathways Ex: sphingosine inhibits phospholipase C; regulation of cAMP by cGMP
• receptor-mediated endocytosis terminates second messenger pathways

LSc 202 Cell Adhesion and Intracellular Junctions Notes Winter 1999

Cell Adhesion and Cell Junctions

• glycoproteins maintain cell adhesions
• critical in stabilizing body form and structure
• many molecules involved in cell adhesion are members of the Immunoglobulin superfamily
• CAMs (Cell Adhesion Molecules):
  • N-CAM (neurons)
  • Ng-CAM (neural/glial)
  • L-CAM (liver)
  • I-CAM (many cell types, binds to the ligand LFA-1)
  • LEC-CAM (leucocytes and other circulatory cells)
• L1 (neural) and J1 (neural/glial)
• cell-cell adhesion important in embryonic development

• Intercellular Junctions – function in adhesion, sealing, and communication
  • Adhesive Junctions:
    • desmosomes – circular or elliptical structures, characterized by dense plaques of protein into which intermediate filaments (tonofilaments, cytoplasmic anchors) in two adjoining cells insert
      present in tissues exposed to shear or lateral stress (Ex: skin, epithelial lining of body cavities)
      desmosomes often disappear in cancer cells allowing metastasis
    • adherens – cytoplasmic face of the plasma membrane attaches to actin microfilaments, form adhesion belts loosely linking adjacent epithelial tissues Ex: heart muscle, thin layers covering organs
    • septate (invertebrates) – contain regularly spaced cross bars or septa in extracellular region
  • Tight Junctions (occluding junctions)
    • form continuous belt around cells lining body cavities
    • seals cells together and prevents leakage
    • outer layer of adjacent plasma membranes fuse along sharply defined ridges
    • ridges partially or completely seal the junction to passage of extracellular fluids
    • more ridges ® tighter the seal
    • block lateral movement of lipids and proteins
    • may contain tightly packed integral membrane proteins in outer leaflet of membranes
    • some are dynamic and regulated, induced by cAMP or Ca²⁺ in the cytoplasm, reduced in the presence of glucose tranporters

      Ex #1: lining the intestinal tract: Na⁺ -ATPase pumps and MHC class I molecules are found on side facing the bloodstream, Na⁺ channels and alkaline phosphatase enzymes found on side facing intestinal cavity

      Ex #2: capillary walls of brain and testis -- prevent blood from mixing with extracellular fluids bladder -- maintain large ionic strength differences between body cavity and tissues

  • Gap Junctions -- communicating junctions important in nutrient exchange, conduction of electrical impulses, development and differentiation, synchronization
    • open channels between the cytoplasm of adjacent cells allows direct passage of small molecules and ions (sealed to extracellular fluid)
    • cylinders (connexons): 4-6 subunits of connexin proteins
    • connexins vary in size and sequence, structure contains four a - helical transmembrane regions connected by cytoplasmic loops
    • connexins similar in the same tissue of different organisms
    • some allow passage of amino acids, second messengers and monosaccharides
    • regulation of gap junctions
      • open and close through conformational changes in the connexins
      • open at low Ca²⁺concentrations, close as Ca²⁺ levels rise
      • voltage
      • phosphorylation by protein kinase A opens connexons more fully
      • role of second messengers
      • can close off broken or leaky cells
    • especially important in heart muscle when cells need to act as a single functional unit
    • not found in skeletal muscle, blood, and most nerve tissues
    • appear as early as the eight-cell stage in mammalian embryonic tissue
    • gap junctions can be increased under certain conditions: Ex: uterus

LSc 202 Extracellular Matrix Notes Winter 1999

extracellular matrix (ECM): complete network of proteins (ex: collagen) and polysaccharides (ex: GAGs) secreted by cells serving as a structural element in tissues

animals: ECM forms diverse structures

• tough, elastic framework of tendons, cartilage, and bone
• cornea of the eye (clear and cystal-like)
• supportive structures in epithelial tissues and other organs

plants and prokaryotes:

• cell walls

Functions of ECM:

• support (primary function)
• regulations of cell division
• adhesion
• motility
• migration
• differentiation during embryonic development
• wound repair
• response to diseases
• filters which exclude larger molecules from some cell layers
• in plants, fungi, algae, and prokaryotes: ECM has recognition sites for extracellular molecules, cell walls can function in cell-cell communication

General composition of ECM:

• long, semicrystalline protein fibers
• fibers are embedded in proteoglycan network
• network and fibers restrict flow of water
• variations in fiber molecules give consistency ranging from soft, watery gels à dense, rock-hard

Collagens

• semicrystalline proteinfibers
• hold cells in place
• tensile strength and elasticity
• found in all animal phyla

Collagen Structure (Fig 8-1)

• insoluble glycoproteins
• (glycine-X-Y)_n
  where X and Y are proline and lysine (modified forms of hydroxyproline and hydroxylysine)
• attached disaccharides made primarily of glucose and galactose
• individual collagen molecules are linear, contain three polypeptide chains (a -chains)
• individual a -chains wind in a left-handed helix
• three a -chains twist together in a right-handed triple helix -- fibrous
• some types of collagen have regions of less rigid structure
• globular amino acid caps form at the ends of the triple helices
• structure maintained by hydrogen bonds
• 25 different closely related a -chains – form (types I-XIV)

 

Different types of Collagen Fibers (Table 8-1)

• types I, II, and III have regular patterns of cross striations
• type IV collagen forms very fine, unstriated fibers, sheetlike supportive meshes, basal laminae or basement membranes

Synthesis and Assembly of Collagen fibers (Fig. 8-7)

• pro- a -chains synthesized on RER
• inserted into ER add carbohydrate groups
• three pro- a -chains wind together
• move to Golgi, sent to plasma membrane
• pro-collagens are secreted from the cell
• extracellular enzymes remove the extra a.a’s forming insoluble collagen fibers

Collagens and Disease

• fibrosis: conditions resulting from excessive production of collagen
  • pulmonary
  • endomyocardial
  • retroperitoneal
• Ehlers-Danlos (rubber-man syndrome : contortionists): results from insufficient collagen production-- mutation in pro-collagen preventing proper enzyme removal of pro sequences
• osteogenesis imperfecta or brittle-bone syndrome: pro-collagen fails to form triple helices -- glycine is replaced with other bulky a.a.’s
• scurvy (caused by vitamin C deficiency): insufficient hydogen bonding to hold triple helix together
• connective tissue disorders – aneurisms, osteoporosis

Proteoglycans: glycoproteins with as much as 95% dry weight comprised of carbohydrates

Structure

• linear polypeptide core
• carbohydrate chains radiating from core like bristles
• vary in core protein length, carbohydrate chain composition and length
• carbohydrates link covalently through serine, threonine, and asparagine residues
• primary carbohydrate type: GAGs (glucosaminoglycans), unbranched chains of glucosamine and galactosamine 2-unit repeats
• GAGs may also link to hyaluronic acid to form larger complexes

Function

• trap and impede flow of water
• resistant to compression
• hyaluronic acid in soluble unattached form lubricates joints, increases fluid viscosity
• low proteoglycan mount – increases rigidity
• higher proteoglycan content – flexible
• mineral crystals in bone
  (calcium hydroxyapatite, calcium carbonate, and citrate)
• temporary or permanent sites of adhesion

Linkage of ECM to the Cell Surface

• direct linkages through surface receptors binding collagen or proteoglycans
• direct anchorage of proteoglycans
• linking glycoproteins (laminin and fibronectin) form non-covalent links crosslinking
• linkers:
  • fibronectins
    • found in all extracellular structures
    • large glycoproteins (95% protein, 5% carbo)
    • 2 fibrous polypeptide chains linked by disulfide bonds at one end
    • V-structure (Fig. 8-11)
    • maintaining cell morphology
  • laminin
    • limited to basal laminae
    • cross-like structure (Fig. 8-12)
    • a , b ₁ and b ₂ polypeptide chains (87%protein, 13% carbohydrates)
    • binding sites for Type IV collagen
    • binds to several GAGs
    • multiple binding sites – interlinker
• cell surface receptors for linkers:
  • integrins Fig 8-13, 8-14: family of transmembrane proteins
    bind cytoskeletal elements and ECM
    • heterodimer of a (11) and b (5) subunits
    • different combinations of a and b
    • a subunit confers binding specificity
    • RGDS (arg-gly-asp-ser) in linkers binds to integrins
    • secondary binding sequences further define specificity

Summary Section in blue p.286
Fig. 8-15 p. 287