Biochemistry Pankaja Naik
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1Structure and Function of the Biomolecules
  • 1. Eukaryotic Cell Structure
  • 2. Carbohydrates
  • 3. Lipids
  • 4. Amino Acids, Peptides, and Proteins
  • 5. Enzymes
  • 6. Hemoglobin
  • 7. Nucleotides and Nucleic Acids2

Eukaryotic Cell StructureCHAPTER 1

 
OVERVIEW
Biochemistry is the study of life on molecular level. Life is based on morphological units known as cells. Cells are the structural and functional units of all living organisms. This chapter offers a survey of the structures and functions of cells. The aim is to provide a biological background for the more detailed mechanisms of biochemistry and molecular biology that follow in subsequent chapters.
 
TYPES OF LIVING CELL
The electron microscope allowed classification of cells into two major groups, prokaryotes and eukaryotes based on the presence and absence of the true nucleus.
  1. Eukaryotes (Greek: Eue = true, karyon = nucleus), which have a membrane enclosed nucleus encapsulating their DNA, (deoxyribonucleic acid). Animals, plants and fungi belong to the eukaryotes. Eukaryotic cells are much larger than prokaryotes. Eukaryotes may be multicellular as well as unicellular, are far more complex than prokaryotes and are characterized by having numerous membrane enclosed organelles (subcellular elements) in their cytoplasm, including:
    • Mitochondria
    • Lysosomes
    • Endoplasmic reticulum
    • Golgi complexes
  2. Prokaryotes have no typical nucleus (Greek: Pro = before) instead consists of nucleoid in which the genome, the complete set of genes, composed of DNA is replicated and stored with its associated proteins. The nucleoid, in bacteria and archaea, is not separated from the cytoplasm by a membrane (Figure 1.1). Bacteria and blue green algae belong to the prokaryotes. Prokaryotes lack membrane enclosed organelles (subcellular elements) in their cytoplasm. Prokaryotes; which comprise the various types of bacteria, have relatively small structures and are invariably unicellular.
Table 1.1 and Figure 1.1 describe some of the major structural features of the prokaryote and eukaryote cells.
Table 1.1   Structural features of prokaryotes and eukaryotes
Organelle
Eukaryotes
Prokaryotes
Nucleus
Present
No define nucleus. DNA present but not separated from rest cell
Plasma membrane
Present
Present
Mitochondria
Present
Absent. Enzymes for oxidation reactions located on plasma membrane
Endoplasmic reticulum
Present
Absent
Ribosomes
Present
Present
Chromosomes
Linear
Circular
Cytoplasm
Contains various membrane bound organelles, such as mitochondria, lysosomes, peroxisomes and Golgi apparatus
Undifferentiated
Reproduction
Mitosis
By binary division
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Figure 1.1: Cell structure of eukaryotic and prokaryotic cell
 
MOLECULAR AND FUNCTIONAL ORGANIZATION OF A EUKARYOTIC CELL
All eukaryotic cells possess characteristic structure and organelles. A cell has three major components.
  1. Plasma membrane (cell membrane).
  2. Cytoplasm with its organelles.
    • Endoplasmic reticulum
    • Golgi apparatus
    • Mitochondria
    • Lysosomes
    • Peroxisomes
  3. Nucleus
Table 1.2 shows biochemical functions of subcellular organelles of the eukaryotic cell.
 
Plasma Membrane
The plasma membrane defines the periphery of the cell, separating its components from the surrounding. It is composed of lipid and protein molecules with small proportion of carbohydrates and forms a thin, tough, pliable hydrophobic barrier around the cell. It is an organized structure consisting of lipid bilayer (Figure 1.2) primarily a phospholipids and penetrated protein molecules forming a mosaic like pattern (Figure 1.3).
  • Each phospholipid molecule consists of head in which phosphoric acid group is located and two thin tails which consists of fatty acid portion (Figures 1.4A and B). The head which is exposed to water is polar and hydrophilic while tail end which is burring is the non-polar and hydrophobic (Figure 1.2).
    Table 1.2   Biochemical functions of subcellular organelles of the eukaryotic cell
    Subcellular organelles
    Function
    Plasma membrane
    Transport of molecules in and out of cell, receptors for hormones and neurotransmitters
    Lysosome
    Intracellular digestion of macromolecules and hydrolysis of nucleic acid, protein, glycosaminoglycans, glycolipids, sphingolipids
    Golgi apparatus
    Post-transcriptional modification and sorting of proteins and export of proteins
    Rough endoplasmic reticulum
    Biosynthesis of protein and secretion
    Nucleus
    Storage of DNA, replication and repair of DNA, transcription and post-transcriptional processing
    Peroxisomes
    Metabolism of hydrogen peroxide and oxidation of long chain fatty acids
    Nucleolus
    Synthesis of rRNA and formation of ribosomes
    Mitochondrion
    ATP synthesis, site for tricarboxylic acid cycle, fatty acid oxidation, oxidative phosphorylation, part of urea cycle and part of heme synthesis
    Smooth endoplasmic reticulum
    Biosynthesis of steroid hormones and phospholipids, metabolism of foreign compounds (cytochrome P450 detoxification)
    Cytosol
    Site for glycolysis, pentose phosphate pathway, part of gluconeogenesis, urea cycle and heme synthesis, purine and pyrimidine nucleotide synthesis
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    Figure 1.2: The basic organization of biological membrane
  • Proteins of the membrane are of two types:
    1. Integral or transmembrane proteins which are either partially or totally immersed in the lipid bilayer.
    2. Peripheral proteins which are attached to the surface of the lipid bilayer.
 
Functions of Plasma Membrane
  • The plasma membrane maintains the physical integrity of the cell by preventing the contents of the cell from leaking into the outside fluid environment and at the same time facilitating the entry of nutrients, inorganic ions and most other charged or polar compounds from the outside.
    zoom view
    Figure 1.3: The fluid mosaic model of cell membrane
    zoom view
    Figures 1.4A and B: Structure of phospholipid: (A) A common glycerophospholipid; (B) Diagrammatic representation of phospholipid
  • The functions of the plasma membrane are coordinated by specialized adhesion receptors called integrins. Integrins are integral transmembrane proteins. Integrins represent important cell receptors that regulate fundamental cellular process; such as attachment, movement, growth and differentiation.
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Cytoplasm and its Organelles
Cytoplasm is the internal volume bounded by the plasma membrane. The clear fluid portion of the cytoplasm in which the organelles are suspended is called cytosol. This contains mainly dissolved proteins, electrolytes and glucose. Five important organelles that are suspended in the cytosol are.
  1. Endoplasmic reticulum
  2. Golgi apparatus
  3. Mitochondria
  4. Lysosomes
  5. Peroxisomes
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Figures 1.5A and B: Structure of endoplasmic reticulum: (A) Rough or granular endoplasmic reticulum; (B) Smooth or a granular endoplasmic reticulum
 
Endoplasmic Reticulum (ER)
The endoplasmic reticulum is the interconnected, folded network of tubular structures in the cytoplasm. A portion of the endoplasmic reticulum has ribosomes bound to it, which give it a rough appearance in contrast with smooth endoplasmic reticulum which is devoid of ribosomes (Figures 1.5A and B).
 
Functions of Endoplasmic Reticulum
  • The rough endoplasmic reticulum is the site for synthesis of proteins that are destined to be exported from the cell. Virtually all integral membrane proteins of the cell, except those located in the membranes of mitochondria are formed by ribosomes bound to the endoplasmic reticulum.
  • The endoplasmic reticulum also has mechanisms for maintaining the quality of the proteins synthesized. The endoplasmic reticulum has three different sensor molecules that monitor the amounts of improperly folded proteins that accumulate.
  • Smooth endoplasmic reticulum is involved in lipid synthesis and contains enzymes termed cytochromes P450 that catalyze hydroxylation of a variety of endogenous and exogenous compounds. These enzymes are important in biosynthesis of steroid hormones and 7removal of toxic substances. Endoplasmic reticulum and Golgi apparatus are involved in formation of other cellular organelles such as lysosomes and peroxisomes.
  • During cell fractionation the endoplasmic reticulum network is disrupted and membrane reseals to form small vesicles called microsomes; which can be isolated by differential centrifugation. Microsomes as such, do not occur in cell.
 
Golgi Apparatus
The Golgi (named for its discoverer Camillo Golgi) apparatus is a flat, membranous sac in which proteins are processed, modified and prepared for export from the cell. It works in association with endoplasmic reticulum, where proteins for certain destinations are synthesized (Figure 1.6).
Functions of Golgi Apparatus: Proteins which are synthesized in the endoplasmic reticulum passed through layers of the Golgi apparatus where enzymes in Golgi membranes catalyze transfer of carbohydrate units to proteins to form glycoproteins or to lipids to make glycolipids, a process that is important in determining the proteins eventual destination. The modified proteins are then sorted, packaged and transported to destination inside or outside the cell. Golgi apparatus plays the role of post office mail sorting room, the mail in this case being newly synthesized proteins.
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Figure 1.6: A Golgi apparatus and its relationship to the endoplasmic reticulum and nucleus
 
Mitochondria (Power House of Cell)
Mitochondria are organelles in eukaryotic cells that supply energy for all cellular metabolic activities. The number of mitochondria in cells varies as do their energy needs. Muscle cells of the heart contain the largest number of mitochondria. Mitochondria are called Power plant of the cell, since they generate most of the cell's energy in the form of ATP.
Erythrocytes are an exception which derive their ATP from glycolysis due to lack of mitochondria. Mitochondria are a double membrane organelle; an outer membrane and an extensive, highly folded inner membrane. The inner membrane is folded into a series of internal ridges called cristae (Figure 1.7). There is two compartments in mitochondria.
  1. The inter membrane space between the outer and the inner membranes.
  2. The matrix, which is bounded by the inner membrane.
The two faces of inner mitochondrial membrane are referred to as the matrix side and the cytoplasmic side. Cytoplasmic side is freely accessible to most small molecules in the cytoplasm. The matrix side and the cytoplasmic side also called the N and P sides respectively, because the membrane potential is negative on the matrix side and positive on the cytoplasmic side.
Mitochondria contain their own DNA, (mtDNA), which in human encodes 13 respiratory chain proteins, as well as small and large ribosomal RNAs and enough tRNAs to translate all codons. However, mitochondria also contain many proteins encoded by nuclear DNA. Mitochondrial DNA is usually described as being circular, but the recent research suggests that the mitochondrial DNA of many organisms may be linear.
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Figure 1.7: Structure of mitochondria
 
Functions of Mitochondria
  • The mitochondrial matrix is the site of most of the reactions of the citric acid cycle and fatty acid oxidation. In contrast oxidative phosphorylation takes place in the inner mitochondrial membrane.
  • The outer membrane is permeable to most small molecules and ions because it contains many mitochondrial porin (pore forming protein) also known as voltage-dependent anion channel (VDAC) that permit access to most molecules. In contrast inner membrane is impermeable to nearly all ions and polar molecules. Many transporters shuttles metabolites such as ATP, pyruvate, and citrate across the inner mitochondrial membrane.
 
Lysosomes
Lysosomes are organelles formed from Golgi Apparatus and dispersed throughout the cytoplasm. The lysosomes are membrane bounded sacs containing hydrolytic enzymes. Lysosomes contain as many as forty different hydrolytic enzymes. The hydrolytic enzymes found in lysosomes include proteases, nucleases, glycosidases, lipases, phosphatases and sulfatases. All these enzymes function at acidic pH, so pH of lysosome matrix is maintain at about 5.
Among all organelles of the cytoplasm, the lysosomes have the thickest covering membrane to prevent the enclosed hydrolytic enzymes from coming in contact with other substances in the cell and therefore prevent their digestive actions. Disruption of the lysosomal membrane within cells leads to cellular digestion. Various pathological conditions such as arthritis, allergic responses, several muscular diseases, and drug induced tissue destruction have been attributed to release of lysosomal enzymes.
 
Functions of Lysosomes
  • Lysosomes are involved in digestion of intra- and extra-cellular substances that must be removed. Substances destined to be degraded are identified and taken up by lysosomes through endocytosis. Products of lysosomal digestion are released from lysosomes and are reutilized by the cell. Indigestible material called residual bodies are removed from the cell by exocytosis.
  • During development lysosomes play an important role in the formation of specialized tissues such as fingers and toes. For example, lysosomes digest the webbed tissues that join fingers and toes in the embryo.
 
Peroxisomes
Peroxisomes (organelles having ability to produce or utilize hydrogen peroxide) are similar to lysosome in that they are membranous sacs containing enzymes. The enzyme content of cellular peroxisome varies according to the need of the tissue. Liver peroxisomes contain three important detoxification enzymes; catalase, urate oxidase and D-amino oxidase.
 
Functions of Peroxisomes
  • Peroxisomes contain enzymes that are used for detoxification rather than for hydrolysis.
  • Peroxisomes also participate in degradation of very long chain fatty acids and synthesis of glycerolipids, plasmalogens and isoprenoids.
  • In peroxisomes a number of molecules which are not metabolized elsewhere are oxidized by enzymes by using molecular oxygen directly and produce hydrogen peroxide (H2O2). Hydrogen peroxide is destroyed further by catalase and peroxidases. By having both peroxide producing and peroxide utilizing enzymes in one compartment, cells protect themselves from the toxicity of hydrogen peroxide.
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Nucleus
Nucleus is the control center of the cell; it contains the DNA organized into chromosomes which carry genetic information. The nucleus is surrounded by a double membrane called nuclear envelope the outer membrane is fused with the endoplasmic reticulum at multiple sites. Nuclear pores occur at points where the outer and inner membranes are connected (Figure 1.8). Nuclear pores facilitate transport of molecules between the cytoplasm and the nucleus.
The space enclosed by the nuclear envelope is called nucleoplasm; within this the nucleolus is present. Nucleolus is an organized structure of DNA, RNA and protein. Nucleolus is a major site of RNA synthesis and the site of assembly of ribosome.
The remaining nuclear DNA is dispersed throughout the nucleoplasm in the form of chromatin fibers). Chromatins are complexes of DNA with specific proteins such as histones. In the nucleus these chromatin fibers are associated with nuclear lamina, a fibrous network made of three proteins, A, B, and C; lying beneath the inner nuclear membranes.
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Figure 1.8: General structure of nucleus
At mitosis chromatin is condensed into discrete structures called chromosomes. The organization of the nuclear envelope, nucleolus, and chromatin is shown in Figure 1.8.
 
Functions of Nucleus
The major functional role of the nucleus is that of replication, synthesis of new DNA and transcription, synthesis of rRNA, tRNA and mRNA. All of the RNA molecules operate functionally outside the nucleus and seem to leave via the nuclear pores.
 
CYTOSKELETON
The cytoplasm of most eukaryotic cells contains network of several types of proteins filaments that interact extensively with each other and with the component of the plasma membrane forming three dimensional meshwork. Such an extensive intracellular meshwork of protein has been called cytoskeleton. Cytoskeleton is not a rigid permanent framework of the cell but is a dynamic, changing structure.
 
Functions of Cytoskeleton
  • The cytoskeleton gives cells their characteristic shape and form, provides attachment points for organelles, fixing their location in cells and also makes communication between parts of the cell possible.
  • It is also responsible for the separation of chromosomes during cell division.
  • The internal movement of the cell organelles as well as cell locomotion and muscle fiber contraction could not take place without the cytoskeleton. It acts as track on which cells can move organelles, chromosomes and other things.
 
Structure of Cytoskeleton
The cytoskeleton is an organized network of three protein filaments; Microfilaments, microtubules and intermediate filaments, differing in width, composition and specific function.
  • Microfilaments consist of long thin strands of protein actin, which is also a main component of muscle. Actin filament form a meshwork just underlying the plasma membrane of many cells and are referred to stress fiber or cell cortex which is labile. They disappear as cell motility increases or upon malignant transformation of cells by chemical or oncogenic viruses.
  • Microtubules are long, thin tubes composed of the protein tubulin. They rapidly assemble into tubular structures and disassemble depending on the needs of cells. Microtubules comprise the spindle fibers that separate chromosomes prior to cell division.
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    Table 1.3   Types of cytoskeleton and their proteins
    Cytoskeleton protein
    Types of protein present
    Microfilaments
    Actin filament
    Microtubules
    Tubulin
    Intermediate filament
    Keratin
    Vimentin
    Desmin
    Glial fibrillary acidic protein (GFAP)
    Peripherin
    Neurofilament
    Lamins
    Centrioles are composed of microtubules and function as the organizing center for the formation of spindle fibers.
  • Intermediate filaments are so-called as their diameter is intermediate between that of microfilaments and of microtubules. These are formed from fibrous protein which cannot be easily disassembled as either the microtubules or the microfilaments can, except lamin. Protein structure of intermediate filaments varies with different tissue type. There are major seven classes of intermediate filaments as indicated in Table 1.3.
 
CELL FRACTIONATION
Investigation of the biochemical properties of organelles requires subcellular fractionation in which the cell is disrupted by mechanical means and the organelles are purified. To obtain purified preparations of organelles the tissue is first carefully broken up in a homogenizing apparatus using isotonic 0.25 M sucrose solution. Before homogenization the tissue is coarsely minced using buffer to maintain the pH at its optimum value for organelle stability. Sucrose solution is used because it is not metabolized in most tissues and it does not pass through membranes readily and thus does not cause inter organelles to swell.
By gently homogenization in an isotonic sucrose solution the cell membrane is ruptured by the shearing forces developed by the rotating homogenizer pestle; keeping most of the internal organelles intact. However, large fragile structures such as the endoplasmic reticulum, is broken into pieces that spontaneously form vesicles called microsomes.
  • Homogenate is strained to remove connective tissue and fragments of blood vessels by a stainless steel sieve.
  • Then homogenate is centrifuged at a series of increasing centrifugal force. In differential centrifugation, the homogenate is subjected to a series of centrifugation steps of increasing time and gravitational force (Flow chart 1.1). The subcellular organelles, e.g. nuclei and mitochondria, which differ in size and specific gravity and thus sediment at different rates in a centrifugal field and can then, be isolated from homogenate by differential centrifugation.
    zoom view
    Flow chart 1.1: Subcellular fractionation of cell by differential centrifugation
    The dense nuclei are sediment first, followed by the mitochondria, and finally the microsomal fraction at the highest forces. After, all the particulate matter has been removed; the soluble remnant is the cytosol.
  • Organelles of similar sedimentation coefficient obviously cannot be separated by differential centrifugation. For example, mitochondria isolated in his way are contaminated with lysosome and peroxisomes. These may be separated by isopyknic centrifugation technique.
 
Isopyknic Centrifugation Technique
In this technique, a density gradient is set up in a centrifuge tube; i.e. the density of the solution in the tube increases from the top to the bottom. Sucrose is often used as a medium. Colloidal materials such as Percoll, which form density gradients with a low osmotic pressure, are often preferred.
Particles are sediment to an equilibrium position at which their density equals that of the medium at that point in the tube (Figure 1.9). Different organelles are thus separated according to their density, their size and shape being immaterial.
After centrifugation to equilibrium, the gradient is fractionated and the separated organelles recovered. Macromolecules, such as large proteins, nucleic acids and nucleoprotein complexes can also be separated by density gradient centrifugation technique.
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Figure 1.9: Separation of organelles by isopyknic centrifugation technique
 
MARKER ENZYMES
The purity of isolated subcellular fraction is assessed by the analysis of marker enzymes. Marker enzymes are the enzymes that are located exclusively in a particular fraction, and thus become characteristic of that fraction.
Analysis of marker enzymes confirms the identity of the isolated fraction and indicates the degree of contamination with other organelles. For example, isolated mitochondria have a high specific activity of cytochrome oxidase but low catalase and acid phosphatase, the catalase and acid phosphatase activities being due to contamination with peroxisomes and lysosomes respectively. Some typical subcellular markers are given in Table 1.4.
Table 1.4   Marker enzymes of subcellular fractions
Fraction
Enzymes
Plasma membrane
5 Nucleotidase, Na+-K+-ATPase
Nucleus
DNA polymerase
RNA polymerase
Endoplasmic reticulum
Glucose-6-phosphatase
Golgi bodies
Galactosyl transferase
Lysosomes
Acid phosphatase
β-glucuronidase
Mitochondria
Succinate dehydrogenase
Cytochrome C oxidase
Peroxisomes
Catalase
Cytosol
Lactate dehydrogenase
Glucose-6-phosphate dehydrogenase
 
ASSESSMENT QUESTIONS
 
Short Notes
1. Diagrammatic representation of cell with functions of the subcellular organelles.
2. Give structure and function of:
  1. Mitochondria
  2. Endoplasmic reticulum
  3. Golgi apparatus
  4. Plasma membrane
  5. Nucleolus
  6. Lysosomes
  7. Peroxisomes.
 
Multiple Choice Questions (MCQs)
1. The following is the metabolic function of ER:
  1. RNA processing
  2. Fatty acid oxidation
  3. Synthesis of plasma protein
  4. ATP-synthesis
2. In biologic membranes, integral proteins and lipids interact mainly by:
  1. Covalent bond
  2. Both hydrophobic and covalent bond
  3. Hydrogen and electrostatic bond
  4. None of the above
3. Plasma membrane is:
  1. Composed entirely of lipids
  2. Mainly made up of proteins
  3. Mainly made up of lipid and protein
  4. Composed of only carbohydrates and lipids
4. Select the subcellular component involved in the formation of ATP:
  1. Nucleus
  2. Plasma membrane
  3. Mitochondria
  4. Golgi apparatus
5. Mitochondrial DNA is:
  1. Maternal inherited
  2. Paternal inherited
  3. Maternal and paternal inherited
  4. None of the above
6. All of the following statements about the nucleus are true, except:
  1. Outer nuclear membrane is connected to ER
  2. It is the site of storage of genetic material
  3. Nucleolus is surrounded by a bilayer membrane
  4. Outer and inner membranes of nucleus are connected at nuclear pores
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7. Golgi apparatus is present in all of the following, except:
  1. RBC
  2. Parenchymal cells
  3. Skeletal muscle cells
  4. Pancreatic cell
8. Peroxisomes arise from:
  1. Golgi membrane
  2. Lysosomes
  3. Mitochondria
  4. Pre-existing peroxisomes and budding off from the smooth ER
9. Na+ - K+ ATPase is the marker enzyme of:
  1. Nucleus
  2. Plasma membrane
  3. Golgi bodies
  4. Cytosol
10. Exocytosis:
  1. Is always employed by cells for secretion
  2. Is used to deliver material into the extracellular space
  3. Take up large molecules from the extracellular space
  4. Allows the salvage of elements of the plasma membrane
11. The approximate number of cells in a normal human body is:
  1. 10
  2. 100
  3. 1014
  4. 10144
12. The largest cell in the human body is:
  1. Nerve cell
  2. Muscle cell
  3. Liver cell
  4. Kidney cell
13. Which one of the following eukaryotic cell structures does not contain DNA?
  1. Nucleus
  2. Mitochondrion
  3. The endoplasmic reticulum
  4. Chloroplast
14. The cytoskeleton includes all of the following, except:
  1. Microtubules
  2. Intermediate filaments
  3. Myosin filaments
  4. Action filaments
15. Ribosomes are found:
  1. Only in the nucleus
  2. In the cytoplasm
  3. Attached to the smooth endoplasmic reticulum
  4. Both b and c
16. The Golgi apparatus is involved in:
  1. Packaging proteins into vesicles
  2. Altering or modifying proteins
  3. Producing lysosomes
  4. All of the above
17. All of the following are functions of the cell membrane, except:
  1. Participating in chemical reactions
  2. Participating in energy transfer
  3. Being freely permeable to all substances
  4. Regulating the passage of materials
18. Who proposed the fluid mosaic model of cell membrane structure in 1972?
  1. Davson and Singer
  2. Frye and Edidin
  3. Brown and Goldstein
  4. Singer and Nicholson
19. Which of the following are involved with the movement or transport of materials or organelles throughout the cell?
  1. Rough endoplasmic reticulum
  2. Cytoskeleton
  3. Smooth endoplasmic reticulum
  4. All of the choices are true
20. Lysosomes are produced by the:
  1. Nucleus
  2. Mitochondria
  3. Golgi apparatus
  4. Ribosomes
Answers for MCQs
1. c
2. c
3. c
4. c
5. a
6. c
7. a
8. d
9. b
10. b
11. c
12. a
13. c
14. c
15. d
16. d
17. c
18. d
19. d
20. c