Items 6-12

The following set of items pertains to cellular membranes and their biochemical composition.

6. The fluid mosaic model of biological membranes states that

(A) All membranes have a core, made up of globular protein subunits surrounded by multiple layers of hydrophobic phospholipids.
(B) Membranes appear to have repeating mosaic patterns because their lipids form micelles that are cross-linked together by a protein backbone.
(C) Membrane proteins can move about within the plane of the membrane because the lipids which from the core of the membrane can diffuse relatively freely with respect to adjacent lipid molecules.
(D) Transfer of molecules across the membranes usually occurs through channel-lined by phospholipids – that form a repeating, or mosaic, pattern.
(E) Protiens can only associate with the lipid core of the membranes by forming covalent linkages with the fatty-acid side chains of constituent phospholipids molecules.

7. Which of the following is TRUE with regard to membrane proteins?

(A) Protiens associated with the plasma membranes of cells are highly hydrophobic.
(B) Membrane proteins that are in contact with the extracellular matrix are rarely if ever glycosylated.
(C) Transmembrane channels, through which charged molecules may pass, are frequently formed by self-associated with the extracellular face of the plasma membrane can usually only be released from the membrane by treatment with proteolytic enzymes.

8. Which term is most accurately used to characterize the interior of the lipid bilayer of the plasma membrane?

(A) hydrophilic
(B) acidophilic
(C) basophilic
(D) amphipathic
(E) hydrophobic

9. All of the following are intergral membrane proteins of the erythrocyte membrane EXCEPT:

(A) glycophorin
(B) band 3 protien
(C) spectrin
(D) glucose translocase
(E) Na+ -K+ ATPase

10. Membrane lipids have all of the following characteristics EXCEPT:

(A) absence of charged groups
(B) amphipathic
(C) fatty acids esterified to glycerol
(D) convalently bound oligosaccharides
(E) Extensive hydrophobic domains

11. All of the following statement concerning membrane composition are true EXCEPT:

(A) Plasma membranes have more lipid than do mitochondrial inner membranes.
(B) Intracellular membranes have less protein than does myelin.
(C) Cholesterol is present in plasma membrane and intracellular membranes.
(D) Cholesterol composition affects membrane fluidity.
(E) Cell membranes have no free carbohydrates moieties.

12. Which of the following membranes has the highest ratio of lipid-to-protien?

(A) red cell membrane
(B) myelin
(C) Golgi membranes
(D) Rough endoplasmic reticulum
(E) Other mitochondrial membrane

ANSWERS AND TUTORIAL ON ITEMSM 6-12

The answers are: 6-C; 7-D; 8-E; 9-10; A; 11-B; 12-B. Cellular membranes are lipid-protein bilayers, consisting of a core bilayer of lipids with which various proteins are associated. The hydrophobic tails of the amphipathic lipids forming the core of the bilayer associate with the another by hydrophobic interactions. These core lipids are weakly associated with one another within the plane of the membrane; hence, while the bilayer membrane strongly resists separation in a direction perpendicular to the membrane plane,lipid-lipid and lipid-protien associations are relatively labile (fluid) within the plane of the membrane.

Proteins that are specifically associated with membranes may be loosely bound, bound firmly via electrostatic interactions, or so tightly bound that they can only be released by treatment with detergents that completely dissolve the membrane. Proteins that can only be released by detergent treatment are referred to as integral membrane proteins. In many cases, such integral membrane proteins extend from one side of the membrane to the other side. Such transmembrane proteins are frequently responsible for selective transport of specific hydrophilic molecules across the membrane, thus facilitating passage of such molecules through the hydrophobic lipid bilayer core. The plasma membrane surrounds each cell and is the boundary between the external and the internal environments of the cell.

The concept of protein turnover is hardly 60 years old. Beforehand, body proteins were viewed as essentially stable constituents that were subject to only minor ‘wear and tear’: dietary proteins were believed to function primarily as energy-providing fuel, which were independent from the structural and functional proteins of the body. The problem was hard to approach experimentally, as research tools were not available. An important research tool that was lacking at that time were stable isotopes. The concept that body structural proteins are static and the dietary proteins are used only as a fuel was challenged by Rudolf Schoenheimer. Schoenheimer escaped from Germany and joined the Department of Biochemistry in Columbia University founded by Hans T. Clarke. There he met Harold Urey who was working in the Department of Chemistry and who discovered deuterium, the heavy isotope of hydrogen, a discovery that enabled him to prepare heavy water, D2O. David Rittenberg who had recently received his Ph.D. in Urey’s laboratory, joined Schoenheimer, and together they entertained the idea of “employing a stable isotope as a label in organic compounds, destined for experiments in intermediary metabolism, which should be biochemically indistinguishable from their natural analog.” Urey later succeeded in enriching nitrogen with 15N, which provided Schoenheimer and Rittenberg with a “tag” for amino acids and as a result for the study of protein dynamics. They discovered that following administration of 15N-labeled tyrosine to rat, only ~50% was recovered in the urine, “while most of the remainder is deposited in tissue proteins. An equivalent of protein nitrogen is excreted.’” They further discovered that from the half that was incorporated into body proteins “only a fraction was attached to the original carbon chain, namely to tyrosine, while the bulk was distributed over other nitrogenous groups of the proteins,” mostly as an NH2 group in other amino acids. These experiments demonstrated unequivocally that the body structural proteins are in a dynamic state of synthesis and degradation, and that even individual amino acids are in a state of dynamic interconversion. Similar results were obtained using 15N-labeled leucine.5 This series of findings shattered the paradigm in the field at that time that: (1) ingested proteins are completely metabolized and the products are excreted, and (2) that body structural proteins are stable and static.

Schoenheimer was invited to deliver the prestigious Edward K. Dunham lecture at Harvard University where he presented his revolutionary findings. After his untimely tragic death in 1941, his lecture notes were edited Hans Clarke, David Rittenberg and Sarah Ratner, and were published in a small book by Harvard University Press. The editors called the book “The Dynamic State of Body Constituents,” adopting the title of Schoenheimer’s presentation. In the book, the new hypothesis is clearly presented: “The simile of the combustion engine pictured the steady state flow of fuel into a fixed system, and the conversion of this fuel into waste products. The new results imply that not only the fuel, but the structural materials are in a steady state of flux. The classical picture must thus be replaced by one which takes account of the dynamic state of body structure.” However, the idea that proteins are turning over was not accepted easily and was challenged as late as the mid-1950s.

For example, Hogness and colleagues studied the kinetics of ß-galactosidase in E coli and summarized their findings: “To sum up: there seems to be no conclusive evidence that the protein molecules within the cells of mammalian tissues are in a dynamic state. Moreover, our experiments have shown that the proteins of growing E. coli are static. Therefore it seems necessary to conclude that the synthesis and maintenance of proteins within growing cells is not necessarily or inherently associated with a ‘dynamic state.’” While the experimental study involved the bacterial ß-galactosidase, the conclusions were broader, including also the authors’ hypothesis on mammalian proteins. The use of the term ‘dynamic state’ was not incidental, as they challenged directly Schoenheimer’s studies.

Now, after more then six decades of research in the field and with the discovery of the lysosome and later the complex ubiquitin-proteasome system with its numerous tributaries, it is clear that the area has been revolutionized. We now realize that intracellular proteins are turning over extensively, that this process is specific, and that the stability of many proteins is regulated individually and can vary under different conditions. From a scavenger, unregulated and non-specific end process, it has become clear that proteolysis of cellular proteins is a highly complex, temporally controlled and tightly regulated process that plays major roles in a broad array of basic pathways. Among these processes are cell cycle, development, differentiation, regulation of transcription, antigen presentation, signal transduction, receptor-mediated endocytosis, quality control, and modulation of diverse metabolic pathways. Subsequently, it has changed the paradigm that regulation of cellular processes occurs mostly at the transcriptional and translational levels, and has set regulated protein degradation in an equally important position. With the multitude of substrates targeted and processes involved, it is not surprising that aberrations in the pathway have been implicated in the pathogenesis of many diseases, among them certain malignancies, neurodegeneration, and disorders of the immune and inflammatory system. As a result, the system has become a platform for drug targeting, and mechanism-based drugs are currently developed, one of them is already on the market.

Deep within cells, there are other (intracellular) membranous systems, including the rough and smooth endoplasmic reticulum, the Golgi apparatus and the nuclear envelope. Individual intracellular organelles such as lysosomes are also surrounded by membranes. Mitochondria have an outer membrane which compartmentalizes the organelle from the cytoplasm. The outer mitochondrial membrane surrounds an inner mitochondrial membrane.

The red cell membrane is on of the most well characterized plasma membranes. In general, integral membrane proteins span the entire thickness of the plasma membrane and can not released from their associated lipids without strong detergent treatment or solvent extraction of membrane. They often have carbohydrate rich portion on the external surface, a middle region rich in hydrophobic amino acids which interact strongly with the hydrophobic domain of membrane lipids and a second hydrophobic domain on the internal (cytoplasmic) surface. The inner hydrophilic domain often interacts with cytoskeletan proteins in the cytoplasmic domain. In the red blood cell, glycophorin is an intergral membrane glycoprotein of unknown function. Band 3 is another intergral membrane protein. It interacts strongly with the cytoskeletal protein ankyrin and may be involved in facilitating diffusion of anions across the plasma membrane.Glucose translocase in an integral membrane protein that actively transports glucose into red blood cells. Spectrin is a cytoplasmic protein that interacts with both gylcophobirin and band 3 protein; spctrin is thought to have a cytoskeletal function, strengthening the cortex of erythrocytes. Ankyrin stablizes the interaction between spectrin and band 3 protein.

Membrane lipids consist largely of phosphoglycerides, sphingomyelin, cholesterol and “other” lipids, e.g., carbohydrates-containing glycolipids such as cerebrosides and gangliosides. The phosphoglycerides, sphingomyelin and glycolipids, are extended amphipathic molecules with hydrophilic charged portions containing phosphate and other water soluble moieties such as choline, ethanolamine, serine and inositol esterified to long, hydrophobic, aliphatic hydrocarbon chains. The amphipathic membrane lipids form a bilayer where the polar heads of one layer interact with the aqueous external milineu and the polar heads of the other half of the bilayer interact with the aqueous cytoplasmic environment. The hydeophobic portions of each half of the bilayer face one another and interact strongly with one another, with bcholesterol and with the hydeophobic portions of integral membrane proteins.

Cholesterol is a compact, hydrophobic, planar compound. It is an important constituent of many membranes even though it is less abundant that phosphogylcerides in the plasma membrane. Cholesterol plays a significant role in determining membrane fluidity. At physiological concentrations, cholesterol limits membrane fluidity. Abnormally high plasma membrane cholesterol levels significantly decrease membrane fluidity. In red blood cells, when the membrane fluidity decrease, these cells become more susceptible to splenic destruction, leading to anemia.

Different cellular membranes have striking differences in their chemical composition. For example, the myelin sheath of nerve fibers (a highly modified plasma membrane) is about 80% lipids and 20% protein. The high lipid content of myelin relates to its insulation function in nerve conduction. In contracts, the inner mitochondrial membrane is about 80% protein and 20% lipid. Here, there are large numbers of enzymes dedicated to electron transport. Most other plasma membranes are just under 50% lipid and just over 50% protein. Similarly, the lipid compositon of different intracellular membranes varies considerably. professional-high-power microscope and cell observation microscope/ cell dissection microscope

The Lysosome and Intracellular Protein Degradation

In the mid-1950s, Christian de Duve discovered the lysosome. The lysosome was first recognized biochemically in rat liver as a vacuolar structure that contains various hydrolytic enzymes which function optimally at an acidic pH. It is surrounded by a membrane that endows the contained enzymes latency that is required to protect the cellular contents from their action. The definition of the lysosome has been broadened over the years. This is because it has been recognized that the digestive process is dynamic and involves numerous stages of lysosomal maturation together with the digestion of both exogenous proteins (which are targeted to the lysosome through receptor-mediated endocytosis and pinocytosis) and exogenous particles (which are targeted via phagocytosis; the two processes are known as heterophagy), as well as digestion of endogenous proteins and cellular organelles (which are targeted by micro- and macroautophagy). The lysosomal/vacuolar system as we currently recognize it is a discontinuous and heterogeneous digestive system that also includes structures that are devoid of hydrolases—for example, early endosomes which contain endocytosed receptor-ligand complexes and pinocytosed/phagocytosed extracellular contents. On the other extreme it includes the residual bodies—the end products of the completed digestive processes of heterophagy and autophagy. In between these extremes one can observe: primary/nascent lysosomes that have not yet been engaged yet in any proteolytic process; early autophagic vacuoles that might contain intracellular organelles; intermediate/late endosomes and phagocytic vacuoles (heterophagic vacuoles) that contain extracellular contents/particles; and multivesicular bodies (MVBs) which are the transition vacuoles between endosomes/phagocytic vacuoles and the digestive lysosomes.