By G. Vibald. Case Western Reserve University.

This condensation reduces the acetyl CoA levels generic 10 mg toradol with visa, which leads to the activation of pyruvate dehydrogenase and inhi- bition of pyruvate carboxylase toradol 10mg with amex. Through such reciprocal regulation, citrate can be continuously synthesized and transported across the inner mitochondrial membrane. In the cytosol, citrate is cleaved by citrate lyase to re-form acetyl CoA and oxaloac- etate. This circuitous route is required because pyruvate dehydrogenase, the enzyme that converts pyruvate to acetyl CoA, is found only in mitochondria and because acetyl CoA cannot directly cross the mitochondrial membrane. The NADPH required for fatty acid synthesis is generated by the pentose phos- phate pathway (see Chapter 29) and from recycling of the oxaloacetate produced by citrate lyase (Fig. Oxaloacetate is converted back to pyruvate in two steps: the reduction of oxaloacetate to malate by NAD -dependent malate dehydrogenase and the oxidative decarboxylation of malate to pyruvate by an NADP+-dependent malate dehydrogenase (malic enzyme) (Fig. The pyruvate formed by malic enzyme is reconverted to citrate. The NADPH that is generated by malic enzyme, along with the NADPH generated by glucose 6-phosphate and gluconate 6-phos- phate dehydrogenases in the pentose phosphate pathway, is used for the reduction reactions that occur on the fatty acid synthase complex (Fig. The generation of cytosolic acetyl CoA from pyruvate is stimulated by elevation of the insulin/glucagon ratio after a carbohydrate meal. Insulin activates pyruvate dehydrogenase by stimulating the phosphatase that dephosphorylates the enzyme to Glucose CO2 NADPH NADP+ Pyruvate malic enzyme Malate – COO NAD+ + Pyruvate cytosolic NADP CO2 NADPH malate CH2 CH3 dehydrogenase NADH H OH C O malic enzyme OAA Acetyl CoA citrate OAA Acetyl CoA – – COO COO lyase Malate Pyruvate ADP + Pi Citrate Citrate ATP Fig. Citrate lyase is also called citrate cleavage enzyme. NADPH is produced by the pentose phosphate pathway and by malic enzyme. The synthesis of malic enzyme, glucose 6-phosphate acetyl CoA ADP + P carboxylase i dehydrogenase, and citrate lyase is induced by the high insulin/glucagon ratio. The ability of citrate to accumulate, and leave the mitochondrial matrix for the synthe- sis of fatty acids, is attributable to the allosteric inhibition of isocitrate dehydroge- O O nase by high energy levels within the matrix under these conditions. The concerted – CH C ~SCoA regulation of glycolysis and fatty acid synthesis is described in Chapter 36. Reaction catalyzed by acetyl CoA Cytosolic acetyl CoA is converted to malonyl CoA, which serves as the immediate carboxylase. CO2 is covalently attached to donor of the 2-carbon units that are added to the growing fatty acid chain on the biotin, which is linked by an amide bond to the fatty acid synthase complex. To synthesize malonyl CoA, acetyl CoA carboxylase -amino group of a lysine residue of the adds a carboxyl group to acetyl CoA in a reaction requiring biotin and adenosine enzyme. Hydrolysis of ATP is required for the triphosphate (ATP) (Fig. Acetyl CoA carboxylase is the rate-limiting enzyme of fatty acid synthesis. Its activity is regulated by phosphorylation, allosteric modification, and induction/ repression of its synthesis (Fig. Citrate allosterically activates acetyl CoA carboxylase by causing the individual enzyme molecules (each composed of 4 sub- units) to polymerize. Palmityl CoA, produced from palmitate (the endproduct of AMP is a much more sensitive indi- fatty acid synthase activity), inhibits acetyl CoA carboxylase. Phosphorylation by cator of low energy levels because an AMP-dependent protein kinase inhibits the enzyme in the fasting state when of the adenylate kinase reaction. The enzyme is activated by dephosphorylation in the fed state The [AMP] to [ATP] ratio is proportional to when energy and insulin levels are high. A high insulin/glucagon ratio also results the square of the [ADP] to [ATP] ratio, so a in induction of the synthesis of both acetyl CoA carboxylase and the next enzyme fivefold change in ADP levels corresponds to in the pathway, fatty acid synthase. Fatty Acid Synthase Complex As an overview, fatty acid synthase sequentially adds 2-carbon units from malonyl CoA to the growing fatty acyl chain to form palmitate. After the addition of each 2-carbon unit, the growing chain undergoes two reduction reactions that require NADPH. CHAPTER 33 / SYNTHESIS OF FATTY ACIDS, TRIACYLGLYCEROLS, AND THE MAJOR MEMBRANE LIPIDS 599 Glucose Citrate Insulin + phosphatase Acetyl CoA Pi + acetyl CoA carboxylase–P acetyl CoA carboxylase (inactive) – ADP ATP AMP-activated Malonyl CoA protein kinase Palmitate Palmitoyl CoA Fig.

POLYMERASE CHAIN REACTION (PCR) PCR is an in vitro method that can be used for rapid production of very large amounts of specific segments of DNA order toradol 10 mg otc. It is particularly suited for amplifying regions of DNA for clinical or forensic testing procedures because only a very small Although only small amounts of sample of DNA is required as the starting material purchase 10mg toradol with amex. Regions of DNA can be ampli- semen were obtained from Vicky fied by PCR from a single strand of hair or a single drop of blood or semen. Tim’s body, the quantity of DNA in First, a sample of DNA containing the segment to be amplified must be iso- these specimens could be amplified by PCR. Large quantities of primers, the four deoxyribonucleoside triphosphates, This technique provided sufficient amounts and a heat-stable DNA polymerase are added to a solution in which the DNA is of DNA for comparison with DNA samples heated to separate the strands (Fig. The primers are two synthetic oligonu- from the three suspects. After multiple heating and cooling cycles, the original strands remain, but most of the DNA consists of amplified copies of the segment (shown in lighter blue) synthesized by the heat-stable DNA polymerase. As the solution is cooled, the oligonucleotides form base pairs with the DNA and serve as primers for the synthesis of DNA strands The DNA polymerase used for PCR by the heat-stable DNA polymerase. The process of heating, cooling, and new is isolated from Thermus aquati- DNA synthesis is repeated many times until a large number of copies of the DNA cus, a bacterium that grows in hot are obtained. The process is automated, so that each round of replication takes springs. This polymerase can withstand the only a few minutes and in 20 heating and cooling cycles, the DNA is amplified heat required for separation of DNA strands. USE OF RECOMBINANT DNA TECHNIQUES FOR DIAGNOSIS OF DISEASE A. DNA Polymorphisms Polymorphisms are variations among individuals of a species in DNA sequences of the genome. They serve as the basis for using recombinant DNA techniques in the diagnosis of disease. The human genome probably contains millions of different polymorphisms. Some polymorphisms involve point mutations, the substitution of one base for another. Deletions and insertions are also responsi- ble for variations in DNA sequences. Some polymorphisms occur within the cod- ing region of genes. Others are found in noncoding regions closely linked to genes involved in the cause of inherited disease, in which case they can be used as a marker for the disease. CHAPTER 17 / USE OF RECOMBINANT DNA TECHNIQUES IN MEDICINE 307 B. Detection of Polymorphisms The mutation that causes sickle cell anemia abolishes a restriction site 1. RESTRICTION FRAGMENT LENGTH POLYMORPHISMS for the enzyme MstII in the -globin gene. The consequence of this mutation is Occasionally, a point mutation occurs in a recognition site for one of the restric- that the restriction fragment produced by tion enzymes. The restriction enzyme therefore can cut at this restriction site in MstII that includes the 5 -end of the -globin DNA from most individuals, but not in DNA from individuals with this mutation. Mutations also can create restriction sites that are not commonly fragments provides a direct test for the muta- present. In this case, the restriction fragment from this region of the genome will tion. In Will Sichel’s case, both alleles for - be smaller for a person with the mutation than for most individuals. These vari- globin lack the MstII site and produce 1. Carriers have both a normal and a mutant In some cases, the mutation that causes a disease affects a restriction site within allele.

In normal subjects cheap 10mg toradol, approximately 55% of this intestinal pool enters the blood through the enterocyte each day toradol 10mg on line. The details of cholesterol absorption from dietary sources was outlined in Chapter 32. Although the absorption of cholesterol from the intestinal lumen is a diffusion- controlled process, there is also a mechanism to remove unwanted or excessive cholesterol and plant sterols from the enterocyte. The transport of sterols out of the enterocyte, and into the lumen, is related to the products of genes that code for the adenosine triphosphate (ATP)-binding cassette (ABC) protein family, ABC1, ABCG5, and ABCG8. These proteins couple ATP hydrolysis to the transport of unwanted or excessive cholesterol and plant sterols (phytosterols) from the entero- cyte back into the gut lumen. Cholesterol cannot be metabolized to CO2 and water and is, therefore, principally eliminated from the body in the feces as unreabsorbed sterols and bile acids. ABC protein expression increases the amount of sterols pres- ent in the gut lumen, with the potential to increase elimination of the sterols into the feces. Patients with a condition known as phytosterolemia (a rare autosomal recessive disease, also known as sitosterolemia) have a defect in the function of either ABCG5 or ABCG8 in the enterocytes, thereby leading to the accumulation of cholesterol and phytosterols within these cells. These eventually reach the bloodstream, markedly elevating the level of cholesterol and phytosterol in the blood. This accounts for the increased cardiovascular morbidity in individuals with this disorder. From these experiments of nature, it is clear that agents that either amplify the expression of the ABC proteins within enterocytes, or block choles- terol absorption from the lumen, have therapeutic potential in the treatment of patients with hypercholesterolemia. Ezetimibe, now available for clinical use, is a compound that is structurally different from the sterols. Its primary action in low- ering serum cholesterol levels is to block cholesterol absorption through a specific but as yet poorly characterized cholesterol absorption mechanism in the brush bor- der of enterocytes. It also may induce ABC protein expression, but this action is relatively unimportant in reducing net cholesterol absorption. The reduction of cholesterol absorption from the intestinal lumen has been shown to reduce blood levels of LDL cholesterol. CHOLESTEROL SYNTHESIS 1 9 2 10 14 15 8 Cholesterol is an alicyclic compound whose basic structure includes the perhy- A drocyclopentanophenanthrene nucleus containing four fused rings (Figure 34. The basic ring structure of sterols; the hydrocarbon chain attached to carbon 17 in the D ring, a methyl group (carbon perhydrocyclopentanophenanthrene nucleus. CHAPTER 34 / CHOLESTEROL ABSORPTION, SYNTHESIS, METABOLISM, AND FATE 623 21 22 24 26 O 20 25 23 CH3 C SCoA 18 17 Acetyl CoA 27 O CH3 C SCoA 19 CoA-SH O O 3 CH C CH C SCoA 3 2 HO Acetoacetyl CoA Fig. O HMG-CoA synthase CH3 C SCoA Approximately one third of plasma cholesterol exists in the free (or unesterified) CoA-SH form. The remaining two thirds exists as cholesterol esters in which a long-chain O fatty acid (usually linoleic acid) is attached by ester linkage to the hydroxyl group C O– at C-3 of the A ring. The proportions of free and esterified cholesterol in the blood β-hydroxy- CH2 can be measured using methods such as high-performance liquid chromatography β-methyl- CH3 C OH glutaryl CoA (HPLC). C Acetyl CoA can be obtained from several sources, including the beta oxidation of O SCoA fatty acids, the oxidation of ketogenic amino acids, such as leucine and lysine, and 2NADPH + 2H+ the pyruvate dehydrogenase reaction. Carbons 1, 2, 5, 7, 9, 13, 15, 18, 19, 20, 22, HMG-CoA 2NADP+ 24, 26, and 27 of cholesterol are derived from the methyl group of acetyl CoA and reductase CoA-SH the remaining 12 carbons of cholesterol from the carboxylate atom of acetyl CoA. The synthesis of cholesterol requires significant reducing power, which is sup- O plied in the form of NADPH. The latter is provided by glucose-6-phosphate dehy- C O– drogenase and 6-phosphogluconate dehydrogenase of the hexose monophosphate CH 2 shunt pathway (see Chapter 29). Cholesterol synthesis occurs in the cytosol, requir- CH3 C OH ing hydrolysis of high-energy thioester bonds of acetyl CoA and phosphoanhydride CH2 bonds of ATP. Stage 1: Synthesis of Mevalonate from Acetyl CoA Mevalonate The first stage of cholesterol synthesis leads to the production of the intermediate Fig. The synthesis of mevalonate is the committed, rate-limiting of acetyl-CoA to mevalonic acid. In this cytoplasmic pathway, two molecules of acetyl CoA condense, forming acetoacetyl CoA, which then condenses with a third mole- cule of acetyl CoA to yield the 6-carbon compound -hydroxy- -methylglutaryl- CoA (HMG-CoA). The HMG-CoA synthase in this reaction is present in the cytosol and is distinct from the mitochondrial HMG-CoA synthase that catalyses HMG-CoA synthesis involved in ketone body production. The committed step and Ann Jeina’s serum total and LDL major point of regulation of cholesterol synthesis in stage 1 involves reduction of cholesterol levels improved only HMG-CoA to mevalonate, a reaction catalyzed by HMG-CoA reductase, an enzyme modestly after 3 months on a Step I embedded in the membrane of the endoplasmic reticulum.

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