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11.4: Glycolysis - Biology

11.4: Glycolysis - Biology


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Learning Objectives

By the end of this section, you will be able to:

  • Explain how ATP is used by the cell as an energy source
  • Describe the overall result in terms of molecules produced of the breakdown of glucose by glycolysis

Even exergonic, energy-releasing reactions require a small amount of activation energy to proceed. However, consider endergonic reactions, which require much more energy input because their products have more free energy than their reactants. Within the cell, where does energy to power such reactions come from? The answer lies with an energy-supplying molecule called adenosine triphosphate, or ATP. ATP is a small, relatively simple molecule, but within its bonds contains the potential for a quick burst of energy that can be harnessed to perform cellular work. This molecule can be thought of as the primary energy currency of cells in the same way that money is the currency that people exchange for things they need. ATP is used to power the majority of energy-requiring cellular reactions.

ATP in Living Systems

A living cell cannot store significant amounts of free energy. Excess free energy would result in an increase of heat in the cell, which would denature enzymes and other proteins, and thus destroy the cell. Rather, a cell must be able to store energy safely and release it for use only as needed. Living cells accomplish this using ATP, which can be used to fill any energy need of the cell. How? It functions as a rechargeable battery.

When ATP is broken down, usually by the removal of its terminal phosphate group, energy is released. This energy is used to do work by the cell, usually by the binding of the released phosphate to another molecule, thus activating it. For example, in the mechanical work of muscle contraction, ATP supplies energy to move the contractile muscle proteins.

ATP Structure and Function

At the heart of ATP is a molecule of adenosine monophosphate (AMP), which is composed of an adenine molecule bonded to both a ribose molecule and a single phosphate group (Figure 1). Ribose is a five-carbon sugar found in RNA and AMP is one of the nucleotides in RNA. The addition of a second phosphate group to this core molecule results in adenosine diphosphate (ADP); the addition of a third phosphate group forms adenosine triphosphate (ATP).

The addition of a phosphate group to a molecule requires a high amount of energy and results in a high-energy bond. Phosphate groups are negatively charged and thus repel one another when they are arranged in series, as they are in ADP and ATP. This repulsion makes the ADP and ATP molecules inherently unstable. The release of one or two phosphate groups from ATP, a process called hydrolysis, releases energy.

Glycolysis

You have read that nearly all of the energy used by living things comes to them in the bonds of the sugar, glucose. Glycolysis is the first step in the breakdown of glucose to extract energy for cell metabolism. Many living organisms carry out glycolysis as part of their metabolism. Glycolysis takes place in the cytoplasm of most prokaryotic and all eukaryotic cells.

Glycolysis begins with the six-carbon, ring-shaped structure of a single glucose molecule and ends with two molecules of a three-carbon sugar called pyruvate. Glycolysis consists of two distinct phases. In the first part of the glycolysis pathway, energy is used to make adjustments so that the six-carbon sugar molecule can be split evenly into two three-carbon pyruvate molecules. In the second part of glycolysis, ATP and nicotinamide-adenine dinucleotide (NADH) are produced (Figure 2).

If the cell cannot catabolize the pyruvate molecules further, it will harvest only two ATP molecules from one molecule of glucose. For example, mature mammalian red blood cells are only capable of glycolysis, which is their sole source of ATP. If glycolysis is interrupted, these cells would eventually die.

Section Summary

ATP functions as the energy currency for cells. It allows cells to store energy briefly and transport it within itself to support endergonic chemical reactions. The structure of ATP is that of an RNA nucleotide with three phosphate groups attached. As ATP is used for energy, a phosphate group is detached, and ADP is produced. Energy derived from glucose catabolism is used to recharge ADP into ATP.

Glycolysis is the first pathway used in the breakdown of glucose to extract energy. Because it is used by nearly all organisms on earth, it must have evolved early in the history of life. Glycolysis consists of two parts: The first part prepares the six-carbon ring of glucose for separation into two three-carbon sugars. Energy from ATP is invested into the molecule during this step to energize the separation. The second half of glycolysis extracts ATP and high-energy electrons from hydrogen atoms and attaches them to NAD+. Two ATP molecules are invested in the first half and four ATP molecules are formed during the second half. This produces a net gain of two ATP molecules per molecule of glucose for the cell.

A Open Assessments element has been excluded from this version of the text. You can view it online here: pb.libretexts.org/fob2/?p=136

Additional Self Check Question

1. Both prokaryotic and eukaryotic organisms carry out some form of glycolysis. How does that fact support or not support the assertion that glycolysis is one of the oldest metabolic pathways?

Answer

1. If glycolysis evolved relatively late, it likely would not be as universal in organisms as it is. It probably evolved in very primitive organisms and persisted, with the addition of other pathways of carbohydrate metabolism that evolved later.

Try It

ATP : (also, adenosine triphosphate) the cell’s energy currency

glycolysis: the process of breaking glucose into two three-carbon molecules with the production of ATP and NADH


4.5 Connections to Other Metabolic Pathways

You have learned about the catabolism of glucose, which provides energy to living cells. But living things consume more than just glucose for food. How does a turkey sandwich, which contains protein, provide energy to your cells? This happens because all of the catabolic pathways for carbohydrates, proteins, and lipids eventually connect into glycolysis and the citric acid cycle pathways (Figure 4.20). Metabolic pathways should be thought of as porous—that is, substances enter from other pathways, and other substances leave for other pathways. These pathways are not closed systems. Many of the products in a particular pathway are reactants in other pathways.

Connections of Other Sugars to Glucose Metabolism

Glycogen, a polymer of glucose, is a short-term energy storage molecule in animals. When there is adequate ATP present, excess glucose is converted into glycogen for storage. Glycogen is made and stored in the liver and muscle. Glycogen will be taken out of storage if blood sugar levels drop. The presence of glycogen in muscle cells as a source of glucose allows ATP to be produced for a longer time during exercise.

Sucrose is a disaccharide made from glucose and fructose bonded together. Sucrose is broken down in the small intestine, and the glucose and fructose are absorbed separately. Fructose is one of the three dietary monosaccharides, along with glucose and galactose (which is part of milk sugar, the disaccharide lactose), that are absorbed directly into the bloodstream during digestion. The catabolism of both fructose and galactose produces the same number of ATP molecules as glucose.

Connections of Proteins to Glucose Metabolism

Proteins are broken down by a variety of enzymes in cells. Most of the time, amino acids are recycled into new proteins. If there are excess amino acids, however, or if the body is in a state of famine, some amino acids will be shunted into pathways of glucose catabolism. Each amino acid must have its amino group removed prior to entry into these pathways. The amino group is converted into ammonia. In mammals, the liver synthesizes urea from two ammonia molecules and a carbon dioxide molecule. Thus, urea is the principal waste product in mammals from the nitrogen originating in amino acids, and it leaves the body in urine.

Connections of Lipids to Glucose Metabolism

The lipids that are connected to the glucose pathways are cholesterol and triglycerides. Cholesterol is a lipid that contributes to cell membrane flexibility and is a precursor of steroid hormones. The synthesis of cholesterol starts with acetyl CoA and proceeds in only one direction. The process cannot be reversed, and ATP is not produced.

Triglycerides are a form of long-term energy storage in animals. Triglycerides store about twice as much energy as carbohydrates. Triglycerides are made of glycerol and three fatty acids. Animals can make most of the fatty acids they need. Triglycerides can be both made and broken down through parts of the glucose catabolism pathways. Glycerol can be phosphorylated and proceeds through glycolysis. Fatty acids are broken into two-carbon units that enter the citric acid cycle.

Evolution Connection

Pathways of Photosynthesis and Cellular Metabolism

Photosynthesis and cellular metabolism consist of several very complex pathways. It is generally thought that the first cells arose in an aqueous environment—a “soup” of nutrients. If these cells reproduced successfully and their numbers climbed steadily, it follows that the cells would begin to deplete the nutrients from the medium in which they lived, as they shifted the nutrients into their own cells. This hypothetical situation would have resulted in natural selection favoring those organisms that could exist by using the nutrients that remained in their environment and by manipulating these nutrients into materials that they could use to survive. Additionally, selection would favor those organisms that could extract maximal value from the available nutrients.

An early form of photosynthesis developed that harnessed the sun’s energy using compounds other than water as a source of hydrogen atoms, but this pathway did not produce free oxygen. It is thought that glycolysis developed prior to this time and could take advantage of simple sugars being produced, but these reactions were not able to fully extract the energy stored in the carbohydrates. A later form of photosynthesis used water as a source of hydrogen ions and generated free oxygen. Over time, the atmosphere became oxygenated. Living things adapted to exploit this new atmosphere and allowed respiration as we know it to evolve. When the full process of photosynthesis as we know it developed and the atmosphere became oxygenated, cells were finally able to use the oxygen expelled by photosynthesis to extract more energy from the sugar molecules using the citric acid cycle.


Lactate Is a Natural Suppressor of RLR Signaling by Targeting MAVS

RLR-mediated type I IFN production plays a pivotal role in elevating host immunity for viral clearance and cancer immune surveillance. Here, we report that glycolysis, which is inactivated during RLR activation, serves as a barrier to impede type I IFN production upon RLR activation. RLR-triggered MAVS-RIG-I recognition hijacks hexokinase binding to MAVS, leading to the impairment of hexokinase mitochondria localization and activation. Lactate serves as a key metabolite responsible for glycolysis-mediated RLR signaling inhibition by directly binding to MAVS transmembrane (TM) domain and preventing MAVS aggregation. Notably, lactate restoration reverses increased IFN production caused by lactate deficiency. Using pharmacological and genetic approaches, we show that lactate reduction by lactate dehydrogenase A (LDHA) inactivation heightens type I IFN production to protect mice from viral infection. Our study establishes a critical role of glycolysis-derived lactate in limiting RLR signaling and identifies MAVS as a direct sensor of lactate, which functions to connect energy metabolism and innate immunity.

Keywords: MAVS RLR signaling glucose metabolism interferon lactate.


Electron Carriers

In living systems, a small class of compounds functions as electron shuttles: they bind and carry high-energy electrons between compounds in pathways. The principal electron carriers we will consider are derived from the B vitamin group and are derivatives of nucleotides. These compounds can be easily reduced (that is, they accept electrons) or oxidized (they lose electrons). Nicotinamide adenine dinucleotide (NAD) (Figure 4.13) is derived from vitamin B3, niacin. NAD + is the oxidized form of the molecule NADH is the reduced form of the molecule after it has accepted two electrons and a proton (which together are the equivalent of a hydrogen atom with an extra electron).

NAD + can accept electrons from an organic molecule according to the general equation:

RH (Reducing Agent) + NAD + (Oxidizing Agent) —-> NADH (Reduced) + R (Oxidized)

When electrons are added to a compound, they are reduced. A compound that reduces another is called a reducing agent. In the above equation, RH is a reducing agent, and NAD + is reduced to NADH. When electrons are removed from compound, it is oxidized. A compound that oxidizes another is called an oxidizing agent. In the above equation, NAD + is an oxidizing agent, and RH is oxidized to R.

Similarly, flavin adenine dinucleotide (FAD + ) is derived from vitamin B2, also called riboflavin. Its reduced form is FADH2. A second variation of NAD, NADP, contains an extra phosphate group. Both NAD + and FAD + are extensively used in energy extraction from sugars, and NADP plays an important role in anabolic reactions and photosynthesis.

Figure 4.13 The oxidized form of the electron carrier (NAD+) is shown on the left and the reduced form (NADH) is shown on the right. The nitrogenous base in NADH has one more hydrogen ion and two more electrons than in NAD+.

Outcomes of Glycolysis

One glucose molecule produces four ATP, two NADH, and two pyruvate molecules during glycolysis.

Learning Objectives

Describe the energy obtained from one molecule of glucose going through glycolysis

Key Takeaways

Key Points

  • Although four ATP molecules are produced in the second half, the net gain of glycolysis is only two ATP because two ATP molecules are used in the first half of glycolysis.
  • Enzymes that catalyze the reactions that produce ATP are rate-limiting steps of glycolysis and must be present in sufficient quantities for glycolysis to complete the production of four ATP, two NADH, and two pyruvate molecules for each glucose molecule that enters the pathway.
  • Red blood cells require glycolysis as their sole source of ATP in order to survive, because they do not have mitochondria.
  • Cancer cells and stem cells also use glycolysis as the main source of ATP (process known as aerobic glycolysis, or Warburg effect).

Key Terms

  • pyruvate: any salt or ester of pyruvic acid the end product of glycolysis before entering the TCA cycle

Outcomes of Glycolysis

Glycolysis starts with one molecule of glucose and ends with two pyruvate (pyruvic acid) molecules, a total of four ATP molecules, and two molecules of NADH. Two ATP molecules were used in the first half of the pathway to prepare the six-carbon ring for cleavage, so the cell has a net gain of two ATP molecules and 2 NADH molecules for its use. If the cell cannot catabolize the pyruvate molecules further (via the citric acid cycle or Krebs cycle), it will harvest only two ATP molecules from one molecule of glucose.

Glycolysis produces 2 ATP, 2 NADH, and 2 pyruvate molecules: Glycolysis, or the aerobic catabolic breakdown of glucose, produces energy in the form of ATP, NADH, and pyruvate, which itself enters the citric acid cycle to produce more energy.

Mature mammalian red blood cells do not have mitochondria and are not capable of aerobic respiration, the process in which organisms convert energy in the presence of oxygen. Instead, glycolysis is their sole source of ATP. Therefore, if glycolysis is interrupted, the red blood cells lose their ability to maintain their sodium-potassium pumps, which require ATP to function, and eventually, they die. For example, since the second half of glycolysis (which produces the energy molecules) slows or stops in the absence of NAD+, when NAD+ is unavailable, red blood cells will be unable to produce a sufficient amount of ATP in order to survive.

Additionally, the last step in glycolysis will not occur if pyruvate kinase, the enzyme that catalyzes the formation of pyruvate, is not available in sufficient quantities. In this situation, the entire glycolysis pathway will continue to proceed, but only two ATP molecules will be made in the second half (instead of the usual four ATP molecules). Thus, pyruvate kinase is a rate-limiting enzyme for glycolysis.


The enzyme triosephosphate isomerase rapidly inter- converts the molecules dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GAP). Glyceraldehyde phosphate is removed / used in next step of Glycolysis.

Details:

GAP is the only molecule that continues in the glycolytic pathway. As a result, all of the DHAP molecules produced are further acted on by the enzyme Triosephosphate isomerase (TIM), which reorganizes the DHAP into GAP so it can continue in glycolysis. At this point in the glycolytic pathway, we have two 3-carbon molecules, but have not yet fully converted glucose into pyruvate.


Cellular Respiration Stage II: The Krebs Cycle

Recall that glycolysis produces two molecules of pyruvate (pyruvic acid), which are then converted to acetyl CoA during the short transition reaction. These molecules enter the matrix of a mitochondrion, where they start the Krebs cycle (also known as the Citric Acid Cycle). The reason this stage is considered a cycle is because a molecule called oxaloacetate is present at both the beginning and end of this reaction and is used to break down the two molecules of acetyl CoA. The reactions that occur next are shown in Figure 4.10.6.

Figure 4.10.6 Reactants and products of the Krebs Cycle.

The Krebs cycle itself actually begins when acetyl-CoA combines with a four-carbon molecule called OAA (oxaloacetate) (see Figure 4.10.6). This produces citric acid, which has six carbon atoms. This is why the Krebs cycle is also called the citric acid cycle.

After citric acid forms, it goes through a series of reactions that release energy. The energy is captured in molecules of NADH, ATP, and FADH2, another energy-carrying coenzyme. Carbon dioxide is also released as a waste product of these reactions.

The final step of the Krebs cycle regenerates OAA, the molecule that began the Krebs cycle. This molecule is needed for the next turn through the cycle. Two turns are needed because glycolysis produces two pyruvic acid molecules when it splits glucose.


11.2.3 Outcomes of Glycolysis

Glycolysis starts with glucose and ends with two pyruvate molecules, a total of four ATP molecules and two molecules of NADH. Since two ATP molecules were invested in the first stage of the pathway, the cell has a net gain of 2 ATP molecules and 2 NADH molecules. If the cell cannot catabolize the pyruvate molecules further, it will harvest only two ATP molecules from one molecule of glucose.

QUICK REVIEW OF GLYCOLYSIS:

    • Splits a six-carbon glucose molecule into 2 three-carbon molecules of pyruvate, using 10 enzyme-catalyzed reactions
    • Yields a net gain of 2 ATP and 2 NADH
    • Takes place in the cytoplasm
    • Can occur without oxygen is an anaerobic process

    Step 7 : Conversion of 1,3-Biphosphoglycerate to 3-Phosphoglycerate

    • The enzyme phosphoglycerate kinase transfers the high-energy phosphoryl group from the carboxyl group of 1,3-bisphosphoglycerate to ADP, forming ATP and 3-phosphoglycerate.
    • This is a unique example where ATP can be produced at substrate level without participating in electron transport chain. This type of reaction where ATP is formed at substrate level is called as Substrate level phosphorylation.

    Materials and Methods

    Strains, media and cultivation conditions

    We used S. cerevisiae strain T73 (CECT 1894) isolated from Alicante (Spain) musts [48], which is commercialized by Lallemand Inc. (Montreal, Canada). This strain has been widely used in several studies and has proven to be a good wine yeast model. This strain was previously genetically modified to T73ura3 [49] to construct other strains given the absence of auxotrophies in wine natural yeasts. These strains are also aneuploids, and have a chromosome number that is not a multiple of the haploid number, and they require several rounds of transformation for deletion gene construction.

    The YEp-TRX2 plasmid was obtained by subcloning a 0.7 kb Eco RI fragment containing the yeast TRX2 gene and promoter in the episomal yeast plasmid Yep352 carrying selectable marker URA3. The TTRX2 strain [2] is a genetically-modified T73ura3 strain following the lithium acetate procedure as modified by [50].

    Strain trx2 was obtained by sequential deletion of the two copies of the TRX2 gene in strain T73ura3. Disruption was carried out by homologous recombination at both ends of the TRX2 open reading frame of an integration cassette carrying a kanR marker gene flanked by loxP sites. Excision of the marker is inducible by the expression of Cre recombinase introduced into the same strain [51] to allow repeated disruptions. Integration of the cassette at the TRX2 locus and further excision of the kanR marker were confirmed by PCR analysis. The absence of any TRX2 gene product was confirmed by northern and western blot analyses. Uracil prototrophy was restored by introducing a 1.1-kb Hind III linear fragment containing the URA3 gene.

    Precultures for industrial biomass propagation experiments were prepared in YPD liquid medium (1% Yeast extract, 2% Peptone, 2% Glucose) and were incubated at 30°C with shaking (250 rpm) for 12 h.

    Molasses medium (diluted to 60 g of sucrose L -1 for the batch phase or 100 g of sucrose L -1 for the fed-batch phase) was supplemented with 7.5 g L -1 of (NH4)2SO4, 3.5 g L -1 of KH2PO4, 0.75 g L -1 of MgSO47H2O, 10 ml L -1 of vitamin solution, and 1 ml L -1 of antifoam 204 (Sigma, St. Louis, Mo.). Molasses and mineral solutions were autoclaved separately. The vitamin solution containing 50 mg L -1 of D-biotin, 1 g L -1 of calcium pantothenate, and 1 g L -1 of thiamine hydrochloride was filter sterilized (0.2-μm pore size) prior to use in the molasses medium.

    The liquid medium YPGF (1% Yeast extract, 2% Peptone, 10% Glucose, 10% Fructose) was used to inoculate fresh yeast and active dry yeast produced under industrial conditions to simulate must sugar content and wine fermentation conditions. YPGF medium was also supplemented with the carbonylation inductor glyoxal (GO) at 5 mM for 1 h to induce protein carbonylation damage [32].

    Industrial production conditions

    Biomass propagation experiments were designed with two growth stages, batch and fed-batch, in a BIOFLO III bioreactor (NBS, New Jersey), and the technical parameters (agitation, pH and feed rate) were established as previously described [1, 3, 29]. Overnight YPD precultures were incubated at 30°C with shaking (250 rpm) (Time 0 h). The bioreactor containing 2 L sterilized molasses medium at pH 4.5 was then inoculated to an initial OD600 nm of 0.05. In the batch phase, cells consumed all the sucrose present in the medium using a fermentative metabolism. When sucrose was exhausted (12-15 h), cells changed their metabolism to respiration, allowing the consumption of the produced ethanol until approximately 40 h of the process. When ethanol was exhausted, the fed-batch phase started by feeding the reactor continuously with molasses medium at the desired flow rate until approximately 80 h, thus avoiding fermentative metabolism in order to gain the highest biomass yield. Three independent production experiments were carried out for the T73, TTRX2 and trx2 strains.

    Biomass drying and rehydration

    At the end of the fed-batch fermentation, biomass was separated by centrifugation from the fermented media and subjected to several washing steps with distilled water. Concentrated biomass (500 mg) was collected in petri plates. Yeast biomass was dehydrated under air flux in a convection oven at 30°C until approximately 8% relative humidity (approximately 24 h) with opened petri plates [3]. Dehydrated biomass was collected in plastic bags and stored under vacuum conditions at room temperature during one week. Rehydration was performed in distilled water at 37°C during 10 min under static conditions and 10 min with shaking at 130 rpm [52].

    Protein extraction and two-dimensional gel electrophoresis

    Cell samples (25 mg) were collected at 0 h, 15 h and 80 h of growth for protein extraction. Cells were resuspended in 150 μL extraction buffer (8 M Urea, 25 mM Tris-HCl pH 8.0), a mixture of protease inhibitors (200 μM phenylmethylsulphonyl fluoride (PMSF), 20 μM TPcK, 200 μM pepstatin A) and 0.2 g of glass beads. Cells were broken in Fast Prep (MP Bio) at 5.0 m/s for 45 sec on 3 occasions. After centrifugation at 12000 rpm for 10 min, the supernatant was sonicated and centrifuged again at 12000 rpm for 10 min. The protein concentration was determined with a Nanodrop ND-1000 UV/Vis spectrophotometer. Among 50-80 μg of protein were diluted in 340 μl of Rehydration Buffer (8 M Urea, 4% CHAPS (w/v), 50 mM DTT and 0.5% ampholytes (v/v) pH 3-10 (Amersham)). Isoelectric focusing (50-100 μg of protein) was performed in immobilised pH gradient strips (3-11 NL Amersham). After the first dimension, strips were incubated for 20 min with 5 ml of a solution containing 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 10% trifluoroacetic acid (TFA). This compound reacts with carbonyl groups in proteins. To stop this reaction, the strip was transferred to a 5 ml solution containing 0.4 M Tris, 6 M Urea, 2% SDS and 20% glycerol. Second dimension SDS-PAGE was performed on 18 × 18 cm 11% polyacrylamide gels.

    Four strips were run in parallel, two strips for the wild type and two strips for the respective mutant strain each time. Gels were either transferred to PVDF membranes for western blot analysis or silver strained and scanned in a GS800 densitometer (Bio-Rad). In both cases, obtained images were analyzed with the PDQuest software (Bio-Rad). Gels were silver-stained using the PlusOne silver staining kit of General Electric Healthcare. PVDF membranes were silver-stained to control the protein load as described elsewhere [53].

    Western blot analysis and carbonyl content quantitation

    Crude extracts were separated in 10% SDS-polyacrylamide gels and transferred to the PVDF membrane from one-dimensional gels. A polyclonal anti-Adh1p antibody was purchased from Acris (R1049) which is able to detect monomer, dimer and tetramer forms of Adh1p under SDS/PAGE conditions as described on the web site http://www.acris-antibodies.com/antibodies/r1049.htm and diluted to 1:1000. The Western blots from two-dimensional gels were prepared as described in the previous section. A 1:5000 dilution of antibody against DNP (Dako) was used. In both cases, a peroxidase-conjugated anti-rabbit antibody was used for detection. Images were acquired in a ChemiDoc XRS System (Bio-Rad) and analyzed with the Quantity One software (Bio-Rad).

    Carbonyl content was quantified using the PDQuest software (Bio-Rad). Carbonylation levels for each protein are calculated by dividing the carbonyl intensity from western 2-D anti DNP (CI) by protein intensity (PI) from 2-D silver-stained gels.

    Protein identification by tryptic digestion and MALDI-TOF

    Protein spots were excised from gels and subjected to in situ digestion with trypsin on a ZipPlate (Millipore). Gel pieces were washed with 25 mM ammonium bicarbonate and dehydrated with acetonitrile followed by (i) reduction of cysteines with 10 mM DTT, (ii) alkylation of free cysteines with 55 mM iodoacetamide, and (iii) in situ digestion with 170 ng of trypsin overnight at 30°C. Peptide extractions and washes were performed on a ZipPlate following the manufacturer's recommendations. Tryptic peptides were recovered in 5 ml of 0.1% TFA, 50% acetonitrile, and spotted onto a MALDI plate in the presence of a-ciano-4-hydroxycynamic acid. Spectra were obtained in an Applied Biosystems voyager DE PRO MALDI-TOF apparatus operating in the reflector mode. The spectra with higher resolutions than 8000 were obtained. External calibration was performed with calibration mixtures from Applied Biosystems. The acquired spectra were processed by Data Explorer (version 4.0). Proteins were identified by peptide mass fingerprinting searching against the Swiss-Prot database using MASCOT. Protein coverage for each spot in the MASCOT analysis of up to 50 percent was considered significant. Alternatively, proteins were identified by gel matching with S. cerevisiae two-dimensional gel electrophoresis maps available in the following databases: YPD (Yeast Proteome Database http://www.proteome.com) YMP (Yeast Mitochondrial Proteome http://www.biochem.oulu.fi/proteomics/ymp.html) and 2-DE S. cerevisiae (IPG6-12) http://www.weihenstephan.de/proteomik/. Functional group analysis was performed using the GOstat and GO term finder (SGD database) online applications with a false discovery rate of 5%. The represented data correspond to the average of three biological replicates.

    ADH immunoprecipitation

    Cells in IP buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 10% glycerol, 0.1% Nonidet-P40) plus 1X protease inhibitors (Roche) and PMSF 0.4 mM were broken with 0.5 g of glass beads in a Fast Prep (MP Bio) at 5.0 m/s for 45 s, 3 times. Lysates were clarified by centrifugation for 15 min at 14.000 rpm and added to 20 μl of Protein A/G PLUS-Agarose beads (Santa Cruz Biotechnology, INC) with bound polyclonal anti-ADH antibody (Acris antibodies). After a 4 h binding step at 4°C, beads were washed three times with IP buffer plus 0.5% TritonX-100 and 0.5 mM NaCl and resuspended in carbonyl derivatization buffer (25 mM Imidazol, 2 mM EDTA, pH 7.0, 6% SDS, 1X protease inhibitor) and boiled 3 min. Sample proteins were derivatized with DNPH and separated in a 9% acrylamide gel (BioRad Laboratories) and incubated with anti-DNP antibody at 1/3500 and anti-rabbit (1/5000).

    Enzyme activities

    Dehydrated cells were inoculated (10 7 cells/mL) in YPGF and incubated at 30°C and 65 rpm for 5 h. Samples were collected at the end of incubation, and cell extracts were prepared using glass beads and assayed as described in the following references: alcohol dehydrogenase [54], enolase [55], pyruvate decarboxylase [56] and catalase [57].


    Watch the video: Overview of glycolysis. Cellular respiration. Biology. Khan Academy (January 2023).