BIL 256 Cell and Molecular Biology Lab Spring, 2007 Background Information Tissue-Specific Isoenzymes A. BIOCHEMISTRY The basic pattern of glucose oxidation is outlined in Figure 3-1. Glucose is split into two molecules of pyruvic acid, each with three carbon atoms. These reactions occur in the cytoplasm by a process called glycolysis. In the presence of oxygen (aerobic conditions), pyruvic acid then enters the mitochondria, where it is completely oxidized to CO 2 and water in the citric acid cycle and in the electron transport chain. During the citric acid cycle, the carbon atoms of the acetyl groups are liberated as CO 2 while the hydrogen atoms (protons + electrons) are transferred primarily to the carrier molecule NAD + which is thereby reduced to NADH + H +. The electrons are then transferred from the NADH + H + to a series of electron carrier molecules that comprise the electron-transport chain. During the course of this transfer, the electrons lose energy which is transferred to ATP. The terminal step in the chain is when the electrons and protons combine with molecular oxygen to form water. The final reaction of anaerobic (without oxygen) glycolysis is the conversion of pyruvate to lactic acid and this reaction is catalyzed by the enzyme lactate dehydrogenase (LDH). In skeletal muscle, where oxygen deprivation is common during exercise, the reaction is efficient and large amounts of lactate can be formed. (See Figure 3-1). In tissues that preferentially oxidize glucose aerobically to CO 2 and water, such as cardiac muscle, the reaction is not efficient and pyruvate is preferentially converted to acetyl CoA which enters the citric acid cycle. In order to understand the differences in efficiency of this reaction in skeletal and heart muscle, it is necessary to explore the structure of the LDH enzyme in different tissues of the body.
Isoenzymes are different molecular forms of the same enzyme, and five major LDH isoenzymes are found in different vertebrate tissues. Each LDH molecule is composed of four polypeptide chains (each is a tetramer) but the subunit composition of the five LDH isoenzymes are different. There are two types of polypeptide chains in LDH called M (for skeletal muscle) and H (for heart muscle) which can be combined into the LDH tetramer in 5 different ways. Each different combination of subunits represents a distinct LDH isoenzyme as illustrated in Figure 3-2. Because the H polypeptide has more acidic amino acid residues than the M polypeptide, the electrophoretic mobilities of the LDH isoenzymes are: LDH 1 > LDH 2 > LDH 3 > LDH 4 > LDH 5. The H and M polypeptides of LDH are encoded by different genes, and the two genes are expressed to different degrees in different tissues. For example, in heart muscle, the gene for the H subunit is more active than the gene for the M subunit. Thus, LDH isoenzyme 1 is the predominant form of the enzyme in cardiac muscle. The reverse is true in skeletal muscle where there is more M than H polypeptide produced, and hence, more of the isoenzyme 5 form of the enzyme. The efficiency of the conversion of pyruvate to lactate increases with the number of M chains. Therefore, the high concentration of LDH 5 (4 M subunits) in skeletal muscle rapidly converts pyruvate to lactate while the high concentration of LDH 1 (4 H subunits) in heart tissue favors conversion of pyruvate to acetyl CoA which enters the citric acid cycle. These tissuespecific differences in LDH isoenzymes can be readily detected by the localization of LDH activity in an agarose gel after electrophoresis of tissue extracts as shown in Figure 3-3. Lactate dehydrogenase, like many other enzymes, is also found in serum where it is derived from
normal cell death since dying and dead cells liberate their cellular enzymes into the bloodstream. The liberation of enzymes into the circulatory system is accelerated during tissue injury and the measurement of LDH isoenzymes in serum has been used extensively for determining the site and nature of tissue injury in humans. For example, when the blood supply to the heart muscle is severely reduced, as during a heart attack, muscle cells die and liberate LDH 1 into the blood stream. Thus, an increase in LDH 1 in serum is indicative of a heart attack. In contrast muscular dystrophy is accompanied by an increase in the levels of LDH 5 which is derived from dying skeletal muscle cells. B. DETECTION OF LDH ACTIVITY IN AGAROSE GELS In this experiment you will prepare tissue extracts from calf thymus and then electrophorese the extracts along with extracts from calf heart, skeletal muscle and serum on agarose gels. The serum and tissue extracts contain hundreds of colorless proteins in addition to LDH. In order to identify the LDH isoenzymes, you will selectively stain the gels after electrophoresis for LDH activity. Each of the LDH isozymes can catalyze the following reaction: LDH Lactate + NAD + Pyruvate + NADH + H + In order to detect the LDH isozyme in an agarose gel after electrophoresis, the above enzymatic reaction is coupled to a color producing reaction: 1) Lactate + NAD + Pyruvate + NADH + H + 2) NADH + PMS NAD + + PMS-H 3) PMS-H + TNBT PMS + TNBT-Formazan NAD - Nicotinamide adenine dinucleotide NADH - Nicotinamide adenine dinucleotide, reduced PMS - Phenazine methosulfate TNBT - Tetranitroblue tetrazolium The highly colored TNBT-Formazan product localizes in the electrophoretic zones of LDH activity and the amount of brown color formed is quantitatively related to the level of LDH isoenzyme present. Objectives: In this experiment, you will first prepare tissue extracts from calf thymus. You will
then characterize the LDH isoenzymes in the tissue extracts and in calf serum. Procedure A. Preparation of the Agarose Gels. 1. Prepare a 1.2% agarose gel 2. While the gel is cooling, prepare the tissue extracts as described below. B. Preparation of the Thymus Extracts. 1. Place 0.5 g of calf thymus into a precooled mortar and cut the tissue into small (l cm) sections with scissors. 2. Add 2 ml of cold extraction buffer and grind the tissue sections with the pestle until a homogenous suspension is formed. The mechanical action of the pestle and the chemical action of a detergent (NP40) that is present in the extraction buffer should disrupt the plasma membrane leaving the nuclear envelope intact. 3. Pour about 1 ml of the homogenate into a small centrifuge tube and centrifuge for 5 minutes to pellet the nuclei. 4. Pour the supernatant into a clean tube, label the tube thymus extract, and then place the tube in an ice bath. C. Electrophoresis 1. To the tube labeled sample buffer, add 50 μl of the thymus extract. 2. Load 15 μl of the following samples into the agarose gel sample wells. Lane Sample 1,5 Calf Serum 2,6 Calf Heart LDH 3,7 Calf Muscle LDH 4,8 Calf Thymus Extract 3. Electrophorese at 170 V until the bromophenol blue in the tissue extract samples has migrated to within 1 mm of the positive electrode end of the gel. 4. Remove the gels from the electrophoresis cell, rinse them in distilled water, and note the blue serum albumin bands in lanes 1 and 5. Some of the bromophenol blue in the sample will remain bound to albumin during the electrophoretic run. IV. Detection of LDH Isoenzymes
1. Add 15ml of the LDH-substrate solution to a gel staining dish containing your gel and incubate in the dark for 30-40 minutes at 37 o C. Data Analysis 1. Rinse the gels with water and examine them on a light box. Locate the bands containing LDH-1, LDH-2, LDH-3, LDH-4, and LDH-5. Figure 3-3 should help in this identification. 2. The amount of brown color is roughly proportional to the amount of an LDH isoenzyme present in a band. In the table below, estimate the amounts of each isoenzyme present in each protein sample. Estimates of the % of Total LDH Activity* SAMPLE LDH-1 LDH-2 LDH-3 LDH-4 LDH-5 Serum Thymus Heart Skeletal Muscle *These percentages can be determined accurately with a gel densitometer. 3. The gels can be stored in standard destain solution containing 10% methanol and 5% acetic acid.