Biotechnology report

Introduction

The Na+, K+-ATPase refers to a ubiquitous enzyme constituted by alpha, beta, and gamma subunits that are responsible for creating and maintaining the sodium and potassium ions gradients on the cell membrane and finishing the cells with three sodium ions and two potassium ions. The main function of the sodium pump is to reproduce the required sodium ions in different body organs. And this regulation of sodium ions in the body is regulated by the tissues specific isoforms. Precise and proper functionality of body cells is enhanced by the regulation of this NKA isozymes hence proper coordination of various physiological responses. It is an essential respirator in the treatment of cognitive heart failure and cardiac arrhythmias.

Living cells have a higher concentration of intercellular potassium ions than intercellular sodium ions (Na+). However, the inverse applies to the condition of the outside cells where the concentrations of intercellular sodium (Na+) are higher than those of intercellular potassium (K+). The difference in concentration between the inner and outer intercellular cells creates a concentration gradient for the loss and gain of sodium ions and potassium ions respectively.

The NKA enzyme makes up the cellular membrane of all mammals thus it is the role of the NKA to preserve and maintain the ionic gradient within the cell membranes and enhance the osmotic and potential equilibrium of the cells. Another essence of the NKA is that it facilitates the body homeostasis response and cell survival since sodium ions gradients are utilized as a source of energy in solute and ions transportation within the cells. The NKA forms the putative component of the cell membrane that is responsible for the reabsorption functions of the kidney and in blood circulation in the heart. The NKA catalyzes the transfer of two potassium ions from extracellular space into the cells and extrusion of the three sodium ions as well as hydrolyzing adenosine triphosphate (ATP) to adenosine diphosphate (ADP). The transportation and flow of these ions across the membrane by the sodium pump process maintains the transmembrane gradient necessary for the driving force for the secondary transportation of metabolic substrates such as glucose and amino acids. Therefore Na+, K+ ATPase, directly or indirectly controls a number of essential cellular body functions. Through regulation of enzymes transportation, it plays crucial roles in the etiology of some pathological processes.

Effects ouabain on Na⁺ K⁺ ATPase activity

One of the Na⁺ K⁺ ATPase function is to catalyze the exchange and transport of sodium and potassium ions across the animals’ plasma membrane. However, the presence of ouabain and other related cardiac glycosides proves to be the greatest inhibitors of the Na⁺ K⁺ ATPase functions. The positive inotropic effects of ouabain on the myocardium are potentially caused by the partial inhibition of the sodium, potassium ions actions resulting in a small increase in alteration in the sarcolemma sodium/calcium ions exchanger thus causing increased intercellular absorption of calcium ions through cell membranes into the tissue cells. The presence of ouabain and together with other related glycosides are the causative agents of cardiac contractility hence becomes the basis of major therapeutic application and recommendation for use of these drugs in the diagnosis of congestive heart failure

The recent experimental research conducted concluded that cardiac glycosides such as ouabain are the source of paracrine hormones in bigger animals hence essential in boosting heart performance and at the same time prevent heart failure. Ouabain also affects the process of proto-oncogenes since the induction of ouabain at early stages induce genes that catalyze the growth of cardiac tissues and hypertrophy.

Aim of the biochemical experiment

The aim and purpose of this biochemical laboratory experiment were to establish the different effects of ouabain and other related cardiac glycosides inhibitor on non- Na⁺K⁺ATPase activity and Na⁺K⁺ATPase activity in animal cells. Out of the experiment, I expected to widen my scope of understanding of the functions of Na⁺K⁺ATPase activity in major organs of mammal such as kidney and liver.

Hypothesis

This report presents a detailed outline of a biochemical laboratory experiment conducted to isolated sodium and potassium ions (Na⁺K⁺ATPase) obtained from kidney tissue and liver tissues were fractionated into the membrane and cytosolic samples for further analysis. The sample was made from kidney and liver and various specific activity analysis was conducted on each sample obtained. The experimental data were recorded for further analysis as explained in the chapters that follow herein were used to distinguish the effects of inhibitor ouabain. The ouabain inhibitor was used to distinguish between non- Na⁺K⁺ATPase activity and Na⁺K⁺ATPase activity in animal cells

Materials and methods

The kidney for the experiment was obtained from a slaughterhouse; this was to ensure that the kidney was fresh with its cells and tissue still functioning. The kidney specimen was sliced from one pole to another with the plane of the cut running through the hilus. The region of medulla was observed in their red dark color. The identification and selection of the inner stripe of the outer medulla are quite simple due to the outstanding color difference between the inner and the outer medulla.

Rongeur, a surgical instrument was used to chop pieces of the outer medulla. The difference between rongeur and scissors only lies on the handle but it has cups that help in medulla selection. The cups of the rongeur were pressed and squeezed inside soft tissues out of the kidney specimen. The process was done through selective extraction because the outer medulla needed to be separated anatomically from the underlying cortex of the fibrous connective tissues of the kidney. However, the process of dissection was not a complicated one since it was partly guided by a sense of touch. We used three curved rongeurs to extract 336 grams of outer medulla tissue from a kidney of 15 kilograms.

To eliminate residual connective tissues and disperse the outer medulla tissue, we used a tissue press. The obtained outer medulla from dissection was then suspended ice-cold buffer made from 250 mmol/L sucrose, 25 mmol/L imidazole, and 1 mmol/L of ethylenediaminetetraacetic acid (EDTA) at 20⁰C and pH of 7.5 to form a half a litter solution. 50 ml portion of the formed suspension was forced through a 2.0-millimeter diameter hole. With each portion containing about 35 grams of tissue. The same procedure was repeated with a plate of 1.5-millimeter diameter holes. This procedure took about four hours to complete and the sieved suspension was stored overnight in a fridge at a temperature of about 5⁰C.

Microsomes preparation by centrifugation

To the five hundred milliliters sieved suspension, another five hundred milliliters of a cold-ISE buffer with strings was added. The resultant mixture was then homogenized into 80 mL ice-cold portions and 60 mL homogenized with Teflon at 200 rpm four times up and down. then 2 liters of cold ISE with strings were added and mixture centrifuged for 30 minutes at 3700 g-average at a temperature of 7⁰C while place in a 1.5 liters capacity rotor. After this process, the supernatant mixture was kept in ice, and pellets were suspended in ISE buffer to a volume of 1.5 liters and then dilution followed then homogenization, and finally, centrifugation procedures were repeated as before. The combined supernatant was then centrifuged for 40 minutes at an 8500 revolution per minute and the pellet was again discarded.  For these procedures, a 1 mL aliquot was used for titration with sodium dodecyl sulfate (SDS), and the small fraction of aliquot and microsomal ware stored at 20⁰C

The Na, K-ATPase activity was then measured as ouabain-sensitive that was associated with the release of phosphate from fro ATP in the presence of sodium ions Na⁺, potassium ions K⁺ and magnesium ions Mg²⁺ and the detergent used for this ions release was 6.0 U/mg protein. The yield was 6700 nmol Pi/min units of activity in 5,7 mg/mL. Lowry et al method of protein measurement was used to measure protein against bovine serum albumin standards without correction for a color factor. When the test was repeated without detergent, activation was found to be at 1.3 U/mg, indicating that ¾ of the enzyme was in tight vesicles. This could be justified by the fact that the substrates in the suspension were in small volume therefore inactive enzyme in the tight vesicles. In the ATP on the right-side-out of the vesicle intravehicular was completely depleted after enzymes had turned over only a few times. Since K+ is a stoichiometric substrate potassium ions were depleted in the case of inside-out vesicles intravascular

Titration and treatment with sodium dodecyl sulfate

Treatment with sodium dodecyl sulfate was performed to remove extraneous proteins while retaining the Na+K+ATPase membrane bonds. The amount of sodium dodecyl sulfate SDS that was added in the process was titrated from aliquots of the enzyme as follows. Microsomes were diluted by the addition of ISE-buffer to a 4.5 mg/L. Then to a 0.08 mL of this diluted microsomal suspension was added 0.002 mL of a buffer having a viable amount of sodium dodecyl sulfate. Then 0.9 mL of sodium chloride NaCl 130 mmol/L, potassium chloride KCl 22 mmol/L, magnesium chloride MgCl2 5 mmol/L and ATP  4 mmol/L was added to 0.5 mL of ISE after incubation for 60 minutes. After 4 minutes, trichloroacetic acid was added at 37⁰C and inorganic phosphate liberated was then measured. The titration process was finalized by the addition of sodium dodecyl sulfate to lower and bring to an end the titration process. From reactions, one of the preparation activities at 0.9 mg SDS/L was established to be 96b percent of the 0.8 mg/L as 0.9 mg SDS/mL was mainly applied for its purification purpose. And the ratio of SDS to protein was established to be o.25 mg SDS/mg protein.

After titration, following treatment with sodium dodecyl sulfate, here two frozen Microsomes preparation was thawed in plenty of water at room temperature and pressure as each preparation was treated separately with SDS. Every microsomal fract6ion obtained was diluted to 4.5 mg/L through the addition of ISE-buffer. Then 60 mL of ISE-buffer containing the estimated amount of SDS that is 275 mg was added to the suspension. ATP was not involved in this reaction or procedure since it adds no advantage to the expected results at this point. The sodium dodecyl sulfate was added to the combined fractions obtained in the previous procedure and suspension left overnight at a temperature of 20⁰C in the presence of SDS.

Centrifugation

The final procedure was the centrifugation process of the treated suspension. The treated suspension was subdivided into two portions and separately centrifuged at 4800 rpm for one hour at approximately 10⁰C. The supernatant suspension was discarded and pellets combined and suspended in 450 mL of ISE-buffer. The process of resuspension and centrifugation was repeated five times. The volume of the last resuspension was adjusted to5mg/mL protein concentration.

Results

The yield of kidney and liver provided 570 grams of outer medulla was about 6500 units in one preparation and the amount of protein was 250 mg and specific activity was 30 nmol Pi/min per mg protein. Nucleotide-binding capacity was obtained to be 3.0 nmol/mg. the molar activity or turnover number was found to be 8700 per minute. Ouabain insensitive ATPase activity was 0.5 to 1.2% of Na, K-ATPase activity. From reactions, one of the preparation activities at 0.9 mg SDS/L was established to be 96b percent of the 0.8 mg/L as 0.9 mg SDS/mL was mainly applied for its purification purpose. And the ratio of SDS to protein was established to be o.25 mg SDS/mg protein

Table 1

The estimated results from kidney and liver ware recorded as outlined in table 2 and table 3.

Table 2: results of cytosolic and membrane fraction of kidney and liver

Table 3: results in cytosolic and membrane of liver and kidney in ration to Lowry Assay values R²

Discussion

The quantity and quality of the specimen used in the experiment can be evaluated by several criteria. For our experiment, we used polyacrylamide gel electrophoresis to detect any element or form of contamination on the specimens (kidney and liver) and Na. K-ATPase. From the result obtained it was observed that the amount of protein varies according to the method of measurement and concentration of substrate used. The specific activity range from 20 nmol/mg/min to 45 nmol/mg/min. Mild injury impairs some partial activities than others. The amount of sodium dodecyl sulfate SDS that was added in the process was titrated from aliquots of the enzyme as follows. Microsomes were diluted by the addition of ISE-buffer to a 4.5 mg/L. Then to a 0.08 mL of this diluted microsomal suspension was added 0.002 mL of a buffer having a viable amount of sodium dodecyl sulfate. Then 0.9 mL of sodium chloride NaCl 130 mmol/L, potassium chloride KCl 22 mmol/L, magnesium chloride MgCl2 5 mmol/L and ATP  4 mmol/L was added to 0.5 mL of ISE after incubation for 60 minutes. After 4 minutes, trichloroacetic acid was added at 37⁰C and inorganic phosphate liberated was then measured

Conclusion

In this experiment, we developed high sensitive immunoaffinity-based mass spectrometry procedures to evaluate and verify both high and low abundance of Na, K+ ATPase activity in complex tissue and cell structures of mammal kidney and liver organs. The experiment also involved a highly sensitive mass spectrometry approach that facilitated the identification of low and high abundance of ATPase activity that uses alpha and beta units and the results obtained from these procedures are recorded in the result section and further elaborated in the discussion section.

The discussion presented and results in interpretations of the experiment was based on observations that the breakdown and uptake of perhaps most proteins occurred in the proximal tubule cells and catabolism occurs in the nephron of the specialized medulla tissues.

In conclusion, our experiment involved series of tests and procedures that enabled the proteomic analysis of the Na+, K+ ATPase activity in the outer medulla tissue of the kidney and liver cortex tissues and at the same time analyzing the effects of ouabain in the whole process.