Int. J. Pharm. Investigation, 2022; 12(3) : 328-333 328 International Journal of Pharmaceutical Investigation, Vol 12, Issue 3, Jul-Sep, 2022 Original Article INTRODUCTION Tese studies examined mutual protective relationships among rabbit muscle aldolase, enolase, phosphofructokinase-1 (PFK-1) and LDH from inhibitions by ascorbate (AA). It was proposed earlier 1-2 that specifc inhibitions of PFK-1 and LDH by AA faciltated glycogen storage in resting muscle by inhibiting glycolysis. A ten step metabolic pathway i.e. Glycolysis is catablozed by the pyruvate that leads to the production of energy i.e. adenosine tri phosphate (ATP ) and nicotine adenine dinucleotide (NADP). Te breakdown of aaldol cleavage is the fourth step of glycolysis in which 6 free carbon of fructise 1,6 bisphosphate in to 2. Te two are glyceraldehyde 3- phosphatte and dihydoxyacetone phosphate. 3 Aldolase were found in various types of bacteria and eukaryotes. It has important role not only in glycolysis but also in gluconeogenesis and metabolism of fructise. 4-5 Earlier studies 4 also showed that activity losses due to inhibition by AA and due to dilution, 5-6 were prevented by the presence of rabbit muscle aldolase. Rexaminations of enolase and other glycolytic enzymes inhibitons by AA were undertaken to determine whether or not inhibitions of enolase by AA were also prevented by aldolase. A report by others 7-8 that a AA-fatty acid derivative inhibited cancer growth efects of other AA-fatty acid derivatives on enolase activity. Mutual protection of PFK-1, aldolase, enolase and LDH from inhibitions were investigated. Comparitive studies of enolase and aldolase efects on PFK-1 activity loses by dilutions, by AA inhibitions, and by AA fatty acid inhibitions were made. MATERIALS AND METHODS Materials Te L-ascorbate (AA), L-ascorbyl dibutyrate (AADB), L-ascorbyl dipalmitate (AADP), L-ascorbyl palmitate (AAP), and L- ascorbyl stearate (AS) are shown in Figure 1 and were obtained from TCI and Alfa Aesar. Enzymes and materials given below were from Sigma (catalog numbers) unless stated otherwise. Methods Unless otherwise stated, all enzymes come from rabbit and all experimental temperatures were 25°C, pH 8.0. Compared to the main enzyme activity, it was determined that enzyme enolase (E0379) contained activities ≥ 0.05% by LDH or by PFK-1; LDH contained ≥ 0.01% enolase activity; and aldolase (A8811) contained ≥ 0.05% PFK-1. AA-fatty acid derivatives were dissolved in ethanol or dimethyl sulfoxide (DMSO, D8418). It can be shown that neither 15% ethanol nor 15% DMSO inhibited any enzymes used in these studies, more than the upper limit of the solvents used. Minimum six experiemnts were performed. Less than ± 10% standard deviation from the mean (SEM) than Data were acceptable. ± 10% error bars represent the SEM. Rabbit Muscle PFK-1 Preparation Purifed rabbit muscle PFK-1 in these experiments was presented previously 5 according to the method of Kemp 9 and stored as a 60% saturated ammonium precipitate until ready for use. A single band in SDS PAGE was used for PFK-1 samples in all muscle of rabbit as shown Inhibition by Ascorbate among Phosphofructokinase-1, Aldolase, Enolase, and Lactate Dehydrogenase in Rabbit Muscle Anita Ricablanca 1 , Ami Abbott 1 , Fatimata Sonago 1 , Montserrat Garcia 1 , Rachel Primacio 1 , Eric Patten 1 , Mudassar Iqbal Arain 2 , Eduardo Fricovsky 2, * 1 Department of Medical Education, School of Medicine University of California, San Diego, La Jolla, CA 92093, USA. 2 Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California San Diego, San Diego, La Jolla, CA, USA. ABSTRACT Background: These studies examined mutual protective relationships among rabbit muscle aldolase, enolase, phosphofructokinase-1 (PFK-1) and LDH from inhibitions by ascorbate (AA). It was proposed earlier that specifc inhibitions of PFK-1 and LDH by AA faciltated glycogen storage in resting muscle by inhibiting glycolysis. Materials and Methods: The L-ascorbate (AA), L-ascorbyl dibutyrate (AADB), L-ascorbyl dipalmitate (AADP), L-ascorbyl palmitate (AAP), and L- ascorbyl stearate (AS) are shown in Figure 1 and were obtained from TCI and Alfa Aesar. Unless otherwise stated, all enzymes come from rabbit and all experimental temperatures were 25°C, pH 8.0. Results: Rabbit muscle enolase was examined for its protective effect on other rabbit muscle glycolytic enzymes against inhibitions by ascorbate (AA) and some AA-faty acid derivatives. The IC 50 values of enolase by ascorbate (AA) and IC 50 values of AA-fatty acid derivatives were compared to estimate inhibition potency. For example, ascorbyl dipalmitate (AADP) was 156 times more inhibitory to enolase than AA. It was previously shown that rabbit muscle aldolase prevented LDH activity loses due to AA inhibition and prevented PFK-1 activity losses due both to dilution and AA inhibition; enolase was found to have the same effects as aldolase. Additionally, PFK-1 prevented enolase and LDH inhibitions by AA. LDH did not prevent enolase or PFK-1 from inhibition by AA. LDH did stimulate enolase activity but not PFK-1 activity. Conclusion: The results suggest that interactions among glycolytic enzyme serve to mutually protect one another from activity losses. The inhibition properties of the AA-fatty acid derivatives are discussed in relation to their possible roles in cancer and diabetes. Keywords: Phosphofructokinase-1, Aldolase, Enolase, Ascorbate, Rabbit. Correspondence Dr. Eduardo Fricovsky Professor of Clinical, University of California, San Diego, La Jolla, CA 92093-0690, U.S.A. Email id: esfricovsky@health.ucsd.edu DOI: 10.5530/ijpi.2022.3.55 Copyright © 2022 Author(s) et al. Exclusive Licensee Phcog.Net. Distributed under a Creative Commons Attribution License (CC BY 4.0). This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike 4.0 License, which allows others to remix, tweak, and build upon the work non-commercially, as long as the author is credited and the new creations are licensed under the identical terms.