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ISOLATION AND PURIFICATION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE FROM THE GUT OF RHINOCEROS LARVA (ORYCTES RHINOCEROS)

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ISOLATION AND PURIFICATION OF 3-MERCAPTOPYRUVATE SULFURTRANSFERASE FROM THE GUT OF RHINOCEROS LARVA (Oryctes rhinoceros)

                                  CHAPTER ONE           1.0. INTRODUCTION AND LITERATURE REVIEW   1.1. INTRODUCTION     One of the major metabolic enzymes that have gained so much interest of scientists is 3-Mercaptopyruvate sulfurtransferase (3-MST). This enzyme occurs widely in nature (Bordo, 2002 and Jarabak, 1981).     It  has  been  reported  in  several  organisms  ranging  from humans  to  rats, fishes and insects. It is a mitochondrial enzyme which has been concerned in the detoxification of cyanide, a potent toxin of the mitochondrial respiratory chain (Nelson et  al.,  2000).  Among  the  several  metabolic  enzymes  that  carry  out xenobiotic  detoxification,  3-mercaptopyruvate  sulfurtransferase  is  of  utmost importance.         3-mercaptopyruvate  sulfurtransferase functions  in  the  detoxifications  of cyanide; mediation of sulfur ion transfer to cyanide or to other thiol compounds. (Vanden et al., 1967). It is also required for the biosynthesis of thiosulfate. In combination with cysteine aminotransferase, it contributes to the catabolism of cysteine and it is important in generating hydrogen sulphide in the brain, retina and vascular endothelial cells (Shibuya et al., 2009). It also acquired different functions  such  as  a  redox  regulation  (maintenance  of  cellular  redox homeostasis)  and  defense against  oxidative  stress,  in  the  atmosphere  under oxidizing conditions Nagahara et al (2005).       Hydrogen sulphide  (H2S)  is  an  important  synaptic  modulator,  signalling molecule, smooth muscle contractor and neuroprotectant (Hosoki et al., 1997). Its  production  by  the  3-mercaptopyruvate  sulfurtransferase  and  cysteine aminotransferase pathways is regulated by calcium ions (Hosoki et al., 1997).       Organisms  that  are  exposed  to  cyanide  poisoning  usually  have  this enzyme  in  them.  This  could  be  in  food as  in  the  cyanogenic glucosides being consumed. It has been studied from variety of sources, which include bacteria, yeasts, plants, and animals (Marcus Wischik, 1998).        Cyanide could be released into the bark of trees as a defence mechanism. There are array of defensive compounds that make their parts (leaves, flowers, stems,  roots  and  fruits)  distasteful  or  poisonous  to  predators.  In  response, however,  the  animals  that  feed  on  them  have  evolved  over  successive generations a range of measures to overcome these compounds and can eat the plant  safely.  The  tree  trunk  offers  a  clear  example  of  the  variety  of  defences available to plants (Marcus Wischik, 1998).       Oryctes rhinoceros larva is one of the organisms that are also exposed to cyanide toxicity because of the environment they are found. 1.2. 3-MERCAPTOPYRUVATE SULFURTRANSFERASE         3-Mercaptopyruvate  sulfurtransferase  (EC.  2.8.1.2),  is  a  member  of  the group,  Sulfurtransferases  (EC  2.8.1.1 – 5),  which  are  widely  distributed enzymes of prokaryotes and eukaryotes (Bordo and Bork, 2002).         3-Mercaptopyruvate  Sulfurtransferase  is  an  enzyme  that  is  part  of  the cysteine  catabolic pathway.  The  enzyme  catalyzes  the  conversion  3- mercaptopyruvate to pyruvate and H2S (Shibuya et al., 2009). The deficiency of this enzyme will result in elevated urine concentrations of 3-mercaptopyruvate as  well  as  of  3-mercaptolactate,  both  in  the  form  of  disulfides  with  cysteine (Crawhall et al., 1969). It catalyzes the chemical reaction: 3-mercaptopyruvate + cyanide à  pyruvate + thiocyanate 3-mercaptopyruvate + thiol   à   pyruvate + hydrogen sulphide (Sorbo 1957).     It  transfers  sulfur-containing  groups  and  participates  in  cysteine metabolism (Shibuya et al., 2013). This enzyme catalyzes the transfer of sulfane sulphur from a donor molecule, such as thiosulfate or 3- mercaptopyruvate, to a nucleophile acceptor, such as cyanide or mercptoethanol. 3-mercaptopyruvate is the  known  sulphur-donor  substrate  for  3-mercaptopyruvate  sulfurtransferase (Porter & Baskin, 1995).         3-mercaptopyruvate  sulfurtransferase  is  believed  to  function  in  the endogenous  cyanide  (CN)  detoxification  system  because  it  is  capable  of transferring sulphur from 3-mercaptopyruvate (3-MP) to cyanide (CN), forming the  less  toxic  thiocyanate  (SCN) (Hylin  and  Wood,  1959). It is  an  important enzyme  for  the  synthesis  of  hydrogen  sulphide  (H2S)  in  the  brain  (Shibuya et al., 2009).       The  systematic  name  of  this  enzyme  class  is 3-mercaptopyruvate: cyanide  sulfurtransferase.  It  is  also  called beta-mercaptopyruvate sulfurtransferase (Vachek and  Wood,  1972). It  is  one  of  three  known  H2S producing  enzymes  in  the  body (Hylin  and  Wood,  1959).  It  is  primarily localised in the mitochondria (Cipollone et al., 2008).        The expression levels of 3-MST in the brain during the fetal and postnatal periods are higher than those in the adult brain (unpublished data) although the promoter  region  shows  characteristics  of  a  typical  housekeeping  gene (Nagahara et al., 2004). The observation is supported by the finding that 3-MST expression  in  the  cerebellum  is  decreased  during  the  adult  period  (Shibuya et al., 2013). On the other hand, its expression level in the lung decreases from the perinatal period. These facts suggest that 3-MST could function in the fetal and postnatal brain. It was reported that serotonin signaling via the 5-HT1A receptor in  the  brain  during  the  early  developmental  stage  plays  a  critical  role  in  the establishment  of  innate  anxiety  during  the  early  developmental  stage (Richardson-Jones et al., 2011).     In  rat,  3-MST  possesses  2  redox-sensing  molecular  switches  (Nagahara and  Katayama,  2005).  A  catalytic-site  cysteine  and  an  intersubunit  disulfide bond serve as a thioredoxin-specific molecular switch (Nagahara et al., 2007). The  intermolecular  switch  is  not  observed  in  prokaryotes  and  plants,  which emerged into the atmosphere under reducing conditions (Nagahara, 2013). As a result, it acquired different functions such as a redox regulation (maintenance of cellular  redox  homeostasis)  and  defense  against  oxidative  stress,  in  the atmosphere under oxidizing conditions (Nagahara et al., 2005).       Moreover,  3-MST  can  produce  H2S  (or  HS−)  as  a  biofactor  (Shibuya et al.,  2009),  which  cystathionine  β-synthase  and  cystathionine  γ-lyase  also  can generate  (Abe  and  Kimura,  1996).  Interestingly  3-MST  can  uniquely  produce SOx in  the  redox  cycle  of  persulfide  formed  at  the  low-redox catalytic-site cysteine (Nagahara et al., 2012). As an alternate hypothesis on the pathogenesis of the symptoms, H2S (or HS−) and/or SOx could suppress anxiety-like behavior, and therefore, defects in these molecules could increase anxiety-like behavior. However,  no  microanalysis  method  has  been  established  to  quantify  H2S  (or HS−) and SOx at the physiological level (Ampola et al., 1969).       MCDU  was  first  recognized  and  reported  in  1968  as  an  inherited metabolic  disorder  caused  by  congenital  3-MST  insufficiency  or  deficiency. Most cases were associated with mental retardation (Ampola et al, 1969) while the pathogenesis remains unknown.       Human  MCDU  was  reported  to  be  associated  with  behavioral abnormalities, mental retardation (Crawhall, 1985), hypokinetic behaviour, and grand mal seizures and anomalies (flattened nasal bridge and excessively arched palate) (Ampola et al, 1969); however, the pathogenesis has not been clarified since MCDU was recognized more than 40 years ago. Macroscopic anomalies were  associated  in  1  case  (Ampola et  al,  1969);  however,  this  could  be  an accidental  combination.  3-MST  deficiency  also  induced  higher  brain dysfunction in mice without macroscopic and microscopic abnormalities in the brain. 3-MST seems to play a critical role in the central nervous system, i.e., to establish normal anxiety (Richardson et al., 2011) 1.2.1. DISTRIBUTION       3-MST  is  widely  distributed  in  prokaryotes  and  eukaryotes  (Jarabak, 1981).  It is  localized  in  the  cytoplasm  and  mitochondria,  but  not  all  cells contain 3-MST (Nagahara et al., 1998). 1.2.2. OCCURRENCE       Human mercaptopyruvate sulfurtransferase (MPST; EC. 2.8.1.2) belongs to  the  family  of  sulfurtransferases (Vanden et  al.,  1967).  These  enzymes MPST  has  a  preference  for  3-mercapto  sulfurtransferase  as  the  sulfur-donor. MPST  plays  a  central  role  in  both  cysteine  degradation  and  cyanide detoxification. In addition, deficiency in MPST activity has been proposed to be responsible  for  a  rare  inheritable  disease  known  as  mercaptolactate-cysteine disulfiduria (MCDU) (Hannestad et al, 2006).

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