Purification, Characterization and Identification of Rat

نویسندگان

  • TOMOO NOGUCHI
  • ETSUO OKUNO
  • YOHSUKE MINATOGAWA
  • RYO KIDO
چکیده

1. Histidine-pyruvate aminotransferase (isoenzyme 1) was purified to homogeneity from the mitochondrial and supernatant fractions of rat liver, as judged by polyacrylamide-gel electrophoresis and isoelectric focusing. Both enzyme preparations were remarkably similar in physical and enzymic properties. Isoenzyme 1 had pI8.0 and a pH optimum of 9.0. The enzyme was active with pyruvate as amino acceptor but not with 2-oxoglutarate, and utilized various aromatic amino acids as amino donors in the following order of activity: phenylalanine >tyrosine >histidine. Very little activity was found with tryptophan and 5-hydroxytryptophan. The apparent Km values were about 2.6mM for histidine and 2.7mM for phenylalanine. Km values for pyruvate were about 5.2mM with phenylalanine as amino donor and 1.1 mM with histidine. The aminotransferase activity of the enzyme towards phenylalanine was inhibited by the addition of histidine. The mol.wt. determined by gel filtration and sucrose-density-gradient centrifugation was approx. 70000. The mitochondrial and supernatant isoenzyme 1 activities increased approximately 25-fold and 3.2-fold respectively in rats repeatedly injected with glucagon for 2 days. 2. An additional histidine-pyruvate aminotransferase (isoenzyme 2) was partially purified from both the mitochondrial and supernatant fractions of rat liver. Nearly identical properties were observed with both preparations. Isoenzyme 2 had pI5.2 and a pH optimum of 9.3. The enzyme was specific for pyruvate and did not function with 2-oxoglutarate. The order of effectiveness of amino donors was tyrosine = phenylalanine> histidine >tryptophan > 5-hydroxytryptophan. The apparent Km values for histidine and phenylalanine were about 0.51 and 1.8mM respectively. Km values for pyruvate were about 3.5mm with phenylalanine and 4.7mM with histidine as amino donors. Histidine inhibited phenylalanine aminotransferase activity ofthe enzyme. Gel filtration and sucrose-densitygradient centrifugation yielded a mol.wt. of approx. 90000. Neither the mitochondrial nor the supernatant isoenzyme 2 activity was elevated by glucagon injection.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

Blood Coagulation Induced by Iranian Saw-Scaled Viper (Echis Carinatus) Venom: Identification, Purification and Characterization of a Prothrombin Activator

  Objective(s): Echis carinatus is one of the venomous snakes in Iran. The venom of Iranian Echis carinatus is a rich source of protein with various factors affecting the plasma protein and blood coagulation factor. Some of these proteins exhibit types of enzymatic activities. However, other items are proteins with no enzymatic activity.   Materials and Methods: In order to study the mechanism ...

متن کامل

A taxonomic study of blue-green algae based on morphological, physiological and molecular characterization in Yazd province terrestrial ecosystems (Iran)

Based on the limited resources available to algal flora in Yazd province in the center of Iran, cultivation, purification and identification of soil algae from seven sites was performed in summer 2013. The soil samples were processed for analysis of Cd, Pb contents. The present paper attempts to analyze survey of soil blue-green algal identification in different types of stations, using morphol...

متن کامل

Purification and Characterization of Milk Clotting Enzyme Produced by Rhizomucor Rmiehei

Milk clotting enzyme (M CE) produced by: Rhizomucor miehei was purified and characterized.The enzyme was purified 220.29-fold with specific activity about 14444.2 U/mg protein byultrafiltration, ammonium sulfate fractionation, Sephacryl S-300 chromatography. The maximumenzyme activity was at 65°C.The milk clotting activity was decreased steadily as pH is increased and indicated maximumactivity ...

متن کامل

Purification and Characterization of a Novel Thermostable and Acid Stable α-Amylase from Bacillus Sp. Iranian S1

This study reports the purification and biochemical characterization of thermostable and acidic-pH-stable α-amylase from Bacillus sp. Iranian S1 isolated from the desert soil (Gandom-e-Beryan in Lut desert, Iran). Amylase production was found to be growth associated. Maximum enzyme production was in exponential phase with activity 2.93 U ml-1 at 50°C and pH 5. The enzyme was purified by isoprop...

متن کامل

RNA-based affinity purification reveals 7SK RNPs with distinct composition and regulation.

Recent studies have uncovered an unanticipated diversity of noncoding RNAs (ncRNAs), although these studies provide limited insight into their biological significance. Numerous general methods for identification and characterization of protein interactions have been developed, but similar approaches for characterizing cellular ncRNA interactions are lacking. Here we describe RNA Affinity in Tan...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره   شماره 

صفحات  -

تاریخ انتشار 2005