Purification and properties of rabbit brain and liver 4-aminobutyrate aminotransferases isolated by monoclonal-antibody immunoadsorbent chromatography

Abstract

The use of a monoclonal-antibody immunoaffinity column for the rapid isolation of 4-aminobutyrate aminotransferases (EC 2.6.1.19) from rabbit brain and liver is described. Homogeneous enzyme protein is eluted from the immunoadsorbent with 100mM-citrate buffer, pH5, and remains stable at 4 degrees C for several days. One such column (bed volume 8 ml) has been used 40 times in a 9-month period to isolate 10-15 units of enzyme activity (specific activity approx. 3.5-7.5 units/mg) per extraction. Kinetic and spectral analysis of the enzymes from the two tissues revealed a close similarity. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis showed the isolated enzyme to have a monomeric Mr of 52 000, and this was confirmed by h.p.l.c. gel exclusion at pH 5.0. The results of Sephadex G-100 chromatography at different pH values are taken to indicate that the enzyme behaves as a dimer at pH 7.0 and above, but as a monomer at pH 5.0. 4-Aminobutyrate aminotransferase isolated from the brain by the procedure of Fowler & John [(1981) Biochem. J. 197, 149-152] is more stable than the immunoaffinity-purified material, and has been shown to contain a contaminant protein of Mr 84 000 that exhibits succinic semialdehyde dehydrogenase activity.

Characterization of Monomeric 4‐Aminobutyrate Aminotransferase at Low pH

“…2, the enzyme was eluted as a monomer over a narrow pH range, i.e., 5.0-5.2, whereas a dimer is predominantly eluted from the column when the pH of the eluant buffer is above pH 5.8. This result confirms the previous observations of Kirby et al [4], who reported the dissociation of dimeric Gaba-t by decreasing the pH of the medium.…”

“…The rotational correlation time of the pyridoxal-P form of the enzyme determined at neutral pH indicates strong interaction between the protomers even on the nanosecond time scale [3]. However, gel filtration experiments conducted at low pH values indicate that the elution profile of the aminotransferase undergoes remarkable changes at pH 5.0 [4].…”

“…The rotational correlation time of the pyridoxal-P form of the enzyme determined at neutral pH indicates strong interaction between the protomers even on the nanosecond time scale [3]. However, gel filtration experiments conducted at low pH values indicate that the elution profile of the aminotransferase undergoes remarkable changes at pH 5.0 [4].Although it has been suggested that a process of dissociation of the dimeric enzyme is responsible for changes in the elution profiles, the effect of low pH on the secondary and tertiary structure of the monomeric species remains to be assessed.The present work deals with a study of the stability of Gaba-t at pH 5.0 using different physical techniques.Circular dichroism is used to probe the secondary structure of the protein and to examine the mode of binding of the cofactor pyridoxal-P, whereas nanosecond fluorescence spectroscopy is applied to the characterization of the rotational correlation time of monomeric species in solution. The analysis of the reversible denaturation of Gaba-t at pH 5.0 is useful for the investigation of the mechanism of folding of the enzyme, since it is Correspondence to J. E. Churchich, Department of Biochemistry, University of Tennessee, Knoxville, TN 37996-0840, USA Abbreviations.…”

Partucularities of g-aminobutyric acid metabolism in the liver of rats during different types of alcohol withdrawal

“…В гепатоцитах были обнаружены полный набор ферментов обмена ГАМК, аналогичный головному мозгу, однако печеночные формы ферментов катаболизма ГАМК являются более неспецифическими [21][22][23]. Так, ГАМК-Т печени способна к переаминированию не только ГАМК, но и b-аланина и пищевых w-аминокислот, а её функции отличаются от функций фермента, локализованного в нервной ткани, поскольку ГАМК не является в печени нейромедиатором [21,22]. Позже было показано, что первично в печени происходит синтез ГАМК-Т, по строению аналогичной ферменту мозгового типа.…”

Candida .delta.-aminovalerate:.alpha.-ketoglutarate aminotransferase: purification and enzymologic properties

“…A few data relative to -aminotransamination are available in mammalian tissues (Roberts & Bregoff, 1953) and microorganisms . Especially 7-aminobutyrate transaminase (Churchich, 1982; Yonaha & Toyama, 1980a,b; White & Sato, 1978;Schousboe et al, 1973;Bloch-Tardy et al, 1974;John & Fowler, 1976; Kirby et al, 1985) and /3-alanine transamination (Yonaha & Toyama, 1980b;Yonaha et al, 1983Yonaha et al, , 1985aNishizuka et al, 1959;Hayaishi et al, 1961) have been investigated. -Aminovalerate transamination has been observed in beef brain (Baxter & Roberts, 1958), pig liver (Buzenet et al, 1978), mouse brain (Schousboe et al, 1973), Pseudomonas SP 126 (Yonaha & Toyama, 1980a), and acetone powder of brain, liver, Escherichia coli and Aspergillus fumigatus (Roberts, 1954), but only one -aminovalerate aminotransferase (2.6.1.48) within our knowledge has been partially purified from lysine-adapted cells of Pseudomonas (Ichihara et al, 1960).…”