ABSTRACT
The presence of catechol-O-methyltransferase (COMT) in the rat brain was studied using a combination of two-dimensional gel electrophoresis (2-DE), protein blotting and a specific antiserum. Two major immunoreactive proteins were identified-one with mol. wt 23 kdalton and an isoelectric point of 5.2, the other of mol. wt 25 kdalton and an isoelectric point of 5.1. In addition, multiple lower molecular weight immunoreactive proteins, possibly corresponding to breakdown products of the enzyme, were also detected. The 23 kdalton form of COMT, which is probably the soluble form of the enzyme, is a major protein visible on silver-stained 2-D gels of rat brain. In contrast, the other proteins recognized by the antiserum were not detected by the silver stain. These results demonstrate, using 2-DE, that at least two distinct forms of catechol-O-methyltransferase are present in rat brain. In addition, since one of these proteins is stained by silver, these results also serve to identify another protein visible on 2-D electrophoretograms of rat brain.
ABSTRACT
The subcellular distribution of proteins normally visible on two-dimension gels of rat brain tissue punches and crude brain homogenate was investigated using two-dimensional gel electrophoresis and computerized scanning densitometry. Seven enriched subcellular fractions (cytosol, mitochondria, microsomes, nucleus, crude synaptic vesicles, myelin and synaptic membrane) were generated from a crude extract of rat brain. Fifty microgram samples of the crude homogenate and each fraction were then taken and the proteins within these samples separated by two-dimensional gel electrophoresis. Proteins were stained with silver and the gels then analyzed by computerized scanning densitometry. Of 136 proteins visible on two-dimension gels of the crude homogenate that were quantitatively examined, a total of 73 (54%) were identified as being primarily located in a single subcellular fraction. The majority of these 73 proteins were found to be located primarily in either the cytosolic or mitochondrial fractions, while fewer proteins were identified as being primarily located in the microsomal, nuclear or crude synaptic vesicular subfractions. In contrast, the myelin and synaptic membrane fractions were found to be the primary location for only a single protein each that is clearly visible in the crude homogenate. In addition, gels of four of the subfractions (mitochondria, cytosol, nucleus and myelin) contained proteins that are not normally visible on gels generated using a crude extract. The subcellular location of a number of proteins found previously to be altered by specific experimental manipulations was also determined, providing further information on these proteins in brain. These results should prove useful in future experiments designed towards isolating and characterizing specific proteins of neurochemical interest.
ABSTRACT
Using a sensitive and specific antiserum, the existence of two subforms of soluble glutamic oxaloacetic transaminase in both rat and human brain has been demonstrated. In the rat, the two proteins each have a molecular mass of 47,000 daltons. The more abundant basic protein has an isoelectric point of 5.9 while the sparsely staining more acidic protein has an isoelectric point of 5.8. In the human the two proteins visualized each have a molecular mass of 43,000 daltons. The more abundant basic protein has an isoelectric point of 5.7 while the sparsely staining more acidic protein has an isoelectric point of 5.6. In both rat and human, it seems reasonable to conclude that the abundant basic protein that reacts with the antiserum is the ? subform of the enzyme, while the sparsely staining more acidic protein is the ? subform of the enzyme. These results are of interest for a number of reasons. First, they establish the existence of at least two subforms of soluble glutamic oxaloacetic transaminase in both rat and human brain. Secondly, they show that this enzyme is a major protein visible on two-dimension gels of rat and human brain and that this enzyme is widely distributed throughout the central nervous system. Finally, they positively identify two more proteins visible on two-dimension gels generated using rat or human brain tissue.
