No abstract

Amounts of glutamate metabolizing enzymes such as glutamate dehydrogenase (GDH), glutamine synthetase (GS), GS-like protein (GSLP), and phosphate-activated glutaminase (PAG) were compared in prefrontal cortex of control subjects and patients with Alzheimer disease (AD). The target proteins were quantified by ECL-Western immunoblotting in extracts from brain tissue prepared by two different techniques separating enzymes preferentially associated with cytoplasm (GDH I and II isoenzymes, GS, and partially GSLP) and membrane (GDH III, PAG, and partially GSLP) fractions. Amounts of all listed enzymes were found significantly increased in the patient group compared with controls. Some links between the measured values were observed in the control, but not in the AD patient group. The results may suggest for the pathological interruption of regulatory relations between distinct enzymes of glutamate metabolism in brain of AD patients.

Glutamine synthetase (GS; EC 6.3.1.2), a key enzyme of glutamate metabolism, and another enzyme possessing high hydroxylamine-L-glutamine transferase activity comparable to that of GS and termed GS-like protein (GSLP) were purified from human brain concurrently. In two-dimensional electrophoresis, GS subunits migrate to at least six different positions (44 Ϯ 1 kDa, pI ϭ 6.4 -6.7), whereas GSLP subunits migrate to at least four different positions (54 Ϯ 1 kDa, pI ϭ 5.9 -6.2). Dependences of enzymatic activity in the transferase reaction on concentrations of Mn 2ϩ and Mg 2ϩ for GS and GSLP are different. High immunological cross-reactivity between GS and GSLP was observed in ELISA. Nevertheless, antisera were raised to GS and GSLP, and a method was developed for the separate detection of GS and GSLP in brain extracts by enzyme-chemiluminescent amplified (ECL) immunoblotting. The distribution of GS and GSLP immunoreactivities between soluble protein and crude mitochondrial fractions indicates tighter association with the particulate fraction for GSLP than for GS. The results from activity measurements suggest that the hydroxylamine-L-glutamine transferase activity measured routinely in protein extracts from brain is the sum of GS and GSLP activities. Similarly, immunoreactivity evaluated by ELISA is a sum of immunoreactivities of GS and GSLP. The relative contributions of GS and GSLP to the total immunoreactivity can be evaluated by ECL-immunoblotting. Key Words: Glutamine synthetase -Hydroxylamine-L-glutamine transferase -Human brain.

The content of casein kinase 2 is considerably decreased in ribosome‐free extracts of the frontal cortex of schizophrenic and Alzheimer's disease patients in comparison to normal brains as has been demonstrated by means of immunoblotting. The activity of casein kinase 2 towards endogenous substrates and casein is also diminished in the cases of mental pathologies examined. This phenomenon may explain the well‐known aberrations in the phosphorylation of structural proteins of human brain which are intrinsic for the mental diseases.

Creatine kinase (CK) is responsible for the creatine/creatine phosphate level which that is known to alter in the brain of patients with schizophrenia. A comparative estimation of CK enzymatic activity and immunoreactivity of CK BB was carried out in readily soluble extracts from frontal cortex, anterior and posterior cingulate cortex, hippocampus and cerebellum from brains of individuals with schizophrenia versus normal controls. CK activity was determined using a commercial diagnostic kit. CK BB immunoreactivity was evaluated by ECL -immunoblotting using monoclonal antibody. A drastic drop of CK activity and CK BB immunoreactivity was observed in all the examined brain areas in schizophrenia patients compared to controls (p<0.01), with the maximum drop in the cerebellum. The reduction was independent of age, postmortem interval or chlorpromazine equivalent. The decreased level of CK BB in schizophrenia was confirmed by purification of CK BB from brains of patients with schizophrenia and control brains: the yield of the purified enzyme was significantly lower in schizophrenia, wherein molecular masses of CK B-subunits were equal. Possible causes and consequences of the decrease in CK BB level observed in brain of patients with schizophrenia are discussed.