Decreased glutamate dehydrogenase protein in spinocerebellar degeneration.

Abstract

SUMMARY A radioimmunoassay system for determining content of glutamate dehydrogenase (GDH) in human leukocytes was established and studied in 14 patients with spinocerebellar ataxia or atypical Parkinsonism. The protein content of leukocyte GDH was decreased in four patients and the reduction in the protein content was proportional to that in the enzyme activity. The ratio of GDH activity to protein content was invariable in healthy controls, diseased controls and patients with reduced GDH activity. These results suggested that at least a portion of the partial GDH deficiency was due to the decreased level of the enzyme protein.Spinocerebellar degeneration is a heterogeneous disease complex, and the majority of the diseases have not been characterised biochemically. Recently, several authors have reported the partial deficiency in glutamate dehydrogenase (GDH; EC 1.4.1.3) activity in leukocytes, fibroblasts, platelets and muscles from patients with nondominant or dominant form of olivopontocerebellar atrophy (OPCA).' -These findings suggested that systemic abnormality of glutamate metabolism might be related to a subgroup of OPCA. However, it remains unclear whether or not GDH deficiency is aetiologically significant, and little is known about the mechanism by which GDH activity is reduced.In this study, we established a radioimmunoassay for determination of GDH protein content in human leukocytes and studied patients with spinocerebellar ataxia. Materials and methodsFourteen patients with spinocerebellar degeneration or atypical Parkinsonism and eight age-matched, healthy controls were studied. Four patients who showed significantly lower level of GDH activity than controls are presented below. No members with similar diseases were known in the families of each case. Decreased glutamate dehydrogenase protein in spinocerebellar degenerationEnzyme assay of leukocyte GDH Leukocytes were separated with 6% dextran from heparinised venous blood, and were disrupted by freeze and thaw cycles. These cells were subjected to enzyme assay and radioimmunoassay. GDH activities were measured fluorometrically by a modification of the method described by Plaitakis et al.2 Aliquots of the homogenate were incubated at 47 5'C for 60 minutes. The activity determined after incubation was considered to represent "heat-stable" GDH. This value was subtracted from the total activity to obtain the activity of "heat-labile" GDH.Radioimmunoassay of leukocyte GDH protein GDH proteins purified from bovine liver, rat liver and bacteria (Proteus species) were purchased from Sigma USA. Rabbits were subcutaneously injected with 1 mg of bovine liver GDH emulsified in Freund's complete adjuvant at 2-week intervals and were bled 7 days after the third injection. The specificity and cross-reactivity of the antiserum was tested by immunoblot analysis with protein extracts from rat cerebellum and liver, and by ELISA using GDH purified from bovine liver, rat liver and bacteria as antigens. Crossreactivity of the antiserum with human GDH was evidenced ...

Important role of Ser443 in different thermal stability of human glutamate dehydrogenase isozymes1

“…These differences are consistent with the electrophoretic characteristics of GDH isozymes purified from human brain [22]. Previous studies showed that GDH activities were differentially altered in neurologic patients [33–38], thus suggesting that these activities are under different genetic control. Since electroretinographic abnormalities have been detected in patients with spinocerebellar ataxia who have a selective deficiency of the heat‐labile GDH [2,38], studies on the molecular basis for the different thermal stability of the GDH isozymes may thus be of importance for understanding the biology of the human nervous systems and the genetic analysis of X‐linked neurodegeneration.…”

Nerve Tissue‐Specific Human Glutamate Dehydrogenase that Is Thermolabile and Highly Regulated by ADP

“…Our work with neurologic patients led to the finding that GDH is present in human tissues in "heat-labile" and "heat-stable" forms and that, in these patients, reduction in GDH activity was largely limited to the heat-labile component (Plaitakis et al, 1984). Similar results have been obtained by other (Konagaya et al, 1986;Kajiyama et al, 1988;Iwattsuji et al, 1989;Abe et al, 1992) but not all (Aubby et al, 1988;Duvoisin et al, 1988) investigators. Additional studies in our laboratory (Colon et al, 1986) showed the presence of two GDH activities in rat brain differing in their relative resistance to thermal inactivation, detergent extractability, and allosteric regulation characteristics.…”

Amino Acid Changes within Antenna Helix Are Responsible for Different Regulatory Preferences of Human Glutamate Dehydrogenase Isozymes

“…Previous studies showed that GDH activities were differentially altered in neurologic patients (29,30), thus suggesting that these activities are under different genetic control. Because electroretinographic abnormalities have been detected in patients with spinocerebellar ataxia who have a selective deficiency of the heat-labile GDH isozymes (10,(31)(32)(33)(34), studies on the molecular basis for the different molecular properties of the GDH isozymes may, thus, be of importance for understanding the biology of the human nervous systems and the genetic analysis of X-linked neurodegeneration. Previously, two types of mitochondrial glutamate carrier (GC1 and GC2) were identified differing in their V max and K m values and in their tissue distribution (35).…”