Atsuko Shimizu

High levels of immunoreactive nervous system-specific enolase in sera of patients with neuroblastoma

Serum levels of nervous system-specific enolase (NSE, gamma gamma form plus alpha gamma form) in patients with neuroblastoma and in control subjects were determined with a sensitive solid-phase sandwich enzyme immunoassay system. Serum levels of NSE in healthy adults ranged from 1.4-5.7 ng/ml (2.87 +/- 1.18 ng/ml, n = 20), and in control children (1-7 years old) from 2.6 to 10.8 ng/ml (5.76 +/- 2.42 ng/ml, n = 20). Serum samples (n = 13) from patients with neuroblastoma contained high levels of NSE, range 13.6 to 330 ng/ml (mean 96 ng/ml); however those (n = 7) from ganglioneuroblastoma patients were within a normal range (3.0-25.0 ng/ml; mean 8.3 ng/ml). These results suggested that the NSE in serum might be a valuable marker substance for screening and therapeutic monitoring of neuroblastoma.

Human Brain‐Type Glycogen Phosphorylase: Quantitative Localization in Human Tissues Determined with an Immunoassay System

Abstract: Glycogen phosphorylase (EC 2.4.1.1) from human brain tissue was purified to homogeneity. Antisera were developed in rabbits with purified phosphorylase as the immunogen. Antibodies were first affinity‐purified with a column of brain phosphorylase‐coupled Sepharose, and then the antibody fraction was adsorbed with a column of muscle phosphorylase‐coupled Sepharose to remove antibodies reactive also with muscle phosphorylase. By using the specific antibodies, a sandwich‐type immunoassay system for measurement of brain phosphorylase was prepared. The assay system consisted of polystyrene balls with immobilized antibrain phosphorylase F(ab′)2 fragments and the same antibody Fab’ fragments labeled with β‐D‐galactosidase from Escherichia coli. The assay was sensitive and specific to the brain phosphorylase. The minimum detection limit of the assay was 0.1 ng/assay tube, and the cross‐reactivity of the assay with muscle phosphorylase was <1%. Tissue concentrations of immunoreactive brain‐type phosphorylase were estimated. The phosphorylase was present in the heart at as high a level as in the brain. The immunoreactivity for brain phosphorylase was distributed widely at a significant concentration in various peripheral tissues, such as the digestive tract, bladder, aorta, liver, and testis. Immunohistochemical localization of brain phosphorylase in the CNS revealed that the enzyme is present in most astrocytes and amyloid bodies, as well as in some neurons in the cerebral cortex and Golgi cells in the cerebellar cortex.

Tissue distribution, developmental profiles and effect of denervation of enolase isozymes in rat muscles

The tissue distribution of muscle-type alpha beta and beta beta enolases in rats were determined with the sandwich-type enzyme immunoassay method which utilized the purified antibodies specific to the alpha and to the beta subunit of enolase, and beta-D-galactosidase from Escherichia coli as label. All the tissues examined contained detectable levels of both alpha beta and beta beta enolases. The beta beta enolase was found at high levels in the skeletal muscle tissues (tongue, esophagus, diaphragm and leg muscles) and in the cartilages (xipoid process and auricular cartilage). The alpha beta enolase was distributed at a relatively high concentration in the heart and in the above-mentioned tissues. The beta beta enolase in the leg muscles, diaphragm and tongue was present on the day of birth at a concentration higher than that of the alpha alpha and alpha beta enolases, and its concentration further increased in a manner apparently related to the functional state of each tissue. Denervation of the leg muscles by cutting the sciatic nerve in adult rats resulted in a drastic change in the isozymes profile. The concentration of beta beta enolase in the tibialis anterior gastrocnemius lateralis and extensor digitorum longus (about 800 pmol/mg protein) decreased to about a half in a few weeks after denervation. In contrast, the concentrations of alpha alpha (2 pmol/mg) and alpha beta (80 pmol/mg) usually showed a slight increase by the treatment (alpha alpha, 7 pmol/mg; alpha beta, 100 pmol/mg after 2 weeks). As compared with these three muscles, the soleus had normally a low enolase level and the effect of denervation was less drastic. These results seem to suggest that the concentration of beta beta enolase is closely correlated with the functional state of the muscle tissue.

Purification of two enolases from human brain using a chromatofocusing column

When a purified preparation of rat alpha gamma enolase (2-phospho-D-glycerate hydrolyase, EC 4.2.1.11) was applied to a chromatofocusing column, the enolase was almost completely dissociated and recombined to form alpha alpha and gamma gamma enolases, which were eluted at different fractions from the column. Using these phenomena, two homo-dimer forms (alpha alpha and gamma gamma) of human brain enolase were purified from a crude preparation of the hybrid alpha gamma enolase by the chromatofocusing, and subsequent chromatography on a QAE-Sephadex column (alpha alpha) or a DEAE-Sephadex column (gamma gamma). Each purified preparation showed a single band on SDS-gel electrophoresis with a relative mobility corresponding to a molecular weight of about 50 000. Amino acid analysis, peptide mapping analysis with a limited proteolysis and immunochemical studies of the purified alpha alpha and gamma gamma enolases revealed that the two subunits, alpha and gamma, are distinct proteins. The antisera to human alpha alpha or gamma gamma enolase cross-related with the respective form of rat enolase.

Hybridization and isolation of human-rat βγ Enolase

Abstract A human-rat hybrid form of enolase (βγ) was prepared by dissociation and reassociation of a mixture of human ββ and rat γγ enolases, followed by isolation of the hybrid form from the parental homodimeric enolases with DEAE-Sephadex column chromatography. The human-rat βγ enolase had a specific activity similar to those of human ββ and rat γγ enolases. The optimal pH, stability against heating, and Km for 2-phosphoglycerate of the hybrid enolase were also similar to those of the homodimeric enolases.