Physico-chemical characteristics and regional distribution studies of GP-350, a soluble sialoglycoprotein from brain.

Abstract

AbstractThe apparent molecular weight of GP‐350, a sialoglycoprotein from calf and rat brain, has been determined by SDS‐polyacrylamide gel electrophoresis. The electrophoretic mobility corresponds to the mobility of a polypeptide with a molecular weight of 11,600 ± 200. On this basis it can be calculated that only one sialic acid residue is present/GP‐350 molecule.From isoelectric focusing experiments it appeared that the isoelectric point of GP‐350 is about 2. The determination of the amide content of the polypeptide chain showed that out of 22.0 acidic amino acid residues of glutamic acid and aspartic acid, only 4.9 residues are amidated. The total amount of the basic amino acid residues lysine and arginine, is 6.5. So, per molecule GP‐350 10.6 acidic amino acid residues are not counteracted by basic amino acid residues. The surplus of the acidic amino acid residues as well as sialic acid result in the pronounced acidic character of GP‐350. This fact is supported by the electrophoretic experiments.The carbohydrate‐polypeptide linkage type has been studied by alkaline sodium borohydride treatment. Two thirds of all the galactosamine was destroyed, whereas the amount of glucosamine remained the same. Amino acid analysis indicated a decrease in serine and threonine with a concomitant small increase in alanine. These data point to the occurrence of linkages between the carbohydrate chain and the polypeptide core of the galactosamineserine or –threonine type. Per molecule GP‐350 about two residues of galactosamine are destroyed, indicating that two carbohydrate chains of this binding type are present. Only one of these chains can be terminated by a sialic acid residue. The other carbohydrate chain may be terminated by fucose.Regional distribution studies showed the presence of GP‐350 in all brain areas studied; in relatively large amounts in the regions rich in ganglia such as caudate nucleus, cerebellar grey matter, pons and medulla oblongata, and in relatively small amounts in the regions poor in ganglia such as corpus callosum, cerebral white, cerebral grey and cerebellar white matter.GP‐350 is also present in the pituitary gland. In the cerebrospinal fluid a glycoprotein is present with the same electrophoretic mobility as GP‐350. However, this glycoprotein gave no precipitin reaction with GP‐350 specific antiserum. Moreover, the amino acid composition was quite different from that of GP‐350.Subcellular distribution study revealed that GP‐350 is present in the soluble cell fraction and in the synaptosomal membrane fraction, whereas it is absent from the purified nuclei, mitochondria, myelin, and also from the microsomal fraction.