No abstract

No abstract

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purifled by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the a carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the posttranslational condensation with the phosphatidylinositolglycan.Membrane proteins vary greatly in the nature and extent of their interactions with the lipid bilayer. A number of diverse cell-surface proteins are anchored in plasma membranes by a phosphatidylinositol-glycan (PI-G) structure that is covalently attached to the carboxyl-terminal amino acid of the mature protein (1-3). Alkaline phosphatase [AP; nonspecific octophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] has been identified as a PI-G-tailed protein (4, 5). The mature enzyme, which is widely distributed in mammalian tissues, can be released from cellular membranes by phosphatidylinositol-specific phospholipase C (6, 7). Studies of AP, biosynthetically radiolabeled in cell culture with components of the putative PI-G moiety (8, 9), further support the initial classification of AP as a PI-Gtailed membrane protein. In higher primates and in man three isozymes of AP are present-namely, intestinal, placental, and the tissue-unspecific form present in liver, bone, kidney, and most other tissues (10,11). The AP isozymes are highly glycosylated homodimers that have subunit molecular masses ranging from 60 to 80 kDa. The cDNA sequences of all three major types of mammalian AP have been deduced (12-17), and they all indicate the presence of a stretch of -20 hydrophobic amino acid residues at the carboxyl terminus of the nascent protein. It is believed, by analogy to the two best understood PI-G-tailed proteins, variant surface glycoprotein of Trypanosoma brucei (18-22) and Thy-1 antigen (23)(24)(25)(26), that after synthesis on membrane-bound ribosomes the nascent form of AP is modified by (i) removal of an amino-terminal signal sequence, (it) addition of N-linked oligosaccharides, (ifi) replacement of a carboxyl-terminal hydrophobic peptide extension with a PI-G moiety that serves as a membrane anchor. The length of the carboxylterminal peptide that is removed from the nascent form of AP and the exact site of PI-G attachment, presumably via an amide bond between the amino group of ethanolamine and the carboxyl group of the carboxyl-terminal amino acid, are not known. To elucidate the post-translational modification of PLAP at its carboxyl terminus, we have purified the carboxyl-terminal chy...

No abstract

Alkaline phosphatase is anchored to the plasma membrane by a carboxyl-terminal phosphatidylinositol glycan moiety. To investigate the biosynthesis of mature alkaline phosphatase, nascent human placental alkaline phosphatase was expressed in a cell-free system and used as substrate for in vitro processing by microsomal extracts. By monitoring the processed product with three site-directed antibodies, it was shown that microsomal extracts from CHO cells that contain other recognized processing activities also remove the carboxyl-terminal signal peptide from the preproenzyme in an apparently selective manner. This peptidaselike cleavage may be brought about by the action of a specific transamidase acting on the nascent protein in the absence of an appropriate phosphatidylinositol glycan cosubstrate.

SUMMARYThe pH-dependent profiles of haemolysis by influenza viruses AHK/8/68 (HK virus) and APR/8/34 (PR8 virus) were found to possess characteristic differences. Both viruses were highly haemolytic at pH 5.0, but while HK virus-induced haemolysis was undetectable above pH 5.5 to 5.6, PR8 virus-induced haemolysis persisted at higher pH values, becoming undetectable above pH 5.7 to 5.8. In order to determine whether these haemolysis profiles were genetically determined, we studied the pH dependence of haemolysis by eight recombinants of HK and PR8 viruses. The pH profile of each recombinant clearly resembled that of one parent or the other, showing that it is an inherited trait. The pH profile was found to be conferred exclusively by the haemagglutinin (HA) gene.

No abstract

No abstract