Purification and characterization of alpha-N-acetylgalactosaminidases I and II from the starfish Asterina amurensis.

Two alpha-N-acetylgalactosaminidases, alpha-N-acetylgalactosaminidase (alpha-GalNAcase) I and II, were purified from the digestive organ of starfish. Purified alpha-GalNAcase I and II gave nearly single protein bands on SDS-polyacrylamide gel electrophoresis, individually. Even the final preparation of alpha-GalNAcase I contained alpha-galactosidase activity, while alpha-GalNAcase II was almost free from that activity with p-nitrophenyl and 4-methylumbelliferyl alpha-N-acetylgalactosaminides as substrates. alpha-GalNAcase I and II both hydrolyzed terminal alpha-N-acetylgalactosaminyl linkages of the natural compounds investigated: Forssman hapten glycolipid, blood group A active oligosaccharide and GalNAc-alpha1-O-serine. On the other hand, oligosaccharides, and glycolipid containing alpha-galactosyl terminals were hydrolyzed by alpha-GalNAcase I but not by alpha-GalNAcase II. The substrate specificities and other enzymatic properties of alpha-GalNAcase I were similar to those of human placental alpha-GalNAcase, but distinct from alpha-GalNAcase II.

Chemical and immunological characterization of the two alpha-N-acetylgalactosaminidases from squid liver.

Based on the inherent alpha-galactosidase activity, squid liver contains two different alpha-N-acetylgalactosaminidases (alpha-GalNAcases): alpha-N-acetylgalactosaminidase I (alpha-GalNAcase I), which typically exhibits the alpha-galactosidase activity and alpha-N-acetylgalactosaminidase II (alpha-GalNAcase II), which is devoid of such activity. The molecular properties of the alpha-GalNAcases that may account for their enzymological differences are as yet unknown. In this study, we have characterized and compared the chemical and immunological properties of alpha-GalNAcase I and alpha-GalNAcase II. Analysis of the N-terminal sequence of the first twenty amino acids revealed the striking homology between alpha-GalNAcase I and alpha-GalNAcase II. Digestion of alpha-GalNAcase I and alpha-GalNAcase II generated the peptide maps that display similarities in peptide pattern, indicating their close relationship in structure. Polyclonal antibodies were generated in rabbits against the purified alpha-GalNAcase I and alpha-GalNAcase II for comparison of the immunological properties. Both Western blot and surface plasmon resonance (SPR) studies showed that the anti-alpha-GalNAcase II antibody reacted with both alpha-GalNAcase I and alpha-GalNAcase II, whereas the anti-alpha-GalNAcase I antibody reacted only with alpha-GalNAcase I, indicating the presence of common as well as unique antigenic determinants on alpha-GalNAcase I and alpha-GalNAcase II. Taken together, these results suggest that alpha-GalNAcase I and alpha-GalNAcase II are closely related with regard to structure and that their nonhomologous domains are possibly responsible for the differences in enzymatic properties.

Identification and characterization of cathepsin D in a highly purified sialidase from starfish A. pectinifera.

A sialidase [EC 3.2.1.18] from the ovary of starfish Asterina pectinifera was isolated and highly purified by preparative PAGE. The SDS-PAGE separation of the purified enzyme revealed two natures of protein bands, upper (50 kDa) and a lower (47 kDa). To identify the protein, N-terminal amino acid sequence of the upper band was done. The sequence matched with the N-terminal amino acid sequence of human lysosomal mature cathepsin D and cathepsin D activity was also found in all the preparation steps. Protease inhibitor pepstatin A inhibited the proteolysis activity of cathepsin D against a synthetic substrate. The two enzymes sialidase and cathepsin D were separated from each other by using high-performance gel-filtration chromatography. The Western blot analysis and isoelectric focusing showed the co-purified cathepsin D is a 50 kDa protein with a PI value of 4.2.

Photoaffinity labeling of sialidase with a biotin-conjugated phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid.

A biotin-conjugated photoactivatable phenylaminodiazirine derivative of 2,3-didehydro-2-deoxy-N-acetylneuraminic acid (DANA) was synthesized to identify sialidase. The free carboxylic group and N-acetyl substituent of sialic acid, which are important for recognition and enzymatic activity of sialidase, were conserved by the photolabeling compound as confirmed using analytical methods. The synthesized compound and DANA competitively inhibited starfish sialidase with a Ki value of 7.6 microM and 4.6 microM, respectively. Photo incorporation of the labeling compound to sialidase increased with irradiation time; 90% photo incorporation was achieved with more than 10-min irradiation, and labeling was completely inhibited by the addition of a competitive inhibitor. Starfish sialidase purified using high-performance gel filtration chromatography was subjected to photoaffinity labeling. A 50-kDa band was revealed to contain the sialidase active site by the photolabeling compound, and labeling was completely hindered in presence of the competitive inhibitor. Labeling specificity was ensured by the addition of the heat-deactivated standard protein chymotrypsinogen A to the reaction mixture.

The presence of ceramidase activity in liver nuclear membrane.

This report presents a demonstration of ceramidase activity in the nuclear membrane or envelope of mammalian livers. The products of ceramidase reaction were identified by means of TLC for released fatty acid and HPLC for sphingosine. The ceramidase activity was maximum over a broad neutral to alkaline region ranging from pH 7.0 to 8.8. This activity was inhibited by N-oleoylethanolamine known as a specific inhibitor for ceramidase and by anandamide to a similar extent. The enzymatic study suggests that the nuclear ceramidase has different properties from other ceramidase reported previously. As sphingomyelinase, one of enzymes involved in the sphingomyelin cycle, are known to be present in the nuclear membrane, it is now evident that at least two enzymes involved in the sphingomyelin cycle are present in the nuclear membrane.

Effects of inorganic anions on the activation of acid sialidases.

An acid sialidase partially purified from porcine liver was activated by incubation at 37 degrees C under acidic pH. This activation was dependent on pH, time and temperature, but not inhibited by amastatin, an inhibitor of aminopeptidase A, in contrast to the case of human placental sialidase. The effects of inorganic anions on the two sialidases from porcine liver and from human placenta were investigated. Among the anions tested, halide ions, especially chloride and bromide ions, markedly enhanced the activation of the two sialidases. However, nitrate, sulfate, sulfite and pyrosulfite ions rarely affected the activation of sialidase from porcine liver, while all of them enhanced the activation of human placental sialidase. The activation of the enzyme from porcine liver was depressed at concentrations of greater than 100 mM of sodium chloride, whereas the enzyme from human placenta was held at maximum activation until 1 M sodium chloride. These results suggest the possibility of the participation of enzyme functions different from that of human placental sialidase in the activation process of sialidase.