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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.

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.

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.

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.

The inhibitory effects of various sulfated compounds on the activities of sialidases purified from porcine liver and human placenta were investigated. Among the sulfated compounds tested, heparin, dextran sulfate, condroitin sulfates and sulfatide significantly inhibited the 4-methylumbelliferyl-alpha-N-acetylneuraminic acid (4-MU-NeuAc) sialidase activities of the two enzyme preparations, but glucose 6-sulfate and glucosamine 6-sulfate did not. Potassium sulfate showed an inhibitory effect only at high concentrations. When the sialidase activities were measured using natural substrates, the sialidase activities for the (alpha2-3) and (alpha2-6) sialyllactoses, and colominic acid, were markedly inhibited by heparin and sulfatide similar to 4-MU-NeuAc, although the fetuin sialidase activity was not significantly influenced by them. The sialidase activity hydrolyzing GM3 was strongly inhibited by heparin, but not by sulfatide.

Sialidase [E.C.3.2.1.18] has previously been purified from porcine liver by procedures including extraction, ammonium sulfate precipitation, concanavalin A-Sepharose adsorption, activation, CM-Sepharose ion exchange chromatography, and HPLC on a Shim pack Diol 300 column. Two sialidase preparations, sialidase I and II, were obtained by CM-Sepharose column chromatography and were eluted with pH 4.5 and 5.0 buffers, respectively. The two enzyme preparations showed the same optimum pH, pH stability, and specificities for natural substrates. The two final preparations contained beta-galactosidase activity and showed three protein components of 64, 30, and 21 kDa with sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which are derived from the beta-galactosidase multimer. The anti-beta-galactosidase multimer antiserum was able to precipitate sialidase activity. It is likely that porcine liver sialidase exists as a multienzyme complex with beta-galactosidase and carboxypeptidase (protective protein).

Lignoceroyl-coenzyme A (CoA) ligase activity was detected in microsomal fractions from chicken liver in the presence of alpha-cyclodextrin as a solubilizing agent of lignoceric acid. Heptakis(2,6-di-O-methyl)-beta-cyclodextrin (dimethyl-beta-cyclodextrin) and hexakis(2,6-di-O-methyl)-alpha-cyclodextrin (dimethyl-alpha-cyclodextrin), among the cyclodextrins tested, were more effective than alpha-cyclodextrin as solubilizing agents. We have characterized lignoceroyl-CoA ligase activity in comparison with palmitoyl-CoA ligase activity. Lignoceroyl-CoA and palmitoyl-CoA ligase activities showed a similar dependency on CoA concentration. However, lignoceroyl-CoA ligase activity exhibited responses to the Mg2+ ion, adenosine triphosphate (ATP), ATP analogues (adenosine monophosphate (AMP) and adenosine diphosphate (ADP)), and heat treatment, which were distinctly different from the responses of palmitoyl-CoA ligase activity. These results are consistent with the idea that lignoceroyl-CoA and palmitoyl-CoA are synthesized by two different enzymes.