An acid sialidase [EC 3.2.1.18] has been purified from human placenta by means of successive procedures including extraction, Con A-Sepharose adsorption, ammonium sulfate precipitation, activation, p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatography and high-performance liquid chromatography on a Shim pack Diol 300 column. The purified enzyme liberated sialic acid residues from sialooligosaccharides, sialoglycoproteins, and gangliosides. In particular, gangliosides GM3, GD1a, and GD1b were hydrolyzed much faster than alpha (2-3) and alpha (2-6)sialyllactoses, and sialoglycoproteins by the enzyme. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme gave five protein bands with molecular weight of 78,000 (78K), 64,000 (64K), 46,000 (46K), 30,000 (30K), and 20,000 (20K). Rabbit antisera were raised against 78K and 46K proteins, and the two antibodies were specifically reactive with the respective component on immunoblot analysis. Both anti-78K protein and anti-46K protein antisera could precipitate sialidase activity. It is likely that the 78K protein and 46K protein are sub-components which are essential for sialidase activity.
An acid sialidase [EC 3.2.1.18], partially purified from human placenta by Con A-Sepharose adsorption and p-aminophenyl thio-beta-D-galactoside-CH-Sepharose (PATG-Sepharose) affinity chromatographies, was activated by incubation at 37 degrees C. This activation showed both time and temperature dependencies, with the most effective activation observed at 37 degrees C in the pH range between 4.3 and 5.2. The influence of various protease inhibitors on its activation was investigated. Among the protease inhibitors tested, amastatin, an inhibitor of aminopeptidase A, significantly inhibited activation. The partially purified enzyme preparation contained aminopeptidase activity, which was inhibited by amastatin. Zinc ions inhibited either the activation of sialidase or the aminopeptidase activity in the enzyme preparation. These results suggest the possibility of participation of aminopeptidase function in the activation process of sialidase.
We have read with interest the letter of Kanazawa 1 about improvement of apraxia of eyelid opening (AEO) with botulinum toxin A.The term AEO defined as an intermittent inability to open the eyes voluntarily is not a specific entity; it is only a clinical sign whose physiopathological origin is variable. It may be caused by an involuntary inhibition of levator palpebrae superioris (IILPS) or a blepharospasm restricted to the pretarsal portion of the orbicularis oculi (OO) called pretarsal blepharospasm (BSP). 2-4 As their clinical appearance are similar, because in both disorders there are not visible contraction of the orbicularis oculi, only a simultaneous electromyographic recording activity from the LPS and pretarsal and orbicular portion of OO can determine its origin. 4,5 In Kanazawa et al.'s cases, the diagnosis was only made by clinical aspect using the ambiguous and unspecific term of AEO.Their patients improved with botulinum toxin A, so the authors suggested that "AEO associated with these neurodegenerative diseases constitutes a form of eyelid dystonia rather than apraxia, even when no overt blepharospasm is observed." Actually, AEO and BSP are not excluding terms; as we stated previously, AEO is only a clinical sign and furthermore can be the result of a pretarsal BSP that can be present even in the absence of a manifest blepharospasm.The best method to disclose the underlying mechanism of impairment of eye lid opening in these three patients would have been an eye-lid electromyography recording. Probably, this EMG record would have showed a pretarsal BSP, whereas an IILPS would have not; this would explain why all of them improved with botulinum toxin A.Therefore, the AEO improvement with botulinum toxin is not related with the association to neurodegenerative diseases, as Kanazawa et al. suggests, but rather with its physiopathological substrate. Finally, we think it would be more appropriate to conclude that botulinum toxin could be a good treatment only for the cases in whom the AEO is caused by BSP but not when the origin is an IILP, as other authors described previously. 4,6 References 1. Kanazawa M, Shimohata T, Sato M, Onodera O, Tanaka K, Nishizawa M. Botulinum toxin A injections improve apraxia of eyelid opening without overt blepharospasm associated with neurodegenerative diseases. Mov Disord 2007;22:597-598. 2. Lepore FE, Duvoisin RC. Apraxia of eyelid opening: an involuntary levator inhibition. Neurology 1985;35:423-427. 3. Elston JS. A new variant of blepharospasm. J Neurol Neurosurg Psychiatry 1992;55:369-371. 4. Aramideh M, Ongerboer de Visser BW, Devriese PP, Bour LJ, Speelman JD. Clinical and electromyographic features of levator palpebrae superioris muscle dysfunction in involuntary eyelid closure. Brain 1994;117:27-38. 5. Esteban A, Traba A, Prieto J. Eyelid movements in health and disease. The supranuclear impairment of the palpebral motility.We appreciate comments by Dr. Valdes, Bilbao-Calabuig, and Posada about our letter regarding the effect of botulinum toxin A injections on a...
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