Thermolysin is a metallopeptidase used to cleave peptide bonds at specific junctions. It has previously been used to cleave specific amino acid sequences found at the junction of the sensory epithelium and underlying stroma of unfixed otolithic organs of the vestibular system. We have used thermolysin to separate sensory epithelium from the underlying stroma in fixed cristae ampullares of mouse, rat, gerbil, guinea pig, chinchilla, and tree squirrel, thus removing the saddle-shaped curvature of the sensory organ and creating a flattened sensory epithelium preparation. This permits visualization of the entire sensory organ in a single mount and facilitates proper morphometric analysis. 1. Introduction Thermolysin, otherwise known as protease type X, is found in Bacillus thermoproteolyticus [1]. Thermolysin is a metallopeptidase and cleaves peptide bonds at the N-terminal of hydrophobic amino acids including alanine, isoleucine, leucine, methionine, phenylalanine, threonine, tryptophan, tyrosine, and valine [2]. Thermolysin was first characterized by Matsubara et al. [3] and subsequently used in unfixed otolith organ tissue to cleave specific amino acid sequences found at the junction of the sensory epithelium and underlying stroma [3, 4]. Suzuki et al. [4] were able to use thermolysin to cleave proteins anchoring the otolithic membrane to the tectorial membrane [4]. After centrifugation, they were able to separate the gelatinous layer from the otolithic stones. Saffer et al. [5] used thermolysin to separate the sensory epithelium from nonsensory epithelium in unfixed utricular maculae of rats [5]. Thermolysin has also been used to separate unfixed human keratinocytes, cultured intestinal epithelial cells, cultured cochlear spiral ganglion neurons, and pancreatic islet cells [6–10]. Thermolysin has not previously been used to separate fixed tissue, and there is concern that the use of fixatives such as glutaraldehyde, formaldehyde, and acrolein may change the protein structure such that it is no longer amenable to cleavage by thermolysin. Despite this concern, we have used thermolysin to separate sensory epithelium from the underlying stroma in fixed cristae ampullares of mouse, rat, gerbil, guinea pig, chinchilla, and tree squirrel (Figure 1). This resulted in removal of the extracellular matrix that imparts the three-dimensional saddle shape of the sensory organ and the creation of a flattened sensory epithelium preparation. This two-dimensional view was desired to improve visualization of the entire sensory epithelium at once and to allow more accurate
References
[1]
J. Feder and J. M. Schuck, “Studies on the Bacillus subtilis neutral-protease- and Bacillus thermoproteolyticus thermolysin-catalyzed hydrolysis of dipeptide substrates,” Biochemistry, vol. 9, no. 14, pp. 2784–2791, 1970.
[2]
G. Allen, “Specific cleavage of the protein,” in Laboratory Techniques in Biochemistry and Molecular Biology, G. Allen, Ed., Sequencing of Proteins and Peptides, chapter 3, pp. 43–71, Elsevier, Amsterdam, The Netherlands, 1981.
[3]
H. Matsubara, A. Singer, R. Sasaki, and T. H. Jukes, “Observations on the specificity of a thermostable bacterial protease ‘thermolysin’,” Biochemical and Biophysical Research Communications, vol. 21, no. 3, pp. 242–247, 1965.
[4]
H. Suzuki, Y. C. Lee, M. Tachibana, K. Hozawa, H. Wataya, and T. Takasaka, “Quantitative carbohydrate analyses of the tectorial and otoconial membranes of the guinea pig,” Hearing Research, vol. 60, no. 1, pp. 45–52, 1992.
[5]
L. D. Saffer, R. Gu, and J. T. Corwin, “An RT-PCR analysis of mRNA for growth factor receptors in damaged and control sensory epithelia of rat utricles,” Hearing Research, vol. 94, no. 1-2, pp. 14–23, 1996.
[6]
L. Germain, M. Rouabhia, R. Guignard, L. Carrier, V. Bouvard, and F. A. Auger, “Improvement of human keratinocyte isolation and culture using thermolysin,” Burns, vol. 19, no. 2, pp. 99–104, 1993.
[7]
A. Gragnani, C. S. Sobral, and L. M. Ferreira, “Thermolysin in human cultured keratinocyte isolation,” Brazilian Journal of Biology, vol. 67, no. 1, pp. 105–109, 2007.
[8]
N. Perreault and J. Beaulieu, “Use of the dissociating enzyme thermolysin to generate viable human normal intestinal epithelial cell cultures,” Experimental Cell Research, vol. 224, no. 2, pp. 354–364, 1996.
[9]
C. Ripoll and G. Rebillard, “A simple technique to efficiently dissociate primary auditory neurons from 5 day-old rat cochleas,” Journal of Neuroscience Methods, vol. 73, no. 2, pp. 123–128, 1997.
[10]
J. Abouaish, M. Graham, P. Bansal-Pakala et al., “Successful isolation and transplantation of nonhuman primate islets using a novel purified enzyme blend,” Transplantation, vol. 92, no. 8, pp. e40–e42, 2011.
[11]
H. Suzuki, M. Furukawa, and T. Takasaka, “Quantitative uronic acid analysis of the otoconial membrane of the guinea pig,” Hearing Research, vol. 114, no. 1-2, pp. 223–228, 1997.
[12]
G. B. Wislocki and A. J. Ladman, “Selective and histochemical staining of the otolithic membranes, cupulae and tectorial membrane of the inner ear,” Journal of Anatomy, vol. 89, no. 1, pp. 3–12, 1955.
[13]
L. F. Bélanger, “Autoradiographic detection of S35 in the membranes of the inner ear of the rat,” Science, vol. 118, no. 3070, pp. 520–521, 1953.
[14]
L. F. Bélanger, “On the intimate composition of membranes of the inner ear,” Science, vol. 123, no. 3207, pp. 1074–1075, 1956.
[15]
R. E. Shrader, L. C. Erway, and L. S. Hurley, “Mucopolysaccharide synthesis in the developing inner ear of manganese deficient and pallid mutant mice,” Teratology, vol. 8, no. 3, pp. 257–266, 1973.
[16]
M. Hultcrantz and D. Bagger-Sj？b？ck, “Inner ear content of glycosaminoglycans as shown by monoclonal antibodies,” Acta Oto-Laryngologica, vol. 116, no. 1, pp. 25–32, 1996.
