We have shown that collagen accounts for approximately 40% of the total protein of the tectorial membrane (TM) of the guinea pig and have estimated several essential parameters of TM composition including dry weight, wet weight, and water content. The major collagenous protein was definitively identified as type II collagen by SDS‐PAGE, CNBr peptide mapping, and immunoblot assays. Quickfreeze, deep‐etch electron microscopy of the guinea pig TM demonstrated a dense meshwork of fibers embedded in a complex microfibrillar matrix which may consist of proteoglycans; the larger fibers were similar in size and appearance to type II collagen fibers of elastic cartilage. Finally, the comparative free'amino acid profiles of TM strongly suggest that the TM is chemically transparent with respect to endolymph. Thus, the TM appears to consist of a highly hydrated matrix mechanically stabilized by type II collagen fibers. The possible physicochemical and functional significance of these findings for sound transduction is discussed.
Evidence is presented that, in contrast to the traditional view, a large proportion of the proteins of the tectorial membrane (TM) consists of collagen, primarily type II: (1) characteristic amino acid composition of TM, including high levels of glycine, hydroxyproline and hydroxylysine; (2) comigration of the main TM protein with appropriate collagen standards in two-dimensional gel electrophoresis; (3) digestion of the main TM protein band following treatment with bacterial collagenase; (4) one- and two-dimensional peptide mapping of cyanogen bromide digests of the TM exhibits patterns characteristic for collagen type II.
According to traditional thinking, the tectorial membrane (TM) is a gel with a matrix of proteins, but containing no collagen. We recently reported patterns of amino acids and proteins of the TM consistent with the presence of substantial amounts of collagen [I. Thalmann et al., J. Acoust. Soc. Am. Suppl. 1 77, S93 (1985)]. Using two-dimensional polyacrylamide gel electrophoresis with extended alkaline range, we have now found that the predominant protein of the TM of the guinea pig virtually superimposes with purified collagen standards. Moreover, peptide mapping of cyanogen bromide digests (“fingerprinting”) results in a pattern closely resembling that of collagen type II. Further procedures for identification and characterization of this protein (effect of collagenase, immunoblotting, etc.) are in progress. At this stage it is not possible to decide to what extent the amino and neutral sugars present in hydrolysates of the TM are part of the alleged collagen molecule. The cupula ampullaris, a vestibular superstructure thought to be analogous to the TM, exhibits a substantially different chemical profile. The functional significance of the findings will be discussed. [Supported in part by NIH.]
The majority of proteins found to date in OC were identified by immunohistological methods. While these techniques are powerful tools for localization of proteins, they frequently lack satisfactory specificity. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) is able to demonstrate the presence in the OC of specific subtypes of certain proteins. In the case of contraction associated proteins, for instance, the presence of nonmuscle actin (beta and gamma), three types of nonmuscle tropomyosin (Tm:4, Tin:5, and Tm:8) and nonmuscle lactic dehydrogenase (LDH B) was documented. Another important role of 2D-PAGE is the demonstration of previously unknown proteins. The presence in the OC of two highly prominent proteins of low molecular weight and strongly acidic pH (assumed to be specific to the OC) was shown. Accordingly, they were termed OCP-I and OCP-II [I. Thalmann et al., Arch. Otorhinolaryngol. 226, 123–128 (1980)]. It was found that small amounts of OCP-I and OCP-II are present in spiral ligament and spiral limbus and more substantial amounts in basilar membrane. Neither protein is detectable in stria vascularis. Likewise, no proteins resembling OCP-I or OCP-II were found in tissues other than inner ear. While some biochemical features of the two proteins have in the meantime been characterized, their functional significance remains obscure. [Work supported by NIH.]
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