Oncomodulin is abundant in the organ of Corti.

A small, acidic Ca(2+)-binding protein (CBP-15) was recently detected in extracts of the mammalian auditory receptor organ, the organ of Corti [Senarita et al. (1995) Hear. Res. 90, 169-175]. N-terminal sequence data for CBP-15 [Thalmann et al. (1995) Biochem. Biophys. Res. Commun. 215, 142-147] implied membership in the parvalbumin family and possible identity with the mammalian beta-parvalbumin oncomodulin. As shown herein, the latter conclusion is supported by strong cross-reactivity between CBP-15 and isoform-specific antibodies to oncomodulin. Moreover, we have succeeded in amplifying the guinea pig CBP-15 coding sequence from organ of Corti cDNA using degenerate oligonucleotide primers based on the rat oncomodulin sequence. The deduced amino acid sequence of guinea pig CBP-15 displays 90%, 92%, and 98% identity with mouse, rat, and human oncomodulin isoforms. Demonstration of the presence of oncomodulin in the organ of Corti is the first documentation of this substance in a postnatal mammalian tissue.

Detection of a beta-parvalbumin isoform in the mammalian inner ear.

A small, acidic calcium-binding protein (CBP-15) has been detected in the guinea pig organ of Corti, the auditory receptor organ. The apparent molecular weight (15,000) and very low isoelectric point (pI approximately 3.1) suggest that CBP-15 is a beta-parvalbumin isoform. Consistent with this hypothesis, CBP-15 exhibits extreme homology to the mammalian oncofetal parvalbumin called oncomodulin. Sequence data have now been obtained for 30 residues in the N-terminal third of CBP-15. Identity with oncomodulin is observed at all 30 positions. This finding could necessitate revision of the assumption that postnatal mammals utilize a single alpha-parvalbumin isoform in muscle and nonmuscle settings alike.

Protein profile of human perilymph: in search of markers for the diagnosis of perilymph fistula and other inner ear disease.

Recent developments in high-resolution two-dimensional polyacrylamide gel electrophoresis, combined with amino acid sequencing and computer-assisted image analysis, have allowed separation of approximately 100 proteins and identification and quantitation of some 30 proteins in human perilymph. The majority of proteins were found to be present in perilymph at levels in basic agreement with the total protein gradient between perilymph and plasma (1:35). However, several striking differences were observed: (1) beta 2-transferrin, known to be absent from normal plasma but present in cerebrospinal fluid, was detected in perilymph at a concentration roughly equal to that in cerebrospinal fluid; and (2) two high-density lipoprotein-associated apolipoproteins--apo D (formerly PLS:33) and apo J or NA1 and NA2 (formerly PSL:29/30), the latter showing identity with SP40/40, or cytolysis inhibitor--were found to be present at concentrations 1 to 2 orders of magnitude higher when examined in terms of total protein and to be comparable with or higher than plasma levels when examined in terms of absolute concentrations. The functional significance of the extremely high levels of the two apolipoproteins is not known at this time. An attempt was made to use beta 2-transferrin, as well as apo D and apo J (NA1/NA2), as markers for the diagnosis of perilymph fistula, one of the most controversial and challenging problems for the otologist today. It was determined that the technique is indeed applicable when relatively pure fistula samples are analyzed. Limitations and potential improvements of the technique are discussed. In addition, the potential usefulness of two-dimensional polyacrylamide gel electrophoresis in other pathologic conditions of the inner ear is discussed briefly.

Partial amino acid sequences of organ of Corti proteins OCP1 and OCP2: a progress report.

Progress in amino acid sequencing of two low-molecular weight acidic proteins of unknown function which are present at high concentrations in the organ of Corti is reported. These two proteins, provisionally termed OCP1 and OCP2, were originally demonstrated by two-dimensional polyacrylamide gel electrophoresis; their presence at high concentrations in the inner ear sensory epithelia strongly suggests that OCP1 and OCP2 serve some important function in the ear. In the present paper, extension of the amino-terminal sequence of OCP2 to 71 residues is described. In addition, the first results on sequencing of OCP1 are presented. Computer algorithms are used to predict important structural features, and the sequences are analyzed for probable phosphorylation, glycosylation, and Ca-binding sites. Projected further studies in immunochemistry and molecular biology of OCP1 and OCP2 based on the amino acid sequencing data are discussed.

Protein profiles of perilymph and endolymph of the guinea pig.

Results of protein separation of guinea pig plasma, perilymph, and endolymph by means of high-resolution two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis are presented. Several proteins are present in perilymph at levels in basic accord with the total protein gradient with respect to plasma; however, others are present in perilymph at levels comparable to plasma levels, and one protein low molecular weight protein, PLS:33, is eight times higher. In addition, a high molecular weight protein is shown to be present at similar levels in the two compartments. These findings indicate that ultrafiltration cannot be the sole mechanism of perilymph production. Endolymph proteins are uniformly five to eight times lower than perilymph levels, essentially following the total protein concentration gradient between the two compartments. This supports the view that endolymph is derived from perilymph rather than directly from blood.

Biochemical features of major organ of Corti proteins (OCP-I and OCP-II) including partial amino acid sequence.

Further biochemical and biophysical characterization of two low-molecular-weight, strongly acidic proteins that are present at extremely high levels in the organ of Corti, tentatively named OCP-I and OCP-II, is presented. The two proteins are also present, although at much lower levels, in the vestibular end-organs and a variety of other inner ear tissues; they have not been observed in other systems. OCP-I and II are highly soluble and do not contain appreciable amounts of carbohydrate. The two proteins, originally described in the guinea pig, are compared electrophoretically with the corresponding proteins in several other mammalian species. Preliminary data on the amino acid composition of the two proteins are presented. Moreover, the amino-terminal sequence of a 22-residue segment of OCP-II is shown and compared to the sequences of known proteins.

Partial amino acid sequence of organ of Corti protein OCP-II.

We have previously described two low-molecular-weight, highly acidic proteins which are present in the organ of Corti in extremely high concentrations. Since the function of these proteins is not known, they have been assigned the tentative names OCP-I and OCP-II. In the hope of obtaining information about their function through homology studies, we have initiated amino acid sequencing of these proteins. We have recently succeeded in obtaining a brief amino-terminal sequence of OCP-II. We now report on a significant extension of the amino-terminal sequence of OCP-II and our first results on sequences of peptide fragments obtained by limited digestion with V8 protease. Together, the sequenced segments account for about one-third of the total sequence. Comparisons with the sequences of known proteins suggest that OCP-II is not a structural protein, but that it may exhibit biologic activity.

Composition and supramolecular organization of the tectorial membrane.

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. Quick-freeze, 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.

Collagen--the predominant protein of the tectorial membrane.

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: characteristic amino acid composition of TM, including high levels of glycine, hydroxyproline and hydroxylysine; comigration of the main TM protein with appropriate collagen standards in two-dimensional gel electrophoresis; digestion of the main TM protein band following treatment with bacterial collagenase; one- and two-dimensional peptide mapping of cyanogen bromide digests of the TM exhibits patterns characteristic for collagen type II.

Prolonged maintenance of endocochlear potential by vascular perfusion with media devoid of oxygen carriers.

A method is described for maintaining the cochlear potentials of the guinea pig via arterial perfusion of the surviving inner ear with an artificial medium devoid of oxygen carriers or oncotic agents. The endocochlear potential (EP) can be maintained at a normal level for periods in excess of 5 h; the responses of the EP to anoxia and to furosemide closely approximate those seen in the intact animal. This preparation may represent a simplified method for carrying out selected arterial perfusion experiments in the surviving inner ear.

Amino acid profiles in inner ear fluids and cerebrospinal fluid.

The levels of 19 amino acids in utricular endolymph, vestibular and cochlear perilymph, and cerebrospinal fluid of guinea pigs were determined using gradient elution reverse phase high performance liquid chromatography of the o-phthaldialdehyde-ethanethiol adducts with fluorescence detection. Aspartate and glutamate were significantly higher in endolymph than in perilymph, in agreement with earlier results on cochlear fluids based on enzymatic fluorometric techniques. All other amino acids tested were significantly lower in the endolymph, in most cases by an order of magnitude. Vestibular perilymph and perilymph of scala vestibuli are virtually identical. Amino acid levels were all higher in perilymph of scala vestibuli than in cerebrospinal fluid; two by an order of magnitude. All differences were statistically significant, with the exception of aspartate. Amino acid levels in perilymph of scala tympani were highly variable dependent upon sampling technique, and no definite values are therefore presented. Comparisons with results from other laboratories, technical pitfalls, and possible implications and interpretations of the results are presented.

Steep gradients of amino acids between cochlear endolymph and perilymph.

Levels of 19 free amino acids in cochlear endolymph and perilymph of scala vestibuli of the guinea pig were determined using reverse phase high performance liquid chromatography (HPLC) of the o-phthaldialdehyde-ethanethiol derivatives with fluorescence detection. Aspartate and glutamate were found to be significantly higher in endolymph than in perilymph, confirming results from another study using a different analytical method. The remaining 17 amino acids were significantly lower in the endolymph, in many cases by an order of magnitude. The data are compared with results on utricular endolymph and vestibular perilymph obtained by HPLC in another study from our laboratory. Possible implications and interpretations of the results are discussed.