Ruth Hogue Angeletti

Relationship between the nerve growth factor-regulated clone 73 gene product and the 58-kilodalton neuronal intermediate filament protein (peripherin).

Abstract: Exposure of PC12 cells to nerve growth factor (NGF) has been shown to induce an rnRNA that encodes a novel neuronal intermediate filament protein. The findings presented here concern the identity of this filament protein. The major protein in NGF‐treated PC12 cell cytoskeletons derived by extraction with 1% Triton X‐100 is of apparent Mr= 58,000, focuses by isoelectric focusing as several closely spaced spots of pl 5.6–5.8, and is elevated relative to non‐NGF‐treated cells. Partial microsequencing of this material reveals 2 internal sequences that are identical to a 14‐residue sequence encoded by the NGF‐regulated clone 73 mRNA, but not to sequences of other known proteins. An antiserum raised against a 19‐residue synthetic peptide corresponding to the deduced C‐terminus of the protein encoded by the NGF‐regulated clone 73 mRNA specifically recognizes the 58,000‐Mr protein. Properties of the 58‐kilodalton protein strongly suggest that it corresponds to an intermediate filament protein (peripherin) previously identified in PC12 cells and in peripheral and certain CNS neurons. Identification of the intermediate filament protein encoded by an NGF‐induced message should facilitate studies of its regulation and function.

A monoclonal antibody to dopamine β-monooxygenase: Detection of biosynthetic intermediates☆

A monoclonal antibody to dopamine beta-monooxygenase (DBH) has been produced by an in vitro immunization technique. This antibody has been found to react with an epitope common to both soluble DBH (SDBH) and membrane-bound DBH (MDBH). A single CNBr fragment contains this antigenic site. Examination of electrophoretograms of chromaffin granule membrane and lysate by the immunoblot procedure revealed additional complexity. A 58-KDa polypeptide in low abundance was stained which could not be detected with the polyclonal antiserum. Chemical deglycosylation of SDBH produces two new polypeptides of 67 and 58 KDa. The entirety of this data suggests that the 58-KDa band is an unglycosylated form of DBH, proteolytically cleaved after biosynthesis.

Catecholamine storage vesicles: topography and function

Abstract The ability of a cell to carry out many specialized functions is achieved by compartmentation of reactions within the cell. Here we review studies of how the functions of the adrenal chromaffin granules relate to their structure and topography.

Antibodies to a Synthetic Peptide, Chromogranin1–14

Chromogranin has a characteristic amino-terminal sequence which is identical in both the bovine and human polypeptides (CG,.,3: Leu-Arg-Val-Asn-Ser-Pro-Met-Asn-LysGly-Asp-Thr-Glu-Val-. Polypeptide chains of similar amino-terminal sequences but differing molecular weights have been identified in the chromafiin vesicle.) It was thus proposed that they compose a family of related proteins. A synthetic peptide corresponding to CG.,, was prepared and used as antigen for production of both polyclonal and monoclonal antibodies. Such probes would facilitate further study of this family of proteins, and provide antibodies that would react across species boundaries. Antibodies were successfully obtained by both methods. The polyclonal antiserum and monoclonal antibodies reacted in immunoblots of bovine, rat, and human tissues with multiple polypeptide chains. In bovine tissues (FIG. lA), these correspond to the multiple chains identified by their parent M, of 125,000, 100,000, 85,000, and 75,000. The M, 65,000 form was, however, not detected. In human tissues (FIG. lB), our previous polyclonal antisera against bovine chromogranin identify several proteins with M, greater than 100,000, plus a lower M, 70,000 band. The antipeptide antibodies also react with these very high molecular weight forms. The antipeptide antibodies reveal some additional polypeptides, however, with molecular weights ranging from 85,000 to 60,ooO. Although the probes were designed to provide a straightforward analysis, they have instead reinforced the complexity of the chromogranin family. It should be emphasized that the immunoreactivity could be completed both by the isolated peptide and by purified chromogranin A. Thus, there are multiple related proteins in each species studied. The precise relationship among them can only be determined by detailed genetic analysis. Furthermore, the antibodies developed by these probes do function across species boundaries. The monoclonal antibodies to CG,-,, have also proved useful for biological studies. We have recently found that chromogranin is located in cells within immune tissues.’ A putative neuroendocrine cell containing chromogranin has been isolated from spleen.5 A second cell type in spleen, the large granular lymphocyte, also appears to contain chromogranin.6 These cells are present in the pellet and buffy fractions, respectively, of Ficoll-separated spleen cells. FIGURE 2 shows immunohistochemical analysis of cytospin preparations of spleen buffy and pellet layers. The CG,.,, monoclonal antibodies specifically detect a single cell type in each cell preparation.

Neuroendocrine Functions in Lymphoid Tissues

The acidic protein chromogranin A has recently been detected in cells within immune tissues using immunohistochemical and immunoblotting analysis (Angeletti and Hickey, 1985). Although other neural and endocrine peptides have also been identified or found to have effects within these tissues (Plotnikoff et al., 1985; Smith et al., 1986; Wybran, 1985), the identification of chromogranin A has somewhat different implications. Individual neuropeptides such as enkephalins, endorphins, vasoactive intestinal peptide, are found in neuronal and endocrine vesicles only in tissue locations appropriate to their functions. Chromogranin A, however, is a pan-neuroendocrine marker protein, found in endocrine and some neurosecretory vesicles, but not in exocrine vesicles (Fischer-Colbrie et al., 1985; Lloyd and Wilson, 1983; O’Conner, 1983).