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Query: EC:3.4.21.1 (
chymotrypsin
)
10,938
document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)
Cleavage at cysteine and
chymotrypsin
digestion were applied to two human neurofilament (NF) subunits, low- and high-molecular-weight NF (NF-L and
NF-H
), to locate the regions reacting with Bodian's silver stain and with several monoclonal antibodies, including NF-specific antibodies and one that recognizes all intermediate filaments (anti-IFA). Our findings indicate that whereas anti-IFA recognizes the highly conserved rod domain, all the NF-specific antibodies, as well as Bodian's silver, react with the carboxy-terminal tailpiece of NF subunits. The silver binding sites in NF-L are located in a carboxy-terminal 12-Kd
chymotrypsin
fragment, a highly charged, unique domain of NF.
...
PMID:Binding of Bodian's silver and monoclonal antibodies to defined regions of human neurofilament subunits: Bodian's silver reacts with a highly charged unique domain of neurofilaments. 241 73
Two monoclonal antibodies that recognize Alzheimer's neurofibrillary tangles (ANTs), AD10 and AB18, have been characterized by immunoblotting against human and calf spinal cord neurofilament (NF) and calf brain microtubule preparations. Both antibodies bind to the 200-kilodalton (kd) (
NF-H
) and 160-kd (NF-M) but not to the 68-kd (NF-L) NF triplet proteins. They also bind to high-molecular-weight microtubule-associated proteins (MAPs) and tau. AD10 immunostains MAP2 and MAP1 families, whereas AB18 stains mainly MAP1 bands. Preincubation of intact filament preparation or nitrocellulose strips containing electroblotted NF proteins with Escherichia coli alkaline phosphatase completely blocks AD10 binding and partially blocks binding of AB18. These results suggest that the determinants recognized by these antibodies are phosphorylated. Immunoblotting of peptide fragments generated by limited proteolysis of NF proteins with
alpha-chymotrypsin
and Staphylococcus aureus V8 protease shows that the localization of the antigenic determinants to AD10 and AB18 in
NF-H
is approximately 100 and 60 kd, respectively, away from the carboxy terminal, a region previously shown to form the NF projection side arm. In NF-M, the antigenic determinants to both antibodies are located also in the projection side arm, in a 60-kd polypeptide adjacent to the alpha-helical filament core. The results show that ANTs contain at least two phosphorylated antigenic sites that are present in NF and MAPs, a finding suggesting that ANTs may be composed of proteins or their fragments with epitopes shared by cytoskeletal proteins.
...
PMID:Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilament, and microtubule-associated proteins. 243 80
It has recently been shown that a monoclonal antibody SM 1-36-2 against connectin, an elastic filament of striated muscles, binds to the "elastic" domain of the molecule, and that the H subunit of neurofilament (
NF-H
), an intermediate filament of nerve cells, shares a homologous domain (Shimizu, T. et al. (1988) Biomed. Res. 9, 227-234 and Itoh, Y. et al. (1988) J. Biochem. 104, 504-508). In order to characterize (1) the intramolecular localization of the domain in the
NF-H
and (2) the effect of the phosphorylation state on the immunoreactivity, the homologous domain in the
NF-H
was analyzed by Western blotting after limited digestion with trypsin or
alpha-chymotrypsin
and dephosphorylation with E. coli alkaline phosphatase. It was found that (1) the epitope was located not in the core region but in the carboxyl-terminal peripheral (cross-bridge) region of
NF-H
and (2) the epitopes in connectin and
NF-H
were not affected by the phosphorylation state.
...
PMID:The immunological homology between two filamentous cross-linker phosphoproteins, connectin and cross-bridge region of neurofilament-H, is not affected by the phosphorylation state. 272 66
A polyclonal antibody against the high molecular weight neurofilament polypeptide (
NF-H
) obtained from cytoskeletal extracts of bovine spinal cord reacted with
NF-H
, with the middle molecular weight neurofilament polypeptide (NF-M) and with a M(r) 51,000 polypeptide, but not with the low molecular weight neurofilament polypeptide (NF-L). The M(r) 51,000 polypeptide corresponded to the glial fibrillary acidic protein (GFAP), which forms the intermediate filaments of astrocytes. The polyclonal antibody affinity-purified with GFAP, reacted with both purified GFAP and
NF-H
. Digestion of
NF-H
with
alpha-chymotrypsin
was used to determine the recognition site of the affinity-purified antibodies. Only fragments of the tail domain of
NF-H
reacted with the antibody. We propose that common epitopes exist between the variable C-terminal domains of
NF-H
and GFAP.
...
PMID:A neurofilament polypeptide and the glial fibrillary acidic protein share common epitopes in the variable region. 750 7
NF-H
has the highest mol. wt. of the three mammalian neurofilament components (NF-L, NF-M,
NF-H
). In spite of its unusually large mol. wt., estimated to be 200 K by gel electrophoresis,
NF-H
contains sequences which identify it as an integral intermediate filament (IF) protein in its amino-terminal region. We have isolated and partially characterized a basic, non-alpha-helical segment located at the amino-terminal end with properties similar to headpieces of other non-epithelial IF proteins. The highly alpha-helical 40-K fragment excised by
chymotrypsin
is now identified by the amino acid sequence of a 17-K fragment. This sequence can be unambiguously aligned with the rod region of other IF proteins and covers about half of the presumptive coiled-coil arrays.
NF-H
and NF-M show 45% sequence identity in this region. The extra mass of
NF-H
in comparison with most other IF proteins arises from a carboxy-terminal extension thought to be responsible for inter-neurofilament cross-bridges in axons. This autonomous domain has a unique amino acid composition characterized by a high content of proline, alanine and particularly of lysine and glutamic acid. The
NF-H
tailpiece extension also carries a large number of serine phosphates, which are not evenly distributed, but are restricted to the amino-terminal part. Having now delineated the intermediate filament-type sequences for all three neurofilament proteins it seems very likely that the three components interact via coiled-coil interactions. They all carry unique carboxy-terminal extensions which increase in length from NF-L to
NF-H
and seem to extend from the filament wall.
...
PMID:Protein-chemical characterization of NF-H, the largest mammalian neurofilament component; intermediate filament-type sequences followed by a unique carboxy-terminal extension. 1645
