A 65-kappaD subunit of active NF-kappaB is required for inhibition of NF-kappaB by I kappaB.

The NF-kappaB transcription factor was affinity-purified from deoxycholate (DOC)-treated cytosol of HeLa cells and shown to contain both a 50-kappaD polypeptide (p50) with a DNA-binding specificity identical to that of nuclear NF-kappaB and a 65-kappaD protein (p65) lacking DNA binding activity. Electrophoretically purified p50, after renaturation, gave rise to a protein-DNA complex that migrated faster than that made by native NF-kappaB. Reconstitution of p50 and p65 together produced a protein that combined with DNA to form a complex with electrophoretic mobility indistinguishable from that of the complex formed by nuclear extracts and DOC-treated cytosolic fractions.

The primary structure of human secretogranin II, a widespread tyrosine-sulfated secretory granule protein that exhibits low pH- and calcium-induced aggregation.

Secretogranin II (previously also called chromogranin C) is a tyrosine-sulfated secretory protein found in secretory granules in a wide variety of endocrine cells and neurons. Here, we have determined the primary structure of human secretogranin II from a full length cDNA clone and have investigated its properties, predicted from the sequence, by studying the behavior of purified secretogranin II under conditions characteristic of the milieu of secretory granules. Analysis of a 2.

I kappa B: a specific inhibitor of the NF-kappa B transcription factor.

In cells that do not express immunoglobulin kappa light chain genes, the kappa enhancer binding protein NF-kappa B is found in cytosolic fractions and exhibits DNA binding activity only in the presence of a dissociating agent such as sodium deoxycholate. The dependence on deoxycholate is shown to result from association of NF-kappa B with a 60- to 70-kilodalton inhibitory protein (I kappa B). The fractionated inhibitor can inactivate NF-kappa B from various sources--including the nuclei of phorbol ester-treated cells--in a specific, saturable, and reversible manner.

Expression, tyrosine sulfation, and secretion of yolk protein 2 of Drosophila melanogaster in mouse fibroblasts.

Tyrosine sulfation is a post-translational modification in the trans Golgi that has been found in all animal species studied. In the preceding paper (Baeuerle, P. A.

Purification of yolk protein 2 of Drosophila melanogaster and identification of its site of tyrosine sulfation.

We have identified the site of tyrosine sulfation in an insect secretory protein, yolk protein 2 of Drosophila melanogaster. Yolk proteins were purified from [35S]sulfate-labeled flies, and yolk protein 2 was separated from yolk protein 1 and yolk protein 3 by preparative two-dimensional polyacrylamide gel electrophoresis. After digestion of yolk protein 2 with trypsin and reversed-phase high performance liquid chromatography, the sulfate label was recovered in two distinct sulfopeptides which, however, had identical NH2-terminal sequences and contained 3 tyrosine residues each.

Activation of DNA-binding activity in an apparently cytoplasmic precursor of the NF-kappa B transcription factor.

In cells that do not express kappa immunoglobulin light chain genes, the kappa enhancer-binding protein NF-kappa B is not evident in either cytoplasmic or nuclear fractions. By denaturation, size fractionation, and renaturation, however, NF-kappa B activity can be revealed in cytosolic fractions, showing that the DNA-binding protein is present but inhibited in its binding activity. By using a variety of protocols involving the dissociating agents sodium desoxycholate and formamide, as much cytosolic NF-kappa B can be found in the fraction from unstimulated 70Z/3 pre-B cells as is found in the nuclear extract from phorbol ester-activated cells.

Tyrosine sulfation is a trans-Golgi-specific protein modification.

The trans-Golgi has been recognized as having a key role in terminal glycosylation and sorting of proteins. Here we show that tyrosine sulfation, a frequent modification of secretory proteins, occurs specifically in the trans-Golgi. The heavy chain of immunoglobulin M (IgM) produced by hybridoma cells was found to contain tyrosine sulfate.

The primary structure of human secretogranin I (chromogranin B): comparison with chromogranin A reveals homologous terminal domains and a large intervening variable region.

We have determined and analyzed the primary structure of human secretogranin I (chromogranin B), a tyrosine-sulfated secretory protein found in a wide variety of peptidergic endocrine cells. A 2.5-kb cDNA clone, hybridizing to an mRNA of similar length, was isolated from a cDNA library of human pheochromocytoma.

Chlorate--a potent inhibitor of protein sulfation in intact cells.

Chlorate is known to be an in vitro inhibitor of ATP-sulfurylase, the first enzyme in the biosynthesis of PAPS which is the ubiquitous co-substrate for sulfation. Here, the effect of chlorate on protein sulfation in intact cells was investigated. Treatment of various cell cultures with 1 mM sodium chlorate in a medium low in sulfate and sulfur-containing amino acids resulted in an inhibition of protein sulfation greater than 95%.

The primary structure of bovine chromogranin A: a representative of a class of acidic secretory proteins common to a variety of peptidergic cells.

We have determined the primary structure of bovine chromogranin A as a first step in the elucidation of the function of this widespread protein. After oligonucleotide screening of a cDNA library of bovine adrenal medulla, a clone (insert length 1.9 kb) containing the entire coding region for chromogranin A was isolated and sequenced.

Tyrosine sulfation of yolk proteins 1, 2, and 3 in Drosophila melanogaster.

Protein sulfation was studied in Drosophila melanogaster after in vivo labeling of flies with inorganic [35S]sulfate. After separation of total fly protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, proteins with sulfated carbohydrates and proteins containing tyrosine sulfate were found in all the molecular weight ranges analyzed. When female and male fly proteins were compared with each other, the electrophoretic patterns of protein-bound carbohydrate sulfate were found to be similar, whereas those of protein-bound tyrosine sulfate were distinct.

Inhibition of N-glycosylation induces tyrosine sulphation of hybridoma immunoglobulin G.

Immunoglobulin G2a (IgG2a) secreted by the hybridoma line M 31 was found to contain covalently linked sulphate. The sulphate was bound to the heavy chain which existed in several isoelectric variants. All variants were sulphated, the more acidic ones being more highly sulphated.

Quantitative data on peroxidatic markers for electron microscopy. With a note on actin identification in Paramecium cells.

Several important points of heme-peptide cytochemistry were quantitatively analyzed, with particular regard to their use in electron microscopic immunocytochemistry. A simple procedure is presented for the preparation of heme-octapeptide (H-8-P) microperoxidase. H-8-P, hemenonapeptide (H-9-P), and various horseradish peroxidase (HRP) isoenzymes were used for coupling with immunoglobulin (Ig)G or the papain-cleavage fragments from IgG (Fab) molecules.