Characterization of rat liver nuclear proteins which recognize the cAMP responsive element.

1. The data herein reveal the existence of cAMP-responsive element (CRE)-binding factors (CRF) in the nuclear extracts from cAMP-treated rat liver. 2. DNAase I and DMS footprinting analysis showed that the CRFs protected the CRE (-77 to -92) in the phosphoenolpyruvate carboxykinase (PEPCK) promoter and the TGACGTCA motif in a consensus oligodeoxynucleotide based on the sequence of the CRE's of 6 cAMP-regulated genes (C32mer). 3. Competition assays indicate that the CRF(s) is a CGTCA-specific, ATF/CREB-like factor(s). 4. Southwestern (SW) blot analysis detected 2 apparent CRFs which have molecular weights of about 30 and 32 kDa, respectively. 5. Based on the comparison of the size and binding specificity of the CRFs with the CREBs reported to date, the CRFs appear to be novel CRE-binding nuclear factors.     

Isolation of mannose-binding proteins from human and rat liver

The interaction of e-aq., CO2-. and one-electron reduced nitroaromatics (RNO2-.) with ascorbate oxidase (AAO) was studied in aqueous solution at pH 6.0 and 7.5 by using the technique of pulse radiolysis. From observations at 330, 410 and 610 nm, interaction of e-aq. and CO2-. with AAO results in non-specific reduction of the protein followed by reduction of Type 1 Cu in a rate-determining intramolecular step. Only a few per cent of the reducing equivalents ultimately results in reduction of Type 1 Cu. With large excesses of reducing equivalents (e-aq. and CO2-.) with respect to the copper concentration, the amount of Type 1 copper reduced never exceeds 50% of the total amount of Type 1 copper after a single radiation pulse. With less-powerful reducing agents, e.g. RNO2-. reduction of Type 1 Cu occurs via a bimolecular step, and there is no evidence for formation of radicals on protein residues. From observations at 330 nm it is evident that Type 2 and/or Type 3 Cu may also be reduced along with Type 1 Cu. Almost stoichiometric reduction of AAO by RNO2-. was observed, e.g. the protein accepts 6-7 reducing equivalents. It is inferred that the various types of redox couples Cu2+/Cu+ are in equilibrium and that intramolecular electron transfer between the different types of Cu is not rate-determining when using RNO2-. as reducing agent.     

Phosphorylation of nuclear proteins in rat regenerating liver.

In studies of the phosphorylated proteins in rat liver and Walker-256, it was established that the ratio of various fractions of P-N linkages to P-O linkages varies from 0.6 to 3.1. In rat regenerating liver nuclei, the ratio of P-N and P-O varies with time after partial hepatectomy. Using [3H]-lysine and 32Pi, it is shown that phosphoryllysine forms in some new and, presumably, some preexisting H1 molecules. Using [3H]histidine and 32Pi, it is shown that phosphohistidine forms exclusively in preexisting H4. The half-life of H4 phosphohistidine appears to be about 2 h.     

Characterization of tyrosylprotein sulfotransferase from rat liver and other tissues

The sulfoconjugation of tyrosyl residues is a widespread post-translational modification of biologically active peptides and proteins. In this paper we describe the characterization of a rat liver tyrosylprotein sulfotransferase that is capable of catalyzing the transfer of a sulfate moiety from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to the synthetic polymer, poly-(Glu6,Ala3,Tyr1) (EAY; Mr 47,000) using a simple filter paper assay. Following sucrose density gradient centrifugation and comparison with known subcellular marker enzyme activities, rat liver tyrosylprotein sulfotransferase activity was shown to have a distribution similar to the Golgi enzyme, galactosyltransferase. Using the enriched Golgi preparation, rat liver tyrosylprotein sulfotransferase displayed a pH optimum of 6.7 and required the presence of 20 mM Mn2+ for maximal activity. Co2+ (20 mM) was able to produce 26% of the maximal stimulation observed with Mn2+, whereas other metal ions, such as Mg2+, Ca2+, and Co2+, were not effective in stimulating tyrosylprotein sulfotransferase activity. Whereas tyrosylprotein sulfotransferase activity was observed in the native membrane-bound state, EAY sulfation was maximally enhanced 3-fold when assayed in the presence of Lubrol Px. Under the optimal conditions for assaying the sulfation of EAY by a rat liver enriched Golgi fraction, significant degradation of the sulfate donor, PAPS, was observed. The addition of both NaF and 5'-AMP to the incubation mixture was found to effectively prevent PAPS degradation and increase the amount of product formed in the assay by 10-fold. Using the optimized conditions for the sulfation of EAY by rat liver tyrosylprotein sulfotransferase, membrane-bound sulfotransferase activity was also observed in the crude microsomal pellets of a variety of rat tissues, including lung, pituitary, and cerebellum, as well as in livers from different species.     

Thiols attached to rat liver non-histone nuclear proteins.

Up to 88% of the total thiol present in isolated rat liver nuclei can be extracted with 8 M urea 50 mM phosphate pH 7.6. There is approx. 5–10% disulphide material present in this extract. When the thiols were labelled with ¹⁴C-N-ethyl maleimide (¹⁴C-NEM) the thiol material co-electrophoresed with the protein material. If a mixed disulphide was formed with ³⁵S-labelled 5-thio-2-nitrobenzoic acid (Ellman's reagent) the thiol compounds could be removed from the protein by isoelectric focusing in polyacrylamide gel. The mixed disulphides obtained could be resolved into at least 10 components on DEAE cellulose. One of the major components had an estimated molecular weight of 3 000 and did not contain peptide material.     

Binding of beryllium to nuclear acidic proteins.

In vitro beryllium (Be) binding to rat liver nuclei has been reassessed (KAss = 2.0 X 10(6) M: n = 17 nmol Be/mg protein). Be also binds to rat liver nucleoli (KAss approx. 4 X 10(6) M: n = 10 nmol Be/mg protein). Examination of rat liver chromatin fractionated on a hydroxyapatite column shows that Be does not bind to histone or to the non-histone protein eluted by 0.05 M sodium phosphate. Be is strongly bound to the non-histone proteins eluted by 0.2 M sodium phosphate (KAss = 1.1 X 10(6) M: n = 55 nmol Be/mg protein) and also to the same extent to the fraction containing DNA which is subsequently eluted from the column. Evidence is provided that the latter binding is not due to DNA. The fractions containing the Be-binding proteins also contain the proteins which are phosphorylated to the greater extent.     

A comparison of nuclear and nucleolar matrix proteins from rat liver

The comparison of the gel electrophoresis patterns of nuclear and nucleolar matrix proteins reveals marked differences between these structures. The nucleolar matrix contains 5 prominent protein bands ( mol.wt.: 8.2; 7.0; 5.6; 4.0 and 3.0 x 10 4 ) which are not found in nuclei. It is suggested that the nucleolar matrix has a distinct structure participating in selective interactions.