[Interaction of Escherichia coli RNA polymerase with eukaryotic TATA-binding protein]. / Vzaimodeistvie RNK-polimerazy Escherichia coli s TATA-sviazyvaiushchim belkom éukariot.

Interaction with eukaryotic TATA-binding protein (TBP) was analyzed for natural Escherichia coli RNA polymerase or the recombinant holoenzyme, minimal enzyme, or its sigma subunit. Upon preincubation of full-sized RNA polymerase with TBP and further incubation with a constant amount of 32P-labeled phosphamide derivative of a TATA-containing oligodeoxyribonucleotide, the yield of the holoenzyme-oligonucleotide covalent complex decreased with increasing TBP concentration. This was considered as indirect evidence for complexing of RNA polymerase with TBP. In gel retardation assays, the holoenzyme, but neither minimal enzyme nor the sigma subunit, interacted with TPB, since the labeled probe formed complexes with both proteins in the reaction mixture combining TBP with the minimal enzyme or the sigma subunit. It was assumed that E. coli RNA polymerase is functionally similar to eukaryotic RNA polymerase II, and that the complete ensemble of all subunits is essential for the specific function of the holoenzyme.

Affinity labeling of RNA-polymerase II in the transcriptionally active complex by a phosphorylating analog of the initiation substrate.

Affinity modification of RNA-polymerase II by a phosphorylating analog of the initiation substrate carrying a zwitterionic 5;-terminal phosphate group with a 4-N,N-dimethylaminopyridine residue (DMAP-pA) was studied during specific transcription initiation controlled by the late adenoviral promotor. Super-selective affinity labeling and standard conditions of affinity modification resulted in labeling a polypeptide with molecular weight corresponding to that of the third subunit of the enzyme, RPB3 (45 kD). The initiation substrate (ATP) protects RNA-polymerase II from modification. The third subunit may be involved in the formation of the substrate-binding site of the enzyme.

[The participation of transmembrane messenger systems in the action of steroid hormones on target cells]. / Uchastie transmembrannykh sistem posrednikov v deistvii steroidnykh gormonov na kletki-misheni.

Estradiol receptors are discovered in nuclei, cytoplasm and plasmatic membranes of the target cells. Estradiol activates the transmembrane polyphosphoinositide system: it stimulates the protein kinase C translocation from cytosol to cell membranes, the membrane protein phosphorylation being elevated. Transmembrane adenylate cyclase system is also activated. The cAMP system stimulation by estradiol is mediated by protein kinase C, phosphorylating a protein of adenylate cyclase complex. Estradiol causes protein kinases A translocation into the cell nuclei and enhances the protein kinase NII activity. The role of protein phosphorylation, activated by steroid hormones, in the transcription and protein synthesis regulation, is discussed.

[Mechanism of the stimulating effect of estradiol on protein kinase C in plasma membranes of target cells]. / Mekhanizmy stimuliruiushchego deistviia éstradiola na proteinkinazu C v plazmaticheskikh membranakh kletok-mishenei.

Using inhibitors and activators of protein kinase C, it was demonstrated that in isolated plasma membranes of target cells estradiol-17 beta selectively stimulates protein phosphorylation by endogenous protein kinase C. In estradiol-dependent tissues, estradiol effectuates the translocation of protein kinase C from the cytosol to the membrane fraction within 10-12 minutes. Estradiol activates protein kinase C in cellular membranes of target tissues via a mechanism which is different from that of phorbol ester (TPA): 3H-estradiol, in contrast with 3H-TPA, it is not bound by protein kinase C and, in contrast with TPA, estradiol-17 beta does not activate purified protein kinase C in vitro. In this case, the specific stimulation of protein kinase C translocation to membranes and the estradiol-induced increase in the phosphorylation of plasma membrane proteins seem to be due to the estradiol-induced activation of the transmembrane system of polyphosphoinositide degradation, eventually resulting in the formation of diacylglycerol, a protein kinase C activator.

[Translocation of protein kinase C from cytosol into cell membranes after treatment with estradiol and enzyme activation in target cells]. / Translokatsiia proteinkinazy C pod deistviem éstradiola iz tsitozolia v kletochnye membrany i aktivatsiia fermenta v kletkakh-misheniakh.

The effect of 17 beta-estradiol on protein kinase C in target cells was studied. It was shown that 10-15 min after injection of ovariectomized animals with estradiol (10 micrograms intraperitoneally) protein kinase C is translocated from the cytosol into the cell membranes of estradiol-dependent mammary gland tumours. A similar effect of estradiol on protein kinase C is observed in uterine tissue. On the contrast, in hormone-independent rat mammary gland tumours estradiol causes no redistribution of protein kinase C between the cytosol and cell membranes. No protein kinase C accumulation in the membranes of hormone-dependent mammary gland tumours is observed 30 min after estradiol injection. However, this period is characterized by the appearance of protein kinase whose activity is not stimulated by Ca2+ or phosphatidylserine and which is eluted from DEAE-cellulose with 0.2 M NaCl. This protein kinase presumably corresponds to the M-fragment, i.e., the catalytic part of protein kinase C formed as a result of protein kinase C proteolysis on the membranes. It seems likely that estradiol, similar to growth factor peptides, realizes its stimulating effect on cell division primarily at the expense of coupling of its membrane receptors with the protein kinase C activation system.