The betagamma subunit of heterotrimeric G proteins interacts with RACK1 and two other WD repeat proteins

A yeast two-hybrid approach was used to discern possible new effectors for the betagamma subunit of heterotrimeric G proteins. Three of the clones isolated are structurally similar to Gbeta, each exhibiting the WD40 repeat motif. Two of these proteins, the receptor for activated C kinase 1 (RACK1) and the dynein intermediate chain, co-immunoprecipitate with Gbetagamma using an anti-Gbeta antibody. The third protein, AAH20044, has no known function; however, sequence analysis indicates that it is a WD40 repeat protein. Further investigation with RACK1 shows that it not only interacts with Gbeta(1)gamma(1) but also unexpectedly with the transducin heterotrimer Galpha(t)beta(1)gamma(1). Galpha(t) alone does not interact, but it must contribute to the interaction because the apparent EC(50) value of RACK1 for Galpha(t)beta(1)gamma(1) is 3-fold greater than that for Gbeta(1)gamma(1) (0.1 versus 0.3 microm). RACK1 is a scaffold that interacts with several proteins, among which are activated betaIIPKC and dynamin-1 (1). betaIIPKC and dynamin-1 compete with Gbeta(1)gamma(1) and Galpha(t)beta(1)gamma(1) for interaction with RACK1. These findings have several implications: 1) that WD40 repeat proteins may interact with each other; 2) that Gbetagamma interacts differently with RACK1 than with its other known effectors; and/or 3) that the G protein-RACK1 complex may constitute a signaling scaffold important for intracellular responses.     

Some new aspects of 17alpha-estradiol metabolism in man

17Alpha-estradiol (1,3,5(10)-estratriene-3,17alpha-diol) together with a tracer dose of the tritium-labeled compound was administered orally and sublingually to male volunteers. The serum concentrations of 17alpha-estradiol (free and liberated by enzymatic hydrolysis) were quantified by GC/MS, and the serum total radioactivity and urinary radioactivity excretion were determined. After oral administration, 17alpha-estradiol was rapidly and intensively conjugated; only tiny quantities of the free steroid (<1% of total) appeared in serum. Sublingual administration resulted in temporary (up to 3 h p.a.) higher serum levels of the free compound. The metabolite patterns obtained by TLC of extracts from serum and urine demonstrated that 17alpha-estradiol is the subject of a poor phase I metabolism in man. A great discrepancy was found in the serum concentrations of 17alpha-estradiol (free + conjugated) determined by GC/MS and the serum radioactivity expressed in 17alpha-estradiol equivalents. By TLC analysis of the steroid conjugates extracted from serum, various 17alpha-estradiol conjugate peaks were found. By enzymatic hydrolysis with beta-glucuronidase/aryl sulfatase from Helix pomatia they were only partially cleaved. Thus, the difference between the serum radioactivity and the 17alpha-estradiol levels determined by GC/MS had to be attributed to an incomplete conjugate hydrolysis. It has been shown with the synthesized 17alpha-estradiol sulfate conjugates that only the 3-sulfate is cleaved by enzymatic hydrolysis, whereas the 17-sulfate group resists enzymatic hydrolysis. The methanolysis procedure (acetyl chloride in MeOH) has proved to be an efficient method for cleaving both the 3-sulfate group and the 17-sulfate group. In contrast to the 17alpha-estradiol conjugates in serum, the urinary conjugates were intensively split by the enzyme preparation. From this, it has to be concluded that the serum conjugates were deconjugated and newly reconjugated before urinary excretion.     

Human testis/sperm-specific histone H2B (hTSH2B). Molecular cloning and characterization

Human sperm, unlike the sperm of other mammals, contain replacement histones with unknown biological functions. Here, we report the identification of the novel human gene coding for a testis/sperm-specific histone H2B (hTSH2B). This variant histone is 85% homologous to somatic H2B and has over 93% homology with the testis H2B of rodents. Using genomic PCR, two genetic alleles of hTSH2B were found in the human population. The hTSH2B gene is transcribed exclusively in testis, and the corresponding protein is also present in mature sperm. We expressed recombinant hTSH2B and identified this protein with a particular H2B subtype expressed in vivo. The subnuclear distribution of H2B variants in sperm was determined using biochemical fractionation and immunoblotting. The H2B variant associated with telomere-binding activity () was solubilized by Triton X-100 or micrococcal nuclease extraction, whereas hTSH2B was relatively tightly bound in nuclei. Immunofluorescence showed that hTSH2B was concentrated in spots located at the basal nuclear area of a subpopulation (20% of cells) of mature sperm. This fact may be of particular importance, because the hTSH2B "positive" and "negative" sperm cells may undergo significantly different decondensation processes following fertilization.     

High-resolution NMR structure of an AT-rich DNA sequence

We have determined, by proton NMR and complete relaxation matrix methods, the high-resolution structure of a DNA oligonucleotide in solution with nine contiguous AT base pairs. The stretch of AT pairs, TAATTATAATTATAATTA, is imbedded in a 27-nucleotide stem-and-loop construct, which is stabilized by terminal GC base pairs and an extraordinarily stable DNA loop GAA (Hirao et al., 1994, Nucleic Acids Res. 22, 576–582). The AT-rich sequence has three repeated TAATTA motifs, one in the reverse orientation. Comparison of the local conformations of the three motifs shows that the sequence context has a minor effect here: atomic RMSD between the three TAATTA fragments is 0.4–0.5 Å, while each fragment is defined within the RMSD of 0.3–0.4 Å. The AT-rich stem also contains a consensus sequence for the Pribnow box, TATAAT. The TpA, ApT, and TpTApA steps have characteristic local conformations, a combination of which determines a unique sequence-dependent pattern of minor groove width variation. All three TpA steps are locally bent in the direction compressing the major groove of DNA. These bends, however, compensate each other, because of their relative position in the sequence, so that the overall helical axis is essentially straight.     

Classification of ultracontinental boreal forests in central Yakutia

Using the Braun-Blanquet approach, five associations of boreal forests were distinguished in central Yakutia, the most continental part of eastern Siberia. Ecological features of the syntaxa were explained with the use of the DCA ordination of 50 relevés. All available data from eastern Siberia were involved in the study for syntaxonomic analysis. Central Yakutian boreal forests were classified into two classes:Rhytidio-Laricetea sibiricae Korotkov etErmakov 1999 — ultracontinental light coniferous hemiboreal forests, andVaccinio-Piceetea Br.-Bl. inBr.-Bl. et al. 1939 — typical coniferous taiga forests of northern Eurasia. A new concept of higher syntaxonomic units of the classVaccinio-Piceetea in eastern Siberia has been developed. Three orders represent the diversity of taiga forests: (1)Cladonio-Vaccinietalia Kielland-Lund 1967 (with alliancesHieracio umbellati-Pinion sylvestris Anenkhonov etChytry 1998 andSaxifrago bronchialis-Pinion sylvestris all. nov.) — light coniferous boreal forests occurring in dry and moderately dry oligotrophic sites in various climatic sectors of Northern Eurasia; (2)Lathyro humilis-Laricetalia cajanderi ord. nov. (with alliancesAulacomnio acuminati-Laricion cajanderi all. nov. andRhododendro daurici-Laricion gmelinii all. nov.) — zonal boreal forests with xeric elements, which are typical of regions of northern Asia with cold, dry ultracontinental climate; (3)Ledo-Laricetalia cajanderi ord. prov. (with allianceLedo-Laricion cajanderi prov.) — North Eurasian boreal forests occurring in cold sites with excessive soil moisture, sometimes water-logged. Phytogeography and ecology of these orders are discussed in comparison with other regions of northern Asia.     

POR structural domains important for the enzyme activity in R. capsulatus complementation system

NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria. To approach molecular factors determining the enzyme activity in a living cell, several mutants of POR from pea (Pisum sativum) with site-directed modifications in different parts of the enzyme were generated. The mutant enzymes were expressed in a R. capsulatus mutant deficient in BChl biosynthesis, and their catalytic activity and ability to integrate in bacterial metabolism were analyzed. Our results demonstrate that in heterologous bacterial cell system, higher plant POR is integrated in the porphyrin biosynthesis network and its activity leads to the formation of photosynthetic chlorophyll-proteins (CPs). The study of POR mutants in R. capsulatus reveals several POR domains important for the association of the enzyme with other subcellular components and for its catalytic activity, including identification of putative enzyme reaction center and substrate binding site. The study also demonstrated that an unknown structural factor is important for the formation of the enzyme photoactive complex in etiolated plants. Moreover, our findings suggest that POR might be directly involved in the regulation of the metabolism of other porphyrins. This revised version was published online in June 2006 with corrections to the Cover Date.     

Characterization of P700 as a photochemical quencher in isolated Photosystem I particles using simultaneous measurements of absorbance changes at 830 nm and photoacoustic signal

The relationship between the redox state of P700, the primary donor of PS I, monitored using absorbance changes at 830 nm and photochemical energy storage in PS I reaction centers assayed with the photoacoustic method (PA) was studied in isolated PS I submembrane particles aspirated onto nitrocellulose filters. Several donors have been used to support the electron transport through PS I. NADPH and NADH demonstrated low rates of electron donation to PS I, while ascorbate and ascorbate plus 2,6-dichlorophenolindophenol (DCIP) couple have been found more effective in both P700⁺ reduction and stimulation of the variable component of the PA signal. A linear relationship was found in isolated PS I particles between the (A_830,max – A_830,steady)/A_830,max and (PA_max – PA_steady)/PA_max ratios, which characterized the relative amount of P700 in the reduced state and the relative magnitude of the variable PA component, respectively. That linear relationship was obtained independently from the nature of electron donor used for the reduction of P700⁺. Such linear relationship was also obtained at various wavelengths of modulated light in the range of 660 to 720 nm, only the slope of the linear fits varied with wavelength. It is concluded that reduced P700 act as a photochemical quencher of absorbed energy. Variable thermal dissipation in PS I reaction centers of isolated submembrane particles linearly depends on the amount of reduced P700 and thus constitutes an appropriate indicator of the redox pressure applied to PS I. This revised version was published online in June 2006 with corrections to the Cover Date.     

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.     

Betam,a structural member of the X,K-ATPase beta subunit family,resides in the ER and does not associate with any known X,K-ATPase alpha subunit

betam, a muscle-specific protein, is structurally closely related to the X,K-ATPase beta subunits, but its intrinsic function is not known. In this study, we have expressed betam in Xenopus oocytes and have investigated its biosynthesis and processing as well as its putative role as a chaperone of X,K-ATPase alpha subunits, as a regulator of sarcoplasmic reticulum Ca(2+)-ATPase (SERCA), or as a Ca(2+)-sensing protein. Our results show that betam is stably expressed in the endoplasmic reticulum (ER) in its core glycosylated, partially trimmed form. Both full-length betam, initiated at Met(1), and short betam species, initiated at Met(89), are detected in in vitro translations as well as in Xenopus oocytes. betam cannot associate with and stabilize Na,K-ATPase (NK), or gastric and nongastric H,K-ATPase (HK) alpha isoforms. betam neither assembles stably with SERCA nor is its trypsin sensitivity or electrophoretic mobility influenced by Ca(2+). A mutant, in which the distinctive Glu-rich regions in the betam N-terminus are deleted, remains stably expressed in the ER and can associate with, but not stabilize X,K-ATPase alpha subunits. On the other hand, a chimera in which the ectodomain of betam is replaced with that of beta1 NK associates efficiently with alpha NK isoforms and produces functional Na,K-pumps at the plasma membrane. In conclusion, our results indicate that betam exhibits a cellular location and functional role clearly distinct from the typical X,K-ATPase beta subunits.