Trimerization and crystallization of reconstituted light-harvesting chlorophyll a/b complex.

The major light-harvesting complex (LHCII) of photosystem II, the most abundant chlorophyll-containing complex in higher plants, is organized in trimers. In this paper we show that the trimerization of LHCII occurs spontaneously and is dependent on the presence of lipids. LHCII monomers were reconstituted from the purified apoprotein (LHCP), overexpressed in Escherichia coli, and pigments, purified from chloroplast membranes. These synthetic LHCII monomers trimerize in vitro in the presence of a lipid fraction isolated from pea thylakoids. The reconstituted LHCII trimers are very similar to native LHCII trimers in that they are stable in the presence of mild detergents and can be isolated by partially denaturing gel electrophoresis or by centrifugation in sucrose density gradients. Moreover, both native and reconstituted LHCII trimers exhibit signals in circular dichroism in the visible range that are not seen in native or reconstituted LHCII monomers, indicating that trimer formation either establishes additional pigment-pigment interactions or alters pre-existing interactions. Reconstituted LHCII trimers readily form two-dimensional crystals that appear to be identical to crystals of the native complex.     

Displacement of housekeeping proteasome subunits by MHC-encoded LMPs: a newly discovered mechanism for modulating the multicatalytic proteinase complex.

The degradation of cytoplasmic antigens to peptides presented by class I MHC molecules is thought to be mediated by the ubiquitin/proteasome pathway. Support for this view came from our observation that the subunit composition of proteasomes can be changed by interferon-gamma (IFN-gamma) treatment. Thereby two subunits, LMP2 and LMP7, which are encoded in the MHC class II region, are incorporated into the proteasomal complex, whereas other subunits disappear. In the experiments reported in this communication we studied the subunit changes occurring in cell lines where the expression of LMP2 or LMP7 can be regulated individually either by IFN-gamma induction or by applying a new system to control the expression of transfected LMPs. In both situations LMP2 induction leads exclusively to the disappearance of housekeeping subunit 2, whereas LMP7 affects only subunit 10. Subunit 2 was found to be 76% homologous to LMP2. Since incorporation of LMP2 into the proteasomal complex prevents processing of the subunit 2 precursor, we conclude that LMP2 displaces subunit 2 during assembly. Subunit displacement is most likely a general mechanism to modulate the catalytic activity of the proteasomal complex without changing its structure. Furthermore, the controlled incorporation of transfected subunits into the complex offers a new approach to study proteasome function in vivo.     

Cloning, expression, and chromosomal localization of the 140-kilodalton subunit of replication factor C from mice and humans.

We have isolated a full-length mouse cDNA encoding a lysine-rich protein of 1,131 amino acids with a calculated molecular mass of 126 kDa. The protein binds in a sequence-unspecific manner to DNA, is localized exclusively in the nucleus, and contains a putative ATP binding site and a stretch of 80 amino acids with homology to the carboxy terminus of prokaryotic DNA ligases. On the basis of the following facts, we conclude that the isolated cDNA encodes the 140-kDa subunit of mouse replication factor C (mRFC140). (i) The sequence around the ATP binding site shows significant homology to three small subunits of human replication factor C. (ii) Polyclonal antibodies raised against the protein encoded by this cDNA cross-react with the 140-kDa subunit of purified human replication factor C (hRFC140) and recognize in mouse cell extracts an authentic protein with an apparent molecular mass of 130 kDa. (iii) Sequence comparison with a human cDNA isolated by using tryptic peptide sequence information from purified hRFC140 revealed 83% identity of the encoded proteins. The mRFC140 gene is ubiquitously expressed, and two mRNAs approximately 5.0 and 4.5 kb long have been detected. The gene was mapped by in situ hybridization to mouse chromosome 5, and its human homolog was mapped to chromosome 4 (p13-p14).     

Mercury (Hg2+) and zinc (Zn2+): Two divalent cations with different actions on voltage-activated calcium channel currents

Summary 1. We examined the actions of mercury (Hg²⁺) and zinc (Zn²⁺) on voltage-activated calcium channel currents of cultured rat dorsal root ganglion (DRG) neurons, using the whole-cell patch clamp technique.2. Micromolar concentrations of both cations reduced voltage-activated calcium channel currents. Calcium channel currents elicited by voltage jumps from a holding potential of –80 to 0 mV (mainly L- and N-currents) were reduced by Hg²⁺ and Zn²⁺. The threshold concentration for Hg²⁺ effects was 0.1 µM and that for Zn²⁺ was 10µM. Voltage-activated calcium channel currents were abolished (>80%) with 5µM Hg²⁺ or 200µM Zn²⁺. The peak calcium current was reduced to 50% (IC₅₀) by 1.1µM Hg²⁺ or 69µM Zn²⁺. While Zn²⁺ was much more effective in reducing the T-type calcium channel current—activated by jumping from –80 to –35 mV—Hg²⁺ showed some increased effectiveness in reducing this current.3. The effects of both cations occurred rapidly and a steady state was reached within 1–3 min. While the action of Zn²⁺ was not dependent on an open channel state, Hg²⁺ effects depended partially on channel activation.4. While both metal cations reduced the calcium channel currents over the whole voltage range, some charge screening effects were detected with Hg²⁺ and with higher concentrations (>100µM) of Zn²⁺.5. As Zn²⁺ in the concentration range used had no influence on resting membrane currents, Hg²⁺ caused a clear inward current at concentrations µM.6. In the present study we discuss whether the actions of both metals on voltage-activated calcium channel currents are mediated through the same binding site and how they may be related to their neurotoxic effects.     

Sequence homology and structure predictions of the creatine kinase isoenzymes

Comparisons of the protein sequences and gene structures of the known creatine kinase isoenzymes and other guanidino kinases revealed high homology and were used to determine the evolutionary relationships of the various guamidino kinases. A CK framework is defined, consisting of the most conserved sequence blocks, and diagnostic boxes are identified which are characteristic for anyone creatine kinase isoenzyme (e.g. for vertebrate B-CK) and which may serve to distinguish this isoenzyme from all others (e.g. from M-CKs and Mi-CKs). Comparison of the guanidino kinases by near-UV and far-UV circular dichroism further indicates pronounced conservation of secondary structure as well as of aromatic amino acids that are involved in catalysis.Abbreviations GuaK guanidino kinase - CK creatine kinase - B-and M-CK brain and muscle cytosolic CK isoenzyme - Mi-CK mitochondrial CK isoenzyme - ArgK arginine kinase - Cr creatine - PCr phosphorylcreatine - PArg phosphorylarginine     

Long lasting anti-adrenergic effect of 7-oxo-prostacyclin in the heart: a cycloheximide sensitive increase of phosphodiesterase isoform I and IV activities

Evidence is accumulating that 7-oxo-prostacyclin (7-oxo-PGI₂) induces a delayed indirect anti-adrenergic and cytoprotective effect on the myocardium, the mechanism of which is still unclear. To demonstrate that a single application of 7-oxo-PGI₂ (50 g/kg i.m.) 48 h prior to starting experiments attenuates the isoprenaline inducible inotropic response and accumulation of cAMP, isolated hearts of pretreated animals were perfused in the Langendorff mode with and without isoprenaline (1 to 100 nM). The late anti-adrenergic effect of the drug was manifested by a significant attenuation in the elevation of cAMP levels as well as in contractile force development. This effect was not due to changes in cAMP generation as there were identical ₁-adrenoceptor densities and affinities (as calculated from [³H]-CGP binding studies), G_i and G_s protein patterns (as taken from Western blots) as well as adenylyl cyclase activity measurements in the hearts studied. The anti-adrenergic potency of 7-oxo-PGI₂, however, was found to be related to a significant rise in cyclic nucleotide hydrolysis by phosphodiesterase (PDE). Using the fast-performance liquid chromatographic separation for PDE isoforms, a significant increase in the activity of PDE isoforms I and IV (260±28 vs 110±12 pmol cGMP/min x enzyme fraction and 77±11 vs 34±3 pmol cAMP/min x enzyme fraction, respectively) was found in the solubilized fraction of cardiac membranes in comparison to untreated controls; PDE IV activity was also increased in the cytosolic fraction (106±14 vs 65±6 pmol cAMP/min x enzyme fraction). The hypothesis that the delayed anti-adrenergic effect of 7-oxo-PGI₂ is initiated by an induction and accelerated synthesis of PDE I and IV in the heart is underlined by the fact that cycloheximide suppresses completely both the rise in PDE activities and the anti-adrenergic effects studied. It is suggested that an inducible predominance of cAMP degradation over its generation may be of relevance in processes related to heart protection.     

A novel human type I hair keratin gene: evidence for two keratin hHa3 isoforms

We present the nucleotide and amino acid sequence for a novel human type I hair keratin, which could be identified through its high sequence homology and strict carboxyterminal length identity as a human ortholog of the murine hair keratin mHa3. Our hHa3 sequence differs, however, from that of a previously described hHa3 hair keratin (published only as an amino acid sequence; [13]) in 24 amino acid positions, 8 of which occur in the middle of the carboxyterminal domain. PCR of genomic DNA from 25 normal human subjects using a primer pair derived from sequence segments located in the 3-region of our hHa3 clone that encode conserved amino acid sequences in both keratins, resulted in the amplification of two distinct products of 0.38 kbp and 1.0 kbp. DNA sequence analysis of the cloned PCR products allowed identification of the 0.38 kb sequence as that originating from Yuet al. [13] and the 1.0 kb sequence as that being derived from our data. The difference in fragment length was due to unique intron 6 sequences, indicating that these two keratin species are encoded by genes of their own. Moreover, extensive Southern blot analyses with DNA from 25 unrelated individuals of different races using a 3-noncoding sequence from our keratin and the intron 6 sequence of the keratin of Yuet al. [13], as hybridization probes showed that both keratin genes are present as single copy sequences occurring ubiquitously and without gross alterations in the human genome. Collectively, these data demonstrate that the human type I hair keratin described in this paper represents an isoform of the previously described hHa3 keratin. We propose that these hHa3 isoforms be named in chronological order of discovery hHa3-I and hHa3-II.