Molecular Mass Species Research Articles

Association of a lower molecular weight protein to the mu-opioid receptor demonstrated by (125)I-beta-endorphin cross-linking studies.

Cross-linking experiments using the (125)I-beta-endorphin revealed the presence of several receptor-related species in cell lines expressing endogenous opioid receptors, including a small molecular mass protein (approximately 22 kDa). Previous reports have suggested that this 22-kDa (125)I-beta-endorphin cross-linked protein could be the degradative product from a higher molecular mass species, i.e., a fragment of the receptor. To determine if this protein is indeed a degraded receptor fragment, (125)I-beta-endorphin was cross-linked to the (His)(6) epitope-tagged mu-opioid receptor (His-mu) stably expressed in the murine neuroblastoma Neuro(2A) cells. Similar to earlier reports with cell lines expressing endogenous receptors, two major bands of 72- and 25-kDa proteins were specifically cross-linked. Initial cross-linking experiments indicated the absolute requirement of the high-affinity (125)I-beta-endorphin binding to the mu-opioid receptor prior to the appearance of the low molecular weight species, suggesting that the 22-kDa protein could be a degraded fragment of the receptor. However, variations in the ratios of these protein bands being cross-linked by several homo- or heterobifunctional cross-linking agents were observed. Although neither the carboxyl terminus mu-opioid receptor-specific antibodies nor the antibodies against the epitope at the amino terminus of the receptor could recognize the 22-kDa protein, this (125)I-beta-endorphin cross-linked species could be coimmunoprecipitated with the receptor antibodies or could be isolated with a nickel resin affinity chromatography. The direct physical association of the 22-kDa protein with the receptor was demonstrated also by the observation that the 22-kDa protein could not bind to the nickel resin alone, but that its binding to the nickel resin was restored in the presence of the His-mu. Taken together, these results suggest that the 22-kDa protein cross-linked by (125)I-beta-endorphin is not a degradative product, but a protein located within the proximity of the mu-opioid receptor, and that it is tightly associated with the receptor.

Cysteines 431 and 1074 Are Responsible for Inhibitory Disulfide Cross-linking between the Two Nucleotide-binding Sites in Human P-glycoprotein

Human wild-type and Cys-less P-glycoproteins were expressed in Pichia pastoris and purified in high yield in detergent-soluble form. Both ran on SDS gels as a single 140-kDa band in the presence of reducing agent and showed strong verapamil-stimulated ATPase activity in the presence of added lipid. The wild type showed spontaneous formation of higher molecular mass species in the absence of reducing agent, and its ATPase was activated by dithiothreitol. Oxidation with Cu(2+) generated the same higher molecular mass species, primarily at 200 and approximately 300 kDa, in high yield. Cross-linking was reversed by dithiothreitol and prevented by pretreatment with N-ethylmaleimide. Using proteins containing different combinations of naturally occurring Cys residues, it was demonstrated that an inhibitory intramolecular disulfide bond forms between Cys-431 and Cys-1074 (located in the Walker A sequences of nucleotide-binding sites 1 and 2, respectively), giving rise to the 200-kDa species. In addition, dimeric P-glycoprotein species ( approximately 300 kDa) form by intermolecular disulfide bonding between Cys-431 and Cys-1074. The ready formation of the intramolecular disulfide between Cys-431 and Cys-1074 establishes that the two nucleotide-binding sites of P-glycoprotein are structurally very close and capable of intimate functional interaction, consistent with available information on the catalytic mechanism. Formation of such a disulfide in vivo could, in principle, underlie a regulatory mechanism and might provide a means of intervention to inhibit P-glycoprotein.

Software (MSPECTRA) for automatic interpretation of triacylglycerol molecular mass distribution spectra and collision induced dissociation product ion spectra obtained by ammonia negative ion chemical ionization mass spectrometry.

Rapid analysis of molecular mass distributions of triacylglycerol (TAG) mixtures and regioisomeric structures of selected molecular mass species is possible using ammonia negative ion chemical ionization mass spectrometry utilizing sample introduction by direct exposure probe. However, interpretation of spectra and calculation of results is time consuming, thus lengthening the total analysis time. To facilitate result calculation a software package (MSPECTRA 1.3) was developed and applied to automatic processing of triacylglycerol molecular mass distribution spectra and collision induced dissociation (CID) product ion spectra. The program is capable of identifying triacylglycerol molecular mass species possessing different ACN:DB (acyl carbon number:number of double bonds) ratios on the basis of m/z values of [M - H](-) ions. In addition to such identification the program also corrects spectra for abundances of naturally occurring (13)C isotopes and calculates relative proportions of triacylglycerol molecular species in the analyzed samples. If several replicate spectra are processed simultaneously the program automatically calculates an average and standard deviation of relative proportions of molecular species. In the case of CID spectra the program identifies fatty acid fragment ions [RCO(2)](-) and the corresponding [M - H - RCO(2)H - 100](-) ions, and calculates the relative proportions of ions in both groups. These proportions are then used automatically to calculate the fatty acid combinations comprising the parent triacylglycerol molecule and the regiospecific positions of fatty acids. Processing of several replicate product ion spectra simultaneously produces averaged proportions of regioisomers comprising the parent triacylglycerol molecular species and the standard deviation of the analysis. The performance of the program was tested by analyzing triacylglycerol samples of human milk, human milk substitutes, human chylomicron and cocoa butter, and by comparing results obtained by automated processing of the data with manually calculated results.

Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor

To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

Role of complex asparagine-linked oligosaccharides in the expression of a functional thyrotropin receptor.

To evaluate the functional role of complex asparagine-linked oligosaccharides of the human thyrotropin receptor (TSHR), a Chinese hamster ovary cell line (JP09) and a K562 cell line (K562-TSHR) expressing this receptor were treated with deoxymannojirimycin (dMM), a mannosidase I inhibitor. dMM blocks the formation of complex-type structures and leads to the formation of high-mannose-type structures. Treatment of cells with dMM led to a decrease in the number of thyrotropin (TSH)-binding sites at the cell surface. Detection of the TSHR at the cell surface using a monoclonal antibody directed against the A subunit showed that this decrease was not due to a decrease in the number of TSHRs expressed at the cell surface. However the recognition of TSHR by a monoclonal antibody directed against the C peptide was greatly decreased. On immunoblotting, after deglycosylation using peptide N-glycanase F, the A subunit was visualized as a doublet (36 and 41 kDa). In control cells the species of higher molecular mass was more abundant whereas after dMM treatment the species of lower molecular mass became more abundant. This difference in molecular mass between the two peptides is compatible with the removal of the C peptide. In conclusion, the results show that inhibition of complex-type structure formation leads to (i) an incapacity for TSHR to bind TSH, without affecting its intracellular transport and (ii) an increase of TSHR susceptibility to proteases that remove the C peptide. We then hypothesized that removal of the C peptide could contribute to the formation of a non-functional TSHR.

Rat tapasin: cDNA cloning and identification as a component of the class I MHC assembly complex.

During the assembly of major histocompatibility complex (MHC) class I molecules transient associations are formed with the endoplasmic reticulum resident chaperones calnexin and calreticulin, ERp57 oxidoreductase, and also with tapasin, the latter mediating binding of the class I molecules to the transporter associated with antigen processing (TAP). We report here the isolation of a cDNA encoding rat tapasin from a DA (RT1av1) library. The cDNA encodes a proline-rich (11.3%) polypeptide of 464 residues with a potential ER-retention KK motif at its COOH-terminus, and a predicted molecular mass of 48 kDa. Matrix-assisted laser-desorption ionisation (MALDI) mass spectrometry of peptides derived from in-gel tryptic digestion of a TAP-associated protein match regions of the predicted translation product. A species of the correct molecular mass and predicted pl was also identified in association with radiolabelled immunoprecipitates of the rat TAP complex analysed by two-dimensional gel electrophoresis. This confirms rat tapasin as a component of the rat MHC class I assembly complex.

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy

A Novel Tissue Inhibitor of Metalloproteinases-3 Mutation Reveals a Common Molecular Phenotype in Sorsby's Fundus Dystrophy

Identification of the 183RWTNNFREY191Region as a Critical Segment of Matrix Metalloproteinase 1 for the Expression of Collagenolytic Activity

Matrix metalloproteinase 1 (MMP-1) cleaves types I, II, and III collagen triple helices into (3/4) and (1/4) fragments. To understand the structural elements responsible for this activity, various lengths of MMP-1 segments have been introduced into MMP-3 (stromelysin 1) starting from the C-terminal end. MMP-3/MMP-1 chimeras and variants were overexpressed in Escherichia coli, folded from inclusion bodies, and isolated as zymogens. After activation, recombinant chimeras were tested for their ability to digest triple helical type I collagen at 25 degrees C. The results indicate that the nine residues (183)RWTNNFREY(191) located between the fifth beta-strand and the second alpha-helix in the catalytic domain of MMP-1 are critical for the expression of collagenolytic activity. Mutation of Tyr(191) of MMP-1 to Thr, the corresponding residue in MMP-3, reduced collagenolytic activity about 5-fold. Replacement of the nine residues with those of the MMP-3 sequence further decreased the activity 2-fold. Those variants exhibited significant changes in substrate specificity and activity against gelatin and synthetic substrates, further supporting the notion that this region plays a critical role in the expression of collagenolytic activity. However, introduction of this sequence into MMP-3 or a chimera consisting of the catalytic domain of MMP-3 with the hinge region and the C-terminal hemopexin domain of MMP-1 did not express any collagenolytic activity. It is therefore concluded that RWTNNFREY, together with the C-terminal hemopexin domain, is essential for collagenolytic activity but that additional structural elements in the catalytic domain are also required. These elements probably act in a concerted manner to cleave the collagen triple helix.

Two rat surfactant protein A isoforms arise by a novel mechanism that includes alternative translation initiation.

A single gene for rat surfactant protein A (SP-A) encodes two isoforms that are distinguished by an isoleucine-lysine-cysteine (IKC) N-terminal extension (SP-A and IKC-SP-A). Available evidence suggests that the variants are generated by alternative signal peptidase cleavage of the nascent polypeptide at a primary site (Cys(-)(1)-Asn(1)) and a secondary site (Gly(-)(4)-Ile(-)(3)). In this study, we used site-directed mutagenesis and heterologous expression in vitro and in insect cells to the examine mechanisms that may lead to alternative signal peptidase cleavage including alternative translation initiation at two in-frame AUGs (Met(-)(30) and Met(-)(20)), a suboptimal context for hydrolysis at the primary cleavage site, or cotranslational protein modifications that expose an otherwise cryptic secondary cleavage site. In vitro translation of a rat cDNA for SP-A resulted in both 28 and 29 kDa primary translation products on SDS-PAGE analysis, while translation of cDNAs encoding Met-30Ala and Met-20Ala mutations resulted in only the single 28 and 29 kDa molecular mass species, respectively. These data are consistent with translation initiation at both Met(-)(30) and Met(-)(20) during in vitro synthesis of SP-A. The Met-30Ala mutation reduced expression of the longer isoform in insect cells, indicating that the Met(-)(30) site also contributes to eucaryotic protein expression. Forcing translation initiation at Met(-)(30) by optimizing the Kozak consensus sequence surrounding that codon or by mutating the Met(-)(20) codon resulted in preferential expression of the longer SP-A isoform but reduced overall expression of the protein almost 10-fold. Both isoforms were generated to some degree whether translation was initiated at the codon for Met(-)(30) or Met(-)(20), indicating that the site of translation initiation is not the sole determinant of isoform generation and suggesting that either the context of the primary cleavage site is suboptimal or that cotranslational modifications affect cleavage. Preventing N-terminal glycosylation at Asn(1) did not affect the site of signal peptidase cleavage. Disruption of interchain disulfide formation at Cys(-)(1) by substitution with serine markedly enhanced cleavage at the Gly(-)(4)-Ile(-)(3) bond, but substitution with alanine enhanced cleavage at the Cys(-)(1)-Asn(1) bond. We conclude that rat SP-A isoforms arise by a novel mechanism that includes both alternative translation initiation at two in-frame AUGs and a suboptimal context for signal peptidase hydrolysis at the primary cleavage site.

The Two Toxoplasma gondii Hypoxanthine-Guanine Phosphoribosyltransferase Isozymes Form Heterotetramers

Two isozymes of the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HGPRT) of the apicomplexan protozoan Toxoplasma gondii are encoded by the single HGPRT gene as a result of differential splicing. Western blotting of total T. gondii protein shows that both isozymes I and II, which differ by 49 amino acids, are expressed. Both form enzymatically active homotetramers when overexpressed in Escherichia coli. The specific activity of HGPRT-I is five times that of HGPRT-II. When both isozymes are co-expressed in E. coli, HGPRT-I.HGPRT-II heterotetramers form. The predominant heterotetramer has enzymatic activity similar to HGPRT-II, and gel filtration chromatography demonstrates that its size is intermediate between the sizes of HGPRT-I and HGPRT-II. Mass spectrometric analysis of cross-linked homo- and heterotetramers reveals species of distinct molecular mass for HGPRT-I, HGPRT-II, and HGPRT-I.HGPRT-II and suggests that the predominant heterotetramer consists of one HGPRT-I subunit and three HGPRT-II subunits. The implications of this finding are discussed.

Reactivity of Freshly Formed Fe(III) in Synthetic Solutions and (Pore)Waters: Voltammetric Evidence of an Aging Process

We report a laboratory and field study that demonstrates that soluble Fe(III) complexed to organic ligands exists in sediment porewaters. Synthetic solutions of Fe(III) with the ligand Tris were used to simulate organic complexation of Fe(III) in natural waters. Size fractionation using gel filtration chromatography shows that at least three different molecular mass species coexist. The molecular masses of FeIIIx−Trisy complexes increase with time, suggesting that aggregation occurs in solution. Soluble Fe(III) is detected by voltammetry at a mercury drop electrode when it is complexed by an organic ligand. Depending on the age of Fe(III), two voltammetric waves can be observed: at ca. −0.3 V for “freshly” formed Fe(III) and at a more negative potential for “aged” Fe(III). A mathematical model was successfully developed to assess a mechanism for this aging process. This model involves oxidation and adsorption of Fe(II), aggregation, and precipitation of Fe(III) complexes. The species formed are extremely reactive. Upon addition of sulfide, reduction of Fe(III) instantaneously produces Fe(II) and FeS in solution, and a nonreductive breakdown of Fe(III) is promoted. The existence of soluble Fe(III) in sediment porewaters has important implications on the mineralization of organic matter. In addition to being highly reactive, Fe(III) can diffuse in and out of sediments, supplying an electron acceptor at locations where hydrous iron oxides are already consumed, potentially shifting local reactions. In addition, reduction of soluble Fe(III) by sulfide and successive formation of aqueous FeS should facilitate the production of pyrite in natural sediments. The use of voltammetry to measure soluble Fe(III) can be applied to any oceanographic or environmental system to determine the fate of Fe(III) in the dissolved phase.

The effect of dexamethasone on body and organ growth of normal and IGF-II-transgenic mice.

The physiological role of IGF-II remains unclear but there is evidence for a role in postnatal growth, the growth of the thymus and bone homeostasis. Glucocorticoids have many effects that are opposite to the effects of IGF-II such as growth retardation, osteoporosis and thymic involution. We therefore wondered whether IGF-II overexpression in transgenic mice might counteract some of the growth inhibitory effects of the glucocorticoid, dexamethasone (DXM). In a dose-finding study in normal mice, 20 microg DXM/day caused a significant growth delay. The various organs had a different susceptibility to the growth inhibitory effects of DXM. Most affected were thymus and spleen, followed by liver, skeletal muscle and lumbar vertebrae. The weights of the kidney, tibia, and humerus were not significantly diminished. In a second experiment, the effects of DXM in normal and IGF-II-transgenic animals were compared. The IGF-II serum levels in the transgenic animals were more than 40-fold increased compared with control mice and were decreased by 35% in the DXM-treated group. IGF-I serum levels were identical in both mouse strains and rose slightly after DXM administration in controls. Transgenic mice had higher levels of IGF binding protein species of apparent molecular masses of 41.5 kDa, 30 kDa, and 26.5 kDa. DXM reduced the 24 kDa band in both mice strains. In addition it reduced the bands at 38.5 kDa and 26.5 kDa but only in the transgenic animals. The effect of DXM on body growth was similar in normal and IGF-II-transgenic mice. The weight reduction of the various organs caused by DXM was similar in both types of mice except for the skeleton. The weight of the tibia and the humerus were significantly higher in the DXM-treated transgenic mice. In conclusion, we speculate that overexpression of IGF-II in mice partially protects bone from the osteopenic effects of glucocorticoids.

Natural Splicing of Exon 2 of Human Interleukin-15 Receptor α-Chain mRNA Results in a Shortened Form with a Distinct Pattern of Expression

We report the existence of eight different interleukin-15 receptor alpha-chain (IL-15Ralpha) transcripts resulting from exon-splicing mechanisms within the IL-15Ralpha gene. Two main classes of transcripts can be distinguished that do or do not (Delta2 isoforms) contain the exon 2-coding sequence. Both classes were expressed in numerous cell lines and tissues (including peripheral blood lymphocytes) at comparable levels and could be transcribed in COS-7 cells, and the proteins were expressed at the cell surface. Both receptor forms displayed numerous glycosylation states, reflecting differential usage of a single N-glycosylation site as well as extensive O-glycosylations. Whereas IL-15Ralpha bound IL-15 with high affinity, Delta2IL-15Ralpha was unable to bind IL-15, thus revealing the indispensable role of the exon 2-encoded domain in cytokine binding. A large proportion of IL-15Ralpha was expressed at the nuclear membrane with some intranuclear localization, supporting a potential direct action of the IL-15.IL-15Ralpha complex at the nuclear level. In sharp contrast, Delta2IL-15Ralpha was found only in the non-nuclear membrane compartments, indicating that the exon 2-encoded domain (which is shown to contain a potential nuclear localization signal) plays an important role in receptor post-translational routing. Together, our data indicate that exon 2 splicing of human IL-15Ralpha is a natural process that might play regulatory roles at different levels.

Characterization of matrix metalloproteinases in human urine: alterations during adolescence.

Matrix metalloproteinases (MMPs) are a family of at least 14 zinc-dependent proteinases that have been implicated in matrix turnover under both normal and pathological conditions. Previous studies have shown that several MMPs are produced in various cell types in the kidney, suggesting that MMPs may be involved in renal morphogenesis and remodelling. Using a variety of techniques, including gelatin and casein zymography, gelatin affinity chromatography, immunoblotting, and immunoprecipitation, we have identified the major gelatinases in human urine as MMP-2 and MMP-9. Latent forms of both enzymes were detected in urine, as well as lower molecular mass species of each, consistent with activated forms of MMP-2 and MMP-9. MMP-2 and MMP-9 were also measured in individual human urine samples (n=40). No significant gender differences in MMP concentrations were detected. However, renal MMP expression appeared to be age dependent; the highest average amounts of urine MMP-2 were detected during adolescence, while the converse was true of urine MMP-9. Together, these findings indicate that under normal conditions, human urine contains MMP-2 and/or MMP-9, suggesting that these two MMPs are normally produced within the kidney, where they may regulate normal renal remodelling and matrix homeostasis in an age-specific manner.

Subcellular distribution of selenium and Se-containing proteins in human liver

Selenium is an essential trace element in many living organisms. In the present paper, the subcellular distribution of selenium and Se-containing proteins in human liver samples, which were obtained from normal subjects who had an accidental death, was investigated by differential centrifugation and column chromatography. Selenium was mainly enriched in nuclei, mitochondria and cytosol. Almost half of Se existed in the nuclei due to their large amount in liver and high Se concentration. 15–30% of Se was found in small compounds with M r<2000 in the liver components separated by dialysis. The average abundance of Se in small molecular mass species of whole-liver was 23.6%, which suggested most of Se associated with biological macromolecules. Eight kinds of Se-containing proteins with molecular mass of 335±20, 249±15, 106±11, 84.6±5.8, 70.5±5.4, 45.6±1.5, 14.8±2.6, 8.5±1.2 kDa were found in the subcellular fractions of human liver. Among them the 335, 84.6 and 8.5 kDa proteins were individually present in one subcellular fraction, whereas the others coexisted in two, three or four subcellular fractions. The most abundant Se-containing proteins, 70.5 and 14.8 kDa, accounted for 33.6% and 48.5% in the whole-liver soluble Se-containing protein, respectively. The former was enriched in cytosol and the latter was mainly present in nuclei and mitochondria.

Direct coupling of ionic high-performance liquid chromatography with electrospray ionization mass spectrometry utilizing a microdialysis junction interface

A method for directly coupling ionic HPLC with electrospray ionization mass spectrometry (ESI-MS) utilizing a microdialysis junction interface is described. HPLC eluent was split postcolumn to allow a ∼5–40 μl/min flow into the microdialysis assembly, which consists of a microbore microdialysis fiber and a larger concentric sheath tubing for the introduction of a counter-current dialysis buffer. The salt-containing sample was desalted on-line and transferred directly to the ESI source of a LCQ ion trap mass spectrometer. A sample flow-rate (inside the microdialysis fiber) of 10–20 μl/min was found optimal for both retaining HPLC resolution and achieving efficient on-line desalting. When performed at 50°C, the microdialysis system could provide complete on-line desalting for LC buffers containing up to 50 m M Tris–HCl and 1 M NaCl. The introduction of an organic sheath liquid with 2% acetic acid enhanced the ESI-MS detection sensitivity by as much as tenfold and a sensitivity in the low pmol range (initial LC injection before flow splitting) was achieved for insulin chain A. Successful on-line desalting was also achieved for a protein mixture consisting of ubiquitin, cytochrome c and lysozyme separated by cation-exchange chromatography and a good quality spectrum was obtained for each component. Limitations of the current system include the working pH range (4–11), the temperature tolerance (<60°C) and the molecular mass cut-off (∼1000) imposed by the microdialysis fiber and the low sensitivity of the LCQ mass spectrometer for high molecular mass species.

The Fate of 14C-Labelled Hurnic Substances in Rice Cells in Culture

Summary Peat and aquatic humic acids covering a molecular mass range from approximately 40,000 to 8,000 were found to stimulate the growth of rice calli and rice cells in suspension culture. Growth stimulation was investigated as a function of humic acid concentration up to 100 ppm and growth time up to 60 days. The humic acids increase calli weight by approximately 80% and cell numbers in culture approximately 160 × after 42 days. The humic acids had no effect on the protein and DNA contents of rice cells; however, by using the radioactive 14 C N-methylamide derivatives of the humic acids it has been found that both rice cell protein and DNA become radiolabelled during culture. The incorporation of 14 C into DNA is approximately 10× that into protein. Extraction of cell DNA followed by hydrolysis with DNAase I and HPLC analysis of the resulting deoxynucleotides has demonstrated that approximately 70% of the 14 C activity is incorporated into the purine deoxyribonucleotides dAMP and dGMP. Analysis of the humic acids in the culture medium during growth showed that while a major proportion of the humic molecules remained, there was some breakdown to lower molecular mass species and possibly some release of the radiolabelled methylamine. However, the asymmetric incorporation of the major proportion of the 14 C into the purine-containing deoxynucleotides did not arise from the putative release of the label from the humic acids, since 14 C-methylamine itself incorporated 14 C evenly into the four deoxynucleotides. It is suggested that aromatic moeties derived from the humic acids could result in phenylalanine and tyrosine entering the metabolic pathways to glycine and aspartate, which are used in the de novo synthesis of the deoxynucleotide bases. The incorporation of 14 C from 14 C-glycine and 14 C-aspartate into rice DNA deoxynucleotides was confirmed in control experiments. The results suggest that rice cells can use humic breakdown products in the synthesis of proteins and DNA.

Oligomerisation of Ca 2+-regulatory membrane components involved in the excitation-contraction-relaxation cycle during postnatal development of rabbit skeletal muscle

The skeletal muscle excitation-contraction-relaxation cycle matures during the first weeks after birth and protein-protein interactions are believed to be essential for proper Ca 2+ regulation. We therefore studied potential changes in the oligomerisation of key components of the Ca 2+-regulatory membrane system during postnatal myogenesis. In contrast to a decrease in calreticulin, the Ca 2+-binding proteins calsequestrin and sarcalumenin increased in abundance in microsomes isolated from muscle between postnatal days 1 and 41. While the expression of the fast Ca 2+-ATPase increased, its slow-twitch isoform decreased. The junctional component triadin, the 53 kDa sarcoplasmic reticulum glycoprotein, as well as the dihydropyridine receptor increased in abundance, while no major changes in the expression of the ryanodine receptor were observed. Crosslinking analysis revealed that the fast Ca 2+-ATPase, α 1-dihydropyridine receptor and calsequestrin exhibit a more pronounced tendency to oligomerise in adult muscle fibres as compared to early postnatal stages. Interestingly, adult calsequestrin exists not only as a 63 kDa form but also as stable molecular species of higher molecular mass. These findings imply that during postnatal development, protein-protein interactions within the Ca 2+-regulatory membrane system become more complex and oligomerisation appears to be an essential prerequisite for the proper physiological functioning of key membrane proteins in matured skeletal muscle fibres.

Defense Mechanism of Rice Plant to Respiratory Inhibition by a Promising Candidate Blasticide, SSF126

A new candidate systemic rice blasticide, SSF126, dose-dependently inhibited NADH oxidation by submitochondrial particles from rice roots. However, oxidation by the root submitochondrial particles was much less susceptible to SSF126 compared to that by submitochondrial particles from mycelial cells ofPyricularia grisea, a pathogen causing rice blast. Interestingly, SSF126 did not completely suppress the respiration by intact rice roots, and the respiratory activity of the roots recovered from inhibition time-dependently even in the presence of SSF126 at concentrations sufficient to fully block oxidation by the submitochondrial particles. This recovery was not due to selective extrusion of SSF126 from the roots, but to switching from the cytochrome pathway to the alternative cyanide-resistant respiratory pathway. In immunoblots of the alternative oxidase, high molecular mass species were detected in the mitochondria from rice roots in addition to low molecular mass species. Quantification of high and low molecular mass species revealed an increase in the amount of a protein corresponding to a 36-kDa species equivalent to a decrease in the amount of a protein corresponding to a 72-kDa species following a 5-h incubation with SSF126. This conversion of the alternative oxidase to the low molecular mass species in the mitochondria was correlated with the respiratory recovery found in intact rice roots, suggesting that the low molecular mass species is the active form of the alternative oxidase and the high molecular mass species is the inactive form. These results suggest that rice plants can block the severe injury caused by limiting the cytochrome pathway by SSF126 through utilization of the alternative pathway promoted by the interconversion of the alternative oxidase protein.

Chromatographic Separation and Sample Preparation in One Step for MALDI Mass Spectrometric Analysis of Subpicomole Amounts of Heterogeneous Protein Samples

A rapid and efficient sample preparation method is described that allows analysis of biopolymer mixtures by matrix-assisted laser desorption/ionization mass spectrometry (MALDI MS). Very low amounts of complex biopolymer mixtures can be analyzed by MALDI MS without any apparent loss of material and with a substantial gain in time by combining chromatographic separation and sample preparation in a single operation. This micropurification method is based on both the formation of a submicroliter reversed-phase chromatographic bed and the use of matrix-containing eluent solutions. In this way, desalting, matrix mixing, and micropurification of a biological sample could be performed in one step. A proteolytic mixture containing Ca2+ ATPase fragments ranging from ∼1000 to ∼30 000 Da, obtained after endoproteinase AspN cleavage of sarcoplasmic reticulum vesicles, was analyzed by MALDI MS. Direct analysis by MALDI MS of this peptidic mixture did not provide any signal for the higher molecular mass species. When o...