Johann Salnikow

The complete amino-acid sequence of the Rieske FeS-precursor protein from spinach chloroplasts deduced from cDNA analysis

SummarySummarySeveral cDNA clones encoding the entire Rieske FeS-precursor protein of the chloroplast cytochrome b6f-complex have been isolated by high density plaque immunoscreening of a phage lambda gt11 cDNA expression library, made from poly A+-RNA of spinach seedlings. The identity of the cDNAs has been confirmed by N-terminal amino acid sequencing of the purified protein. The nucleotide sequence indicates a protein of 247 amino acid residues including a putative transit sequence of 68 amino acids corresponding to molecular masses of 26.3 kDa (precursor) and 18.8 kDa (mature protein; 179 amino acid residues). Alignteins of the sequence with sequences from Rieske FeS-proteins of respiratory electron transport chains, two of bacterial and three of mitochondrial origin, shows little sequence homology, but remarkable similarity in secondary structure including a putative N-terminal transmembrane segment of about 25 residues and the peptides CTHLGCV and CPCHGS in the C-terminal region of the protein that are involved in the binding of the Fe2S2-cluster.

Comparison of two-dimensional electrophoresis patterns of heat shock protein Hsp27 species in normal and cardiomyopathic hearts

Heat shock protein Hsp27 occurs in a complex pattern in human myocardial tissue. Normal and failing explanted human heart from patients with dilated cardiomyopathy (DCM) or ischemic heart failure (IHF), respectively, were analyzed by high resolution two‐dimensional electrophoresis (23×30 cm) and Hsp27 immunostaining. Twelve Hsp27 spots in DCM samples were significantly altered in intensity and ten of these were significantly changed in IHF. Four spots (h1, h2, h4, h5) in DCM samples and three spots (h2, h4, h5) in IHF at a molecular mass of 28 kDa were decreased in intensity. In this study, investigating left ventricles of human myocardium, spot h4 was only detected in normal heart samples. On the other hand, spots with a lower molecular mass of 27 kDa (h14, h15, h17, h20, h21) and 22—23 kDa (46, h47, h50) increased in intensity in failing hearts, suggesting that some form of Hsp27 degradation occurs during heart failure.

Proteins and amino acids in beers, their contents and relationships with other analytical data

Abstract Fluorometry, ion-exchange chromatography, electrophoretic separations and Fourier transform–infrared (FT–IR) spectra were used to determine and characterize amino acids and proteins in 15 different beer samples. Proteins precipitated by ammonium sulfate yielded complex electrophoretic patterns. The major bands corresponded to 45–40 kDa as determined by a two-dimensional gel electrophoresis (2-DE). Proteins and some amino acids are partially responsible for nutritional value and stability of beer. Therefore, electrophoretic analysis revealed that protein characterization of beer during all technological stages might be useful in its quality. FT–IR protein spectra showed the presence of I, II and III amide bands. Protein distribution and amino acid composition of beer differ significantly, depending on the raw materials and enzymatic reactions used in beer technology. Concentrations of histamine (3.02–3.23 mg/l), proline (1.60–3.13 mg/l) and tyramine (3.61–7.4 mg/l) increased during beer fermentation. Statistically significant change was registered in the protein content of the final product, which was less than that in wort ( p p

Isolation and identification of a fourth subunit in the membrane part of the chloroplast ATP-synthase

The subunit composition of highly active purified ATP‐synthase from chloroplasts, CF0F1, was investigated by SDS gel electrophoresis. An additional subunit of CF0, was detected with an apparent molecular mass of 20 kDa. It is stained weakly with Coomassie blue but very strongly with silver. This subunit was isolated on a preparative SDS gel and the N‐terminal amino acid sequence analyzed. It shows that the 20 kDa protein is identical with the protein encoded by the spinach chloroplast gene atpI, called subunit IV [(1986) Mol. Genet. 203, 117–128]. However, in comparison to the gene‐derived sequence, the first 18 amino acids are missing, indicating N‐terminal processing.

Amino-acid sequence of the large subunit of d-ribulosebisphosphate carboxylase/oxygenase from Nicotiana tabacum☆

Abstract The complete amino acid sequence of the large subunit of the ribulosebisphosphate carboxylase (3-phospho- d -glycerate carboxy-lyase (dimirizing), ED 4.1.1. 39) from Nicotiana tabacum has been determined by alignment of tryptic, chymotryptic and cyanogen bromide fragments. The sequence is — except for positions 284 (glycine instead of cysteine) and 377 (valine instead of glutamic acid) — in agreement with the one deduced from gene sequencing (Shinozaki, K. and Sugiura, M. (1982) Gene 20, 91–102). However, the protein chemical determination yields additional information not evident from nucleotide sequencing: (1) The amino terminus is proteolytically processed and appears inhomogeneous; (2) The amino acid sequence is dimorphic in positions 394 and 405 (and possibly in position 23). The latter observation proves that for the large subunit of ribulosebisphosphate carboxylase from N. tabacum there exist at least two (if not more) slightly different genes.

A two-dimensional electrophoresis database of rat heart proteins.

More than 3000 myocardial protein species of Wistar Kyoto rat, an important animal model, were separated by high‐resolution two‐dimensional gel electrophoresis (2‐DE) and characterized in terms of isoelectric point (pI) and molecular mass (Mr). Currently, the 2‐DE database contains 64 identified proteins; forty‐three were identified by peptide mass fingerprinting (PMF) using matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), nine by exclusive comparison with other 2‐DE heart protein databases, and in only 12 cases of 60 attempts N‐terminal sequencing was successful. We used the Make2ddb software package downloaded from the ExPASy server for the construction of a rat myocardial 2‐DE database. The Make2ddb package simplifies the creation of a new 2‐DE database if the Melanie II software and a Sun workstation under Solaris are available. Our 2‐DE database of rat heart proteins can be accessed at URL http://gelmatching.inf.fu‐berlin.de/˜pleiss/2d.

Improved automated solid-phase microsequencing of peptides using DABITC

Abstract Attachment studies with EDC (1-ethyl-3-dimethylaminopropyl carbodiimide) have revealed that peptides, ranging from 4 to 31 residues, can be linked to aminopropyl glass with satisfactory yield, if optimal conditions are obeyed. The attachment yields are, however, significantly lower with peptides possessing carboxyl terminal lysine. High-sensitivity solid-phase sequencing of immobilized peptides can be performed if DABITC (4- N, N -dimethylaminoazobenzene 4′-isothiocyanate), an Edman reagent with a covalently linked chromophore, is used in conjunction with phenyl isothiocyanate. Technical improvements have been introduced by utilization of intermittent nitrogen flushes, pumping of the DABITC reagent in a stepwise manner, and incorporation of an automated conversion device based on instant aequous dilution of the trifluoroacetic acid effluent. Solid-phase microsequencing constitutes a method of general applicability for peptides in the 1- to 10-nmol range, with up to 30–35 identifiable degradation steps.

Chromosomally and plasmid-encoded gene clusters for CO2 fixation (cfx genes) in Alcaligenes eutrophus

SummaryTwo sets of structural genes (cfxL, cfxS) for ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) were localized in the genome of Alcaligenes eutrophus H16 by means of hybridization probes. One set is encoded on a chromosomal 12 kb EcoRI fragment, the other on an 11 kb EcoRI fragment of the megaplasmid pHG1. These fragments have previously been shown to carry the chromosomal and plasmid copies of the also reiterated phosphoribulokinase (PRK) gene (cfxP). Restriction mapping of the cloned fragments showed that, on each of the two genetic entities, the three cfx genes are located in a very similarly organized cluster. The closely linked Rubisco genes are separated from the downstream PRK gene by about 3.8 kb. The two cfx regions are highly homologous as deduced from cross-hybridization experiments. The Rubisco genes were found to be cotranscribed into a 2.1 kb cfxL-cfxS mRNA. Their expression is regulated at the transcriptional level. Rubisco and PRK genes were subcloned and expressed in Escherichia coli under the control of the lac promoter of pUC plasmids. Since active recombinant enzymes with the structural properties of the authentic enzymes were formed, the ribosome binding sites of the genes are recognized in E. coli. A low level of expression of the Rubisco genes occurred even without lac promoter control, indicating some activity of the cfxL-cfxS promoters in the foreign host.

An iterative calibration method with prediction of post-translational modifications for the construction of a two-dimensional electrophoresis database of mouse mammary gland proteins.

Protein databases serve as general reference recources providing an orientation on two‐dimensional electrophoresis (2‐DE) patterns of interest. The intention behind constructing a 2‐DE database of the water soluble proteins from wild‐type mouse mammary gland tissue was to create a reference before going on to investigate cancer‐associated protein variations. This database shall be deemed to be a model system for mouse tissue, which is open for transgenic or knockout experiments. Proteins were separated and characterized in terms of their molecular weight (Mr) and isoelectric point (pI) by high resolution 2‐DE. The proteins were identified using prevalent proteomics methods. One method was peptide mass fingerprinting by matrix‐assisted laser desorption/ionization‐mass spectrometry. Another method was N‐terminal sequencing by Edman degradation. By N‐terminal sequencing Mr and pI values were specified more accurately and so the calibration of the master gel was obtained more systematically and exactly. This permits the prediction of possible post‐translational modifications of some proteins. The mouse mammary gland 2‐DE protein database created presently contains 66 identified protein spots, which are clickable on the gel pattern. This relational database is accessible on the WWW under the URL: http://www.mpiib‐berlin.mpg.de/2D‐PAGE.

Identification of two binding sites of the D-ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach for D-ribulose 1,5-bisphosphate and effectors of the carboxylation reaction.

Abstract Fluorimetric studies of the binding of d -ribulose 1,5-bisphosphate (RuP 2 ) and the effectors 6-phosphogluconate and fructose 1,6-bisphosphate to the d -ribulose 1,5-bisphosphate carboxylase/oxygenase from spinach were correlated with the functions of these sugar phosphates in the carboxylation reaction. These agents compete for two binding sites of the enzyme. At relatively low concentrations they bind to an allosteric site, where 6-phosphogluconate and fructose 1,6-bisphosphate display their stimulating effect on the fixation of CO 2 . At higher concentrations these compounds inhibit the carboxylation reaction and compete with RuP 2 for the reaction center of the carboxylase. Preincubation of the enzyme with low concentrations of RuP 2 (0.1–5 μ m ) inhibits the activity of these effectors as well as the effector-induced fluorescence changes of the enzyme-2- p -toluidinonapthalene-6-sulfonate (TNS) complex by competition for the regulatory center which could be identified as the high affinity binding site of the enzyme for RuP 2 with a K D = 0.6 μm. The deactivation of the carboxylase which is observed on preincubation of the enzyme with RuP 2 in the absence of bicarbonate and Mg 2+ cannot be correlated to the binding of RuP 2 to the effector site. The deactivation process occurs in an RuP 2 concentration range similar to that for CO 2 fixation.