The light-harvesting chlorophyll a/b-binding protein (LHCP) is largely protected against protease (except for about 1 kD on the N terminus) in the thylakoid membrane; this protease resistance is often used to assay successful insertion of LHCP into isolated thylakoids in vitro. In this paper we show that this protease resistance is exhibited by trimeric light-harvesting complex of photosystem 11 (LHCII) but not by monomeric LHCll in which about 5 kD on the N terminus of LHCP are cleaved off by protease. When a mutant version of LHCP that is unable to trimerize in an in vitro reconstitution assay is inserted into isolated thylakoids, it gives rise to only the shorter protease digestion product indicative of monomeric LHCII. We conclude that more of the N-terminal domain of LHCP is shielded in trimeric than in monomeric LHCll and that this difference in protease sensitivity can be used to distinguish between LHCP assembled in LHCll monomers or trimers. The data presented prove that upon insertion of LHCP into isolated thylakoids at least part of the protein spontaneously binds pigments to form LHCII, which then is assembled in trimers. The dependence of the protease sensitivity of thylakoid-inserted LHCP on the oligomerization state of the newly formed LHCll justifies caution when using a protease assay to verify successful insertion of LHCP into the membrane.

Δ5-3β-Ηydroxysteroid dehydrogenase (Δ5-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ5-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ5-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed.

Lanatoside 15′-O-acetylesterase (LAE) from in-vitro-cultivated cells of Digitalis lanata Ehrh. was isolated and partially sequenced. The enzyme was extracted with citrate buffer from acetone dry powder. It was purified in a two-step chromatographical procedure including Phenyl Sepharose hydrophobic interaction chromatography followed by CM Sepharose cation-exchange chromatography to more than 330 μmol · s−1 · (g protein)−1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified protein showed a major band at 39 kDa. The protein was identified by correlation of band intensity on SDS-PAGE and enzyme activity of CM Sepharose column fractions. Size-exclusion chromatography on Sephacryl 200 revealed a single activity peak with an apparent molecular mass of about 85 kDa. Electrophoresis under nondenaturating conditions of purified LAE showed only one band with esterase activity. The intensity of this band was correlated with that of the 39-kDa band after SDS-PAGE. About 30% of the protein, including the N-terminus and several fragments obtained by Lys-C protease digestion, was sequenced. A fragment obtained by Lys-C digestion showed partial homology to other hydrolases and apoplasmic proteins. It included the probable location of an active-site histidine. The activity of LAE was high in non-morphogenic D. lanata cell strains selected for high activities in the chemical transformation of cardenolides, but rather low in the proembryogenic masses of the embryogenic cell strain VIII. It increased during the development of somatic embryos. The LAE activity in leaves of D. lanata plants was in the range 4–24 nmol · s−1 · (g protein)−1.

A three-step chromatographic procedure was developed for purification of cardenolide 16′-O- glucohydrolase (CGH) from Digitalis lanata Ehrh. leaves, including Phenyl-Sepharose hydrophobic interaction chromatography followed by SP-Sepharose cation exchange and Q-Sepharose anion-exchange chromatography. Starting with acetone dry powder the purification resulted in an 760-fold enrichment of CGH. Molecular weight, substrate specificity, pH optimum and temperature stability of CGH were determined. Antibodies against CGH were prepared in rabbits. The SDS gel electrophoresis of protein extracts from leaves of D. lanata and other D. species showed bands at 70␣kDa and 36 kDa reacting with the antibodies. The 70-kDa protein is the main protein stained with CGH antibodies in freshly prepared extracts of D. lanata. It may represent undegraded CGH. The 36-kDa protein is enriched in aged CGH preparations. It is probably a degradation product. Proteins related to 70-kDa and 36-kDa bands also occur in crude protein preparations from leaves of D. heywoodii P. et M. Silva, D. mariana Boiss., D. purpurea L., and D. thapsi L. indicating that CGH is also present in these species. Purified CGH was digested with proteases V8 and Lys-C and the resulting fragments obtained were sequenced. One fragment had the typical amino-acid sequence of the catalytic center of family-1 glycosyl hydrolases (EC 3.2.1.x). Cardenolide 16′-O-glucohydrolase, like the other members of this enzyme family, appeared to have a glutamic acid residue directly involved in glycosidic bond cleavage as a nucleophile.

The use of diode-array detection allows the non-destructive identification of tryptophan and tyrosine residues in complex peptide mixtures separated by capillary electrophoresis. Second-order derivative spectra of both amino acids show significant differences while zero-order spectra are overlapping to a great extent. A mixture of peptides containing tryptophan and/or tyrosine residues was used to evaluate this method. Tryptic peptide maps of carbonic anhydrase and of the bacterial chaperonin protein GroEL, and of an autodigest of trypsin were characterized for tryptophan- and tyrosine-containing peptides. Automated spectra library search was performed successfully.

Molecular chaperones are an abundant class of proteins that have apparently evolved to modulate the folding of a variety of other proteins in cells. The focus of this review is one group of molecular chaperones, the chaperonins, that comprise a sequence-related family of proteins, initially found in prokaryotes and in certain cellular organelles that derived from prokaryotes. These proteins possess an intriguing oligomeric molecular architecture, and mechanistically are perhaps the best understood of the molecular chaperones. The discovery, identification, and isolation of chaperonin proteins (and their co-chaperonins) from prokaryotic and eukaryotic cells is described. In addition, the role of chaperonins in facilitating protein folding and suppressing aggregation is discussed. In many microorganisms, chaperonins are also heat- and stress-induced proteins. Therefore, an understanding of the molecular details of their role(s) in protein folding could resolve the enigma of their cellular function during a physiological stress response.

Publisher Summary This chapter focuses on amino acid analysis on microscale from electroblotted proteins. Amino acid analysis is routinely used to determine the amount and the composition of proteins, peptides, and free amino acids. Proteins at present often are isolated by sodium dodecyl sulfate-PAGE and electroblotting onto a chemically inert membrane like polyvinylidenfluoride. Amino acid analysis involves two major steps, complete hydrolysis of proteins and peptides, followed by quantification of the amino acids liberated. Hydrolysis is the most crucial step, especially susceptible to contamination and to loss of sample. With submicrogram amounts of samples, it has to be performed in gas phase, because of reduced contamination compared with liquid hydrolysis. However, all reverse-phase HPLC systems used for amino acid analysis are very sensitive to minimal changes in buffer concentrations, gradient shape, and temperature.

Nature 363, 644-648 (1993) THE following correction concerns only the contribution to this paper of E.M., R.G. and E. Schafer. We purified a protein of MT 60,000 (60K) from oat seedlings that possesses an oligomeric structure of 600K and is partially similar to the cytosolic TCP1 proteins from certain eukaryotes.

Type 1 phytochrome from etiolated oat seedlings was digested with V-8 protease. Microsequencing of a 13 kDa fragment yielded a sequence of 31 amino acids. The fragment starting with the alanine residue at position 427 of the entire phytochrome amino acid sequence revealed a heterogeneity (threonine, alanine and asparagine) at position 10. This demonstrates that the phytochrome type A genes AP3, 4 and 5 are expressed as proteins.

Abstract— –Phototransformation of oat type I phytochrome in vitro from the red‐light‐absorbing form (Pr) to the far‐red‐light‐absorbing form (Pfr) at physiological temperature (24°C) was investigated with a multichannel transient spectrum analyser. Four sequential intermediates were detected between Pr and Pfr. Absorption spectra of these intermediates suggested that three of them corresponded with the intermediates lumi‐R, meta‐Ra and meta‐Rc detected earlier at low temperature spectroscopy. A new intermediate named meta‐Rb was found in the pathway between meta‐Ra and meta‐Rc. The new intermediate is not identical with meta‐Rb previously detected at low temperature. The rate constant of Pfr appearance in isolated oat phytochrome dissolved in buffer containing 5% (vol/vol) glycerol was similar to that of etiolated pea epicotyl tissue.

A protein-kinase activity which is co-purified with phytochrome from etiolated oat seedlings was investigated in some detail. Whereas phytochrome was always phosphorylated in solution (together with some contaminating protein bands), radioactive phosphate was not found in the phytochrome band after native gel electrophoresis and incubation of the entire gel with labeled ATP. Since protein kinases are usually autophosphorylated under these conditions, the result shows that the kinase activity does not reside in the phytochrome molecule itself. Radioactivity was exclusively detected in a band with the apparent molecular weight 450 kDa; sodium-dodecyl-sulfate gel electrophoresis revealed an apparent molecular weight of 60 kDa for the phosphorylated subunit. The N-terminal amino-acid sequence A L E S A (G) K Q (L) V P W was determined for this subunit which is a potential candidate for the protein kinase. The optimum conditions (pH, metal ion concentration) and kinetics of the phosphorylation reaction were determined. The presumed connection between proteinkinase activity and the signal chain leading from the far-red-absorbing form of phytochrome to physiological responses still awaits elucidation.

Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543–8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.

The cross-reactivity of diverse monoclonal antibodies against phytochrome from Zea and Avena was tested by enzyme-linked immunosorbentassay (ELISA) and by immunoblotting. About 40 antibodies were selected by means of nondenatured phytochrome; all of them reacted with sodium dodecyl sulfate denatured homologous antigen on immunoblots. The epitopes for 14 antibodies (4 raised against Avena and 10 against Zea phytochrome) were localized in 6 regions of the phytochrome molecule by means of Western blot analysis of proteolytic fragments of known localization. Results of studies on the inhibition of antibody binding by other antibodies were largely compatible with these latter findings. Except in a few cases, inhibition occurred when antibodies were located on the same or a closely adjacent region. As demonstrated by 16 species, cross-reactivity with phytochromes from other Poaceae was high. Greater losses in cross-reactivity were observed only with antibodies recognizing an epitope in the vicinity of the carboxyl terminus of 118-kg · mol-1 phytochrome. Cross-reactivity with phytochrome from dicotyledons was restricted to a few antibodies. However, phytochrome(s) from plants illuminated for 24 h or more could be detected. One of the antibodies that recognized phytochrome from dicotyledons was also found to recognize phytochrome or a protein of 120–125 kg·mol-1 from several ferns, a liverwort and mosses. This antibody (Z-3B1), which was localized within a 23.5-kg·mol-1 section of Avena phytochrome (Grimm et al., 1986, Z. Naturforsch. 41c, 993), seems to be the first antibody raised against phytochrome from a monocotyledon with such a wide range of reactivity. Even though epitopes were recognized on different phytochromes, the strength of antibody binding indicated that these epitopes are not necessarily wholly identical.

Phytochrome from etiolated oat seedlings was digested under carefully controlled conditions with trypsin. A dodecapeptide which contains the blocked amino‐terminus of phytochrome was isolated from the digestion mixture and analyzed. According to amino acid analysis and the results obtained in FAB mass spectrometry, the sequence of mature phytochrome starts with N‐acetylserine, corresponding to serine‐2 derived from the DNA sequence.

Phytochrome from etiolated oat seedlings was digested with subtilisin. A 16 kDa fragment was isolated and investigated by microsequencing. Its amino‐terminal sequence SLPGGSMEV/ML revealed a heterogeneity (valine and methionine) at position 9. This proves expression of several phytochrome genes on the protein level. One can conclude from the ratio valine/methionine that the isophytochrome derived from AP5 (containing methionine) is a major gene product besides isophytochromes derived from genes AP4 and AP3 (containing valine).

Abstract Native (124 kDa) phytochrome has been isolated from etiolated oat seedlings in 25% yield. The procedure includes only one chromatographic step namely a hydroxylapatite column, besides several centrifugation steps. Large scale preparations with 3 kg of fresh tissue are completed within 16 h. The purity of isolated phytochrome is as good as or even better than that of previous preparations as deduced from SDS gelelectrophoresis and from the absorbance ratio A667 / A280 = 0.99