Wolf F. Brandt

Isolation and characterization of a D-7 LEA protein from pollen that stabilizes glasses in vitro

A heat-soluble protein present in substantial quantities in Typha latifolia pollen was purified to homogeneity. The protein was subjected to cyanogen bromide cleavage, and the peptides produced were separated by HPLC chromatography and sequenced. The two sequences determined were found to be related to the putative D76 LEA protein from Brassica napus seeds and one of them to the D-7 LEA protein from upland cotton. This suggests the pollen protein to be a member of the LEA group III family of proteins. The secondary structure of the protein in solution and in the dry state was investigated using Fourier transform IR spectroscopy. Whereas the protein in solution was highly unordered, being largely in a random coil conformation, the conformation was largely alpha-helical after fast drying. Slow drying reversibly led to both alpha-helical and intermolecular extended beta-sheet structures. When dried in the presence of sucrose, the protein adopted alpha-helical conformation, irrespective of drying rate. The effect of the protein on the stability of sucrose glasses was also investigated. The dehydrated mixture of sucrose and the LEA protein had higher glass transition temperatures and average strength of hydrogen bonding than dehydrated sucrose alone. We suggest that LEA proteins may play a role together with sugars in the formation of a tight hydrogen bonding network in the dehydrating cytoplasm, thus conferring long-term stability.

Response of the Leaf Cell Wall to Desiccation in the Resurrection Plant Myrothamnus flabellifolius

The Myrothamnus flabellifolius leaf cell wall and its response to desiccation were investigated using electron microscopic, biochemical, and immunocytochemical techniques. Electron microscopy revealed desiccation-induced cell wall folding in the majority of mesophyll and epidermal cells. Thick-walled vascular tissue and sclerenchymous ribs did not fold and supported the surrounding tissue, thereby limiting the extent of leaf shrinkage and allowing leaf morphology to be rapidly regained upon rehydration. Isolated cell walls from hydrated and desiccated M. flabellifolius leaves were fractionated into their constituent polymers and the resulting fractions were analyzed for monosaccharide content. Significant differences between hydrated and desiccated states were observed in the water-soluble buffer extract, pectin fractions, and the arabinogalactan protein-rich extract. A marked increase in galacturonic acid was found in the alkali-insoluble pectic fraction. Xyloglucan structure was analyzed and shown to be of the standard dicotyledonous pattern. Immunocytochemical analysis determined the cellular location of the various epitopes associated with cell wall components, including pectin, xyloglucan, and arabinogalactan proteins, in hydrated and desiccated leaf tissue. The most striking observation was a constitutively present high concentration of arabinose, which was associated with pectin, presumably in the form of arabinan polymers. We propose that the arabinan-rich leaf cell wall of M. flabellifolius possesses the necessary structural properties to be able to undergo repeated periods of desiccation and rehydration.

The predominant polyphenol in the leaves of the resurrection plant Myrothamnus flabellifolius, 3,4,5 tri-O-galloylquinic acid, protects membranes against desiccation and free radical-induced oxidation

The predominant (>90%) low-molecular-mass polyphenol was isolated from the leaves of the resurrection plant Myrothamnus flabellifolius and identified to be 3,4,5 tri-O-galloylquinic acid using 1H and 13C one- and two-dimensional NMR spectroscopy. The structure was confirmed by mass spectrometric analysis. This compound was present at high concentrations, 44% (by weight) in hydrated leaves and 74% (by weight) in dehydrated leaves. Electron microscopy of leaf material fixed with glutaraldehyde and caffeine demonstrated that the polyphenols were localized in large vacuoles in both hydrated and dehydrated leaves. 3,4,5 Tri-O-galloylquinic acid was shown to stabilize an artificial membrane system, liposomes, against desiccation if the polyphenol concentration was between 1 and 2 microg/mug phospholipid. The phase transition of these liposomes observed at 46 degrees C was markedly diminished by the presence of 3,4,5 tri-O-galloylquinic acid, suggesting that the presence of the polyphenol maintained the membranes in the liquid crystalline phase at physiological temperatures. 3,4,5 Tri-O-galloylquinic acid was also shown to protect linoleic acid against free radical-induced oxidation.

A simple modification converts the spinning cup protein sequencer into a vapour-phase sequencer

Spinning cup protein sequencer Vapour‐phase sequencer Protein structure

Comparison of the N-terminal amino acid sequences of histone F3 from a mammal, a bird, a shark, an echinoderm, a mollusc and a plant.

It has often been stated that histone F2al (IV) and F3 (III) are evolutionarily extremely stable proteins [ l-3,5]. This was based on the observation that these histones had a virtually identical electrophoretic mobility regardless of the source [ 1,2] . The tryptic fingerprint maps of the two proteins from calf and pea were shown to be very similar [S] and in the case of histone F2al from pea and calf the primary structures had been established and found to differ by only two amino acid residues out of a total of lb2 residues [6]. The assumption of evolutionary constancy of F3 throughout the biological world is mainly based on electrophoretic studies [ 1,2] . Only recently the sequences of histone F3 isolated from calf [7], chicken [8,9] and carp [4] have been established. The latter two were found to be identical but differed in their sequence from the calf histone with respect to residue Ser 96 which is replaced by Cys in calf. In order to establish the evolutionary relationships of histone F3 over a more extended range in the biological world we isolated this histone from a number of organisms separated widely on the evolutionary scale, namely from a shark, an echinoderm, a mollusc and a plant. Their electrophoretic mobility, amino acid composition and the N-terminal 40-50 amino acid sequence were compared to those of histone F3 from calf [7] and chicken [8,9].

Sequence of the cysteine-containing portion of histone F2al from the sea urchin Parechinus angulosus

Histone F2al has been shown to be evolutionarily a very stable protein showing only two conservative changes in organisms as widely separated on the evolutionary scale as calf and pea [ 11. The F2al from echinoderms, in contrast to both calf and pea, has been shown to contain cysteine [2]. We have isolated F2al from the sea urchin and placed in sequence 52 of the 102 amino acids. Cysteine was found to replace threonine at position 73.

Chapter 12 Fractionation of Histones on Molecular Sieve Matrices

Publisher Summary This chapter describes the methods for fractionation of histones on molecular sieve matrices. Methods for the isolation and fractionation of histones are governed by two properties of the nuclear proteins inherent to their biological function: their strong positive charge and their tendency to aggregate. Purified nuclei facilitate the subsequent histone fractionation as a source of nucleoprotein. Histones are dissociated from DNA for the purpose of fractionation of the total complement or the purification of individual histones. A fraction containing the total complement of histones either as hydrochlorides or sulfates, if not contaminated with other proteins, neutral polysaccharides, or polyanionic polysaccharides, can be fractionated on molecular sieve matrices like Bio-gel or Sephadex. To minimize the aggregation of histones dilute HCl is introduced as a solvent for the fractionation of histones on molecular sieves. The purification of histones on molecular sieve matrices in dilute acid in the presence or absence of NaCl is successful in conjunction with the selective procedures of extraction. Gel electrophoresis is used as a sensitive and rapid method to establish the purity of histone fractions.

The complete amino acid sequence of histone F3 from chicken erythrocytes

We wish to report the complete sequences of the 136 amino acids of the only cysteine containing histone isolated from chicken erythrocytes. For the first time, to our knowledge, all the positions of the amino acids, in the course of establishing the primary structure of a protein, have been assigned without the use of overlapping sequences. To achieve this, specific chemical cleavages rather than enzymatic degradations were chosen and applied, first to the original protein chain, and subsequently to the generated polypeptides to yield sets of not more than 3 peptides in any single cleavage. Their relative position in the protein or polypeptides became evident after comparison of the Nand C-terminal amino acid in the cleavage products and the uncleaved starting material. The simplicity of the peptide mixture after each cleavage, resulting in easy separation of the peptides, together with the highly efficient Edman degradations of automatic sequencing, allowed us to perform a rapid and relatively non-laborious primary structure determination of histone F 3 .

Proteolytic degradation of histones and site of cleavage in histone F2a1 and F3

Histones are susceptible to proteolytic degradation during their isolation [ 1,2]. This degradation can be minimized by protease inhibitors like diisopropylphosphofluoridate and by performing all operations at low temperatures [ 11. Subsequently it has been shown that a trypsin-like protease is closely associated with isolated chromatin [2-41. This enzyme is coextracted with histones in dilute acid and may autolyse such a preparation near neutral pH [2,5]. The enzyme has its optimal activity at pH 8 with nucleoprotein or free histones as substrate [2,3]. The disc electrophoretic pattern of the histone fraction isolated from degraded nucleoprotein reveals that only a limited number of fragments are formed [ 1,2], and that the 5 histone groups show different susceptibility to the proteolysis [2]. Although the enzyme has been partially purified [3], its specificity has not yet been determined. We now wish to report the purification and characterization of two histone degradation products isolated from cycad pollen nucleoprotein. The results of the determination of the N-terminal sequence of these fragments have allowed us to identify them as breakdown products of histone F3 and F2al respectively. A number of interesting features of the cleavage sites became apparent. Possible biological implications are briefly discussed.

Purification and characterization of the cysteine‐containing F3 histone from chicken erythrocyte

Knowledge of the amino acid sequence of histones will assist in the understanding of the molecular mechanisms of their function in the cell nucleus. Up to now only histones of group F3 have been shown to contain cysteine giving to this histone group particular properties with respect to protein-protein interaction. The structure of F3 histones is therefore of special interest. A new method has been developed to isolate highly purified F3 (Arg-rich) histone from chicken erythrocytes in gram amounts. The protein was characterized by its molecular weight, amino acid composition and end groups. Evidence was obtained that the microheterogeneity of this protein is due to fractional acetylation. The three CNBr-cleavage fragments were isolated and their relative sequence in the protein determined. Amino acid analysis of these fragments revealed clustering of acidic and basic amino acids.