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A polyphenol-rich extract of the medicinal resurrection plant Myrothamnus flabellifolia was shown to inhibit viral (M-MLV and HIV-1) reverse transcriptases. Fractionation and purification of this extract yielded the major polyphenol, 3,4,5 tri-O-galloylquinic acid, as the main active compound. A sensitive, ethidium bromide based fluorescent assay, was developed and used to monitor the kinetics of M-MLV and HIV-1 reverse transcriptases in the presence and absence of 3,4,5 tri-O-galloylquinic acid. Kinetic monitoring of these enzymes in the presence of 3,4,5 tri-O-galloylquinic acid revealed non-competitive inhibition with IC(50) values of 5 μM and 34 μM for the M-MLV and HIV-1 enzymes, respectively. We propose that 3,4,5 tri-O-galloylquinic acid and related polymers have potential as indigenous drugs for anti-viral therapy.

International audience; Vegetative desiccation tolerance occurs in a unique group of species termed ‘resurrection plants’. Here, we review the molecular genetic, physiological, biochemical, ultrastructural and biophysical studies that have been performed on a variety of resurrection plants to discover the mechanisms responsible for their tolerance. Desiccation tolerance in resurrection plants involves a combination of molecular genetic mechanisms, metabolic and antioxidant systems as well as macromolecular and structural stabilizing processes. We propose that a systems-biology approach coupled with multivariate data analysis is best suited to unraveling the mechanisms responsible for plant desiccation tolerance, as well as their integration with one another. This is of particular relevance to molecular biological engineering strategies for improving plant drought tolerance in important crop species, such as maize ( Zea mays) and grapevine ( Vitis vinifera).

Myrothamnus flabellifolia (Welw.) is a relatively large resurrection plant, a woody shrub between 0.5 m and 1.5 m tall (Sherwin et al., 1998) that grows on rock inselbergs (Porembski and Barthlott, 2000) throughout southern Africa (Weimarck, 1936; Van Wyk et al., 1997; Glen et al., 1999). The plant was first recorded in 1859 by Friedrich Welwitsch, who named the plant Myrothamnus (myron meaning aromatic and thamnos meaning bush) flabellifolia (meaning fan-like leaves) (Puff, 1978a; Glen et al., 1999), the leaves having a balsamic-like odour (Puff, 1978a; Glen et al., 1999). Weiss (1906) was the first to note the ‘miraculous manner’ with which the desiccated plant revived when supplied with water (Fig. 1A, B). Myrothamnus flabellifolia occupies an important position in traditional African folklore and medicine (Watt and Breyer-Brandwijk, 1962; Hutchings, 1996; Van Wyk et al., 1997). The Zulu name for the plant is ‘uvukwaba- file’ (wakes from the dead). The reviving ability is believed to be passed on to the ill person during treatment (Hutchings, 1996; Van Wyk et al., 1997). The plant is a geophyte possessing an extensive root system which extends into the crevices of the rocky slopes upon which it grows (Child, 1960; Glen et al., 1999). Myrothamnus flabellifolia can dehydrate its vegetative tissue, in particular its leaves, to an air-dry state. In this state, the leaves and stem segments curl and change colour from green to dullbrown (Farrant et al., 1999; Glen et al., 1999). When water is provided to the roots the plant re-hydrates its desiccated tissue and returns to its original colour and shape (Glen et al., 1999; Farrant et al., 2003). Since the last review on M. flabellifolia was written many years ago (Puff, 1978a) and since considerable work has been published in the last decade, this review focuses on recent advances in the understanding of the physiology, biochemistry and chemistry of M. flabellifolia.

Incorporation of the cell wall LEA-like stress response protein Hsp12p from the yeast Saccharomyces cerevisiae into the carbohydrate polymer agarose was found to reduce the gel strength of the agarose matrix. Solutes known to stimulate Hsp12p production in yeast were found to increase the gelation temperature, the turbidity and the gel strength of the agarose matrix. Increased gel strength brought about by solute incorporation was found to be reversed by co-incorporation of Hsp12p or by the chaotropic solute urea. These results suggested a mode of action for Hsp12p, assuming that agarose is a reasonable macroscopic model system for investigating the effects of cell wall proteins on the properties of the yeast cell wall.

A tissue culture protocol, suitable for transformation, was established for the pasture grass Eragrostis curvula. Callus was generated in the dark from leaf and seed tissues on a medium comprising MS salts supplemented with 2 mg/l 2,4-D, 0.01 mg/l BAP and 2% sucrose. Plant regeneration occurred via organogenesis on the same medium with 6% and 3% sucrose for shoot and root formation, respectively. Optimal regeneration (50 plantlets per callus) occurred when light of 45 micromol/m2 per s was used. The yeast Saccharomyces cerevisiae Hsp12 gene was cloned into the Sac1 of the pCAMBIAUbeeQ vector and callus was transformed by biolistic bombardment. Best transformation (30%) occurred when the target tissue was bombarded twice at a distance of 70 mm using a bombardment pressure of 9,100 kPa. Although successful transformation and transcription of the Hsp12 gene occurred, no Hsp12 protein was found present in tissue extracts of transformed grass.

The aim of the present study was to analyze mass spectra of scorpions belonging to the genus Parabuthus (Pocock 1890) by means of matrix-assisted laser desorption time-of-flight mass spectrometry (MALDI-TOFMS) and to construct a species-specific venom code for species identification. The venom compositions of sixteen Parabuthus species, occurring in southern Africa, were characterized using representative peaks in the molecular mass range of 6400-8400 Da. This mass range is characteristic for the typical long-chain neurotoxins influencing sodium channels. Only a few of these peptides have been sequenced up to now. The impetus for development of these species-specific profiles was the observation of unique, highly reproducible mass spectral peaks within a specific species. An identification label for all the different species could be found using a minimum number of peaks. MALDI-TOFMS is therefore proposed as a complementary method to morphological and behavioural characteristics for species and ultimately subspecies discrimination.

Summary Chloroplasts isolated from the resurrection plant Myrothamnus flabellifolius using trehalose gradients had a higher buoyant density than chloroplasts isolated from another resurrection plant Craterostigma wilmsii . The latter had the same buoyant density as those isolated from the desiccation-sensitive (DS) plant Pisum sativum . The increased buoyant density in M. flabellifolius was ascribed to the unusual ultrastructure of the thylakoid membranes. Standard chloroplast isolation protocols resulted in membrane damage in both resurrection plants. Trehalose rather than sucrose gradients were required for isolation of intact chloroplasts. Immunological studies showed that epitopes related to the desiccation stress protein dsp 21 from Craterostigma plantagineum were present in M. flabellifolius . Several small (10–15 kDa) unique stromal proteins were also present. Polyphenolics, including anthocyanins, accumulated in leaves and chloroplasts of M. flabellifolius during drying. Envisaged functions for these are maintenance of membrane integrity, chlorophyll masking and antioxidant protection. Metal (Ca 2+ , Cu 2+ , Fe 2+ , Mg 2+ and Mn 2+ ) concentrations declined and thylakoid membranes separated upon drying. We propose that this might be a mechanism to put a stasis on photosynthesis and minimise photo-oxidation damage under water stress conditions.

The somatic and testis isoforms of angiotensin-converting enzyme (ACE) are both C-terminally anchored ectoproteins that are shed by an unidentified secretase. Although testis and somatic ACE both share the same stalk and membrane domains the latter was reported to be shed inefficiently compared with testis ACE, and this was ascribed to cleavage at an alternative site [Beldent, Michaud, Bonnefoy, Chauvet and Corvol (1995) J. Biol. Chem. 270, 28962-28969]. These differences constitute a useful model system of the regulation and substrate preferences of the ACE secretase, and hence we investigated this further. In transfected Chinese hamster ovary cells, human somatic ACE (hsACE) was indeed shed less efficiently than human testis ACE, and shedding of somatic ACE responded poorly to phorbol ester activation. However, using several analytical techniques, we found no evidence that the somatic ACE cleavage site differed from that characterized in testis ACE. First, anti-peptide antibodies raised to specific sequences on either side of the reported cleavage site (Arg1137/Leu1138) clearly recognized soluble porcine somatic ACE, indicating that cleavage was C-terminal to Arg1137. Second, a competitive ELISA gave superimposable curves for porcine plasma ACE, secretase-cleaved porcine somatic ACE (eACE), and trypsin-cleaved ACE, suggesting similar C-terminal sequences. Third, mass-spectral analyses of digests of released soluble hsACE or of eACE enabled precise assignments of the C-termini, in each case to Arg1203. These data indicated that soluble human and porcine somatic ACE, whether generated in vivo or in vitro, have C-termini consistent with cleavage at a single site, the Arg1203/Ser1204 bond, identical with the Arg627/Ser628 site in testis ACE. In conclusion, the inefficient release of somatic ACE is not due to cleavage at an alternative stalk site, but instead supports the hypothesis that the testis ACE ectodomain contains a motif that activates shedding, which is occluded by the additional domain found in somatic ACE.

We have isolated a 72-amino acid peptide from extracts of sinus glands of the South African rock lobster, Jasus lalandii, and identified it, functionally and immunologically, as a hyperglycemic hormone. This is the second peptide with hyperglycemic activity found in this palinurid species and, because it occurs in smaller quantities (approximately 3 pmol/sinus gland) than the previously identified hyperglycemic hormone [14], this minor isoform is designated Jala cHH-II. The complete elucidation of the primary structure of cHH-II, as determined by automated Edman degradation of the N-terminus enzymatic digests of the non-reduced peptide, chemical cleavage and mass spectrometry, is presented here. Jala cHH-II (molecular mass of 8357 Da) is more hydrophobic than Jala cHH-I (8380 Da). The two cHHs have a free N-terminus a blocked C-terminus; and share 90% sequence homology. We also present structural data of a further two peptides isolated from sinus gland extracts that were immunopositive to cHH antisera. These peptides, with masses of 7665 and 7612 Da, structurally represent C-terminally truncated forms of the major and the minor Jala cHH peptides, respectively, but do not have any hyperglycemic activity in vivo. We demonstrate that the prevalence of these truncated forms can be reduced by the addition of proteases to the homogenization buffer during preparation of the tissues.

We identified and chemically characterized the two major forms of sulfakinins from an extract of 800 corpora cardiaca/corpora allata complexes of the American cockroach, Periplaneta americana. Bioactivity during the purification was monitored by measuring heart beat frequency in a preparation in situ. By Edman degradation analysis and MS, these main forms were identified as having the primary structures Pea-SK [EQFDDY(SO(3)H)GHMRFamide] and Lem-SK-2 [pQSDDY(SO(3)H)GHMRFamide]. The sulfation was confirmed by UV, MS and peptide synthesis. In addition, post-translationally modified sulfakinins of both major forms were isolated and identified. Firstly, nonsulfated forms of these peptides are present in considerable amounts in the corpora cardiaca/allata. Secondly, the N-terminally blocked Pea-SK and the nonblocked Lem-SK-2 occur naturally in neurohaemal release sites. Thirdly, modified Pea-SK with O-methylated glutamic acid occurs which is not an artefact of peptide purification. The major forms of the sulfakinins were shown to be highly active on both the heart and hindgut with threshold concentrations of approximately 5 x 10(-10) M (heart) and 2 x 10(-9) M (hindgut).

LEA group I, II and III antibodies all recognised soluble proteins present in an extract of yeast (Saccharomyces cerevisiae). The smaller protein of the two recognised by the group I antibody displayed identical migration on SDS-PAGE to the pea seed LEA group I protein against which the antibody was raised. However, the antibody failed to recognise the predominant protein present after heating the extract at 80 °C for 10 min. This predominant protein, which also displayed identical migration on SDS-PAGE, was purified from the supernatant of the extract heated at 80 °C for 10 min. Peptide sequencing after CNBr cleavage identified the isolated protein as the heat shock protein HSP 12. Despite a previous report that HSP 12 is a heat shock protein, HSP 12 was found to increase in yeast grown at 37 °C compared with growth at 30 °C . However, increased amounts of HSP 12 were present in yeast after entry into stationary phase; this was enhanced by growth in the osmolytes NaCl and mannitol.

The ketones 3-pentanone, 4-methyl-2-pentanone, 5-methyl-3-hexanone, 4-methyl-3-hexanone, and the esters ethyl propanoate, 2-methylpropyl acetate, ethyl 3-methylbutanoate, and 2-methylpropyl propanoate were identified as the only volatile organic constituents of the preorbital secretion of the klipspringer, Oreotragus oreotragus. These compounds are considerably more volatile than those found in the preorbital secretions of other antelopes belonging to the tribe Antelopini. More than 50% of the preorbital secretion consists of proteinaceous material, which probably acts as a controlled-release substance in retarding the evaporation of the volatile constituents of the secretion from territorial marks.

The LEA-like protein previously isolated from a homogenate of pea (Pisum sativumL.) embryonic axes heated at 80°C for 10 min (Russouwet al., 1995) was purified without exposure to heat. Peptides produced by trypsin digestion were separated by HPLC and sequenced. The protein was identified as a member of the LEA group I family. The conformation of the protein was compared before and after heat treatment by antibody affinity, circular dichroism spectroscopy, fluorescence spectroscopy and 8-anilino-1-naphthalenesulfonic acid binding. No differences could be detected, demonstrating that the protein was not irreversibly denatured by exposure to high temperature.

An LEA-like protein has been isolated and characterized from pea (Pisum sativum) embryos. It is the most prevalent protein in a homogenate of pea axes heated for 10 min at 80°C and then centifuged for 10 min at 17000g(80°C supernatant fraction). It has a molecular mass of 11 kDa and is very rich in hydrophilic amino acids, notably aspartate, glutamate and glycine. The protein is not recognized by an antibody to the group II dehydrin C-terminal consensus sequence. Antibodies to the isolated protein recognize a number of larger proteins in the 80°C supernatant fraction of pea and other legumes (chick pea, soybean and bean) as well as gramineous seeds (wheat, maize and barley). The protein undergoes a substantial increase in α-helical content at high ionic strength but does not dimerize.

We describe a simple and effective chemical protocol for attaching polyallylamine to polyvinylidene difluoride (PVDF) membranes. The underivatized PVDF discs were first etched in 0.5 M alcoholic KOH for 1.5 min and subsequently reacted with polyallylamine of high molecular weight under alkaline conditions. The covalently attached amino groups were reacted with 1,4-phenylene diisothiocyanate (DITC), thus converting the amino-modified PVDF discs to DITC-functionalized discs. The substitution level of the DITC-functionalized discs was found to be lower than that of the equivalent discs available commercially. Lysine-containing polypeptides were immobilized on these DITC-membrane discs and then subjected to either solid-phase manual Edman degradation or automated gas-phase sequencing. The method was found to be well suited for the sequencing of 32P-labeled phosphopeptides by employing the manual solid-phase technique.

1. 1. Nerve growth factor from Bitis arietans venom was isolated in high yield and purified to homogeneity using a rapid two-step procedure involving gel exclusion chromatography and reversed-phase HPLC. 2. 2. On polyacrylamide gel electrophoresis in SDS, the NGF migrates as a 25 kDa homodimer and is thus atypical of other Viperid NGFs. 3. 3. Evidence suggests that, unlike mammalian βNGFs, the subunits of the Bitis arietans homodimer are covalently linked by a disulphide bond(s). 4. 4. Partial sequence analysis shows that only 6 out of the first 21 amino acids are identical with those of cobra NGF including cys-14 and val-21 which are known to be important for NGF activity.

The mannose/glucose-binding lectin (ML) from the nodules of peanut ( Arachis hypogaea L.) was found to be antigenically identical to ML from hypocotyls, stems, leaves and roots but cotyledon ML was antigenically different. The different tissue lectins had relative molecular weights similar to nodule ML and were each composed of two identical or nearly identical subunits. There were no differences between the set of multiple bands that each lectin formed during isoelectric focusing. The antigenically different cotyledon ML and nodule ML had similar carbohydrate specificities but differed in their sensitivity to haemagglutination inhibition by sugars and certain glycoproteins. In contrast, the antigenically identical leaf ML was identical to nodule ML in its reaction with sugars and glycoproteins. Nodule and cotyledon ML both contained mannose, galactose, fucose, xylose and acetylglucosamine although the proportions and content of sugars differed. The amino acid composition of nodule and cotyledon ML was similar but slight differences in their amino-terminal amino acid sequences suggested that they are produced by alleles of the same gene.

†Background Myrothamnus flabellifolia is unique as the only woody resurrection plant. It is an important plant in southern Africa because of its widespread occurrence and usage in African medicine and traditional culture. Many reports have investigated facets of its biology and the mechanisms associated with its desiccation tolerance. †Scope The general biology of the woody resurrection plant Myrothamnus flabellifolia is reviewed. The review focuses on the geography and ecology, systematic placement, evolution, morphology and reproductive ecology of M. flabellifolia as well as the wood anatomy and re-filling mechanism. In addition, the desiccation tolerance, ethnobotanical importance and medicinal properties of the plant are reviewed. Also, future research avenues are suggested, in particular the necessity to research the biogeography and systematics of the species and the role of the polyphenols present, as well as the molecular basis of the plant’s desiccation tolerance.

ASP53, a 53 kDa heat soluble protein, was identified as the most abundant protein in the mature seeds of Acacia erioloba E.Mey. Immunocytochemistry showed that ASP53 was present in the vacuoles and cell walls of the axes and cotyledons of mature seeds and disappeared coincident with loss of desiccation tolerance. The sequence of the ASP53 transcript was determined and found to be homologous to the double cupin domain-containing vicilin class of seed storage proteins. Mature seeds survived heating to 60°C and this may be facilitated by the presence of ASP53. Circular dichroism spectroscopy demonstrated that the protein displayed defined secondary structure, which was maintained even at high temperature. ASP53 was found to inhibit all three stages of protein thermal denaturation. ASP53 decreased the rate of loss of alcohol dehydrogenase activity at 55°C, decreased the rate of temperature-dependent loss of secondary structure of haemoglobin and completely inhibited the temperature-dependent aggregation of egg white protein.

Light microscopy and low-temperature scanning electron microscopy were used to systematically compare the surface and internal ultrastructures of hydrated and desiccated leaves of the resurrection plant Myrothamnus flabellifolia (Welw.). This revealed that leaf tissue underwent considerable shrinkage and collapse on desiccation but was supported by a framework of vascular and sclerenchymous tissue, which is responsible for the fan-like shape of the leaves. In addition, the leaf ribs were covered with wax and an internal wax cuticle was observed. Biochemical analysis showed that the cyanidin 3-glucoside content increased on desiccation as did the trehalose and sucrose contents. Salt deposits were observed at the apices of desiccated leaves in the proximity of hydathode-like structures. We propose that this might regulate the leaf salt content since decreased intracellular cation concentration was observed in desiccated leaves. We believe that these unique adaptations contribute to the remarkable desiccation-tolerance properties of this plant.

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 polyphenol contents and compositions in desiccated leaves of Myrothamnus flabellifolius plants collected in various locations in Namibia and South Africa were analyzed using UV spectroscopy and high-performance liquid chromatography-mass spectrometry. A study of the genetic relatedness of these populations was also performed by determination of the DNA sequence of the intergenic spacer region between the psbA and the trnH genes in the chloroplast genome. Namibian M. flabellifolius plants contained significantly more polyphenols than South African plants. Namibian plants essentially contained a single polyphenol, 3,4,5-tri-O-galloylquinic acid, whereas South African plants contained a variety of galloylquinic acids including 3,4,5-tri-O-galloylquinic acid together with higher molecular weight galloylquinic acids. Sequence analysis revealed a 1.4% divergence between Namibian and South African plants corresponding to the separation of these populations of approximately 4 x 10(6) years. The significance of the poly-phenol content and composition to the desiccation tolerance of the two populations is discussed.

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 μg/μg phospholipid. The phase transition of these liposomes observed at 46 °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.

Yeast cells Saccharomyces cerevisiae, late embryogenic abundant-like stress response protein Hsp 12 (heat-shock protein 12) were found by immunocytochemistry to be located both in the cytoplasm and in the cell wall, from where they could be extracted with dilute NaOH solutions. Yeast cells with the Hsp 12 gene disrupted were unable to grow in the presence of either 12 mM caffeine or 0.43 mM Congo Red, molecules known to affect cell-wall integrity. The volume of yeast cells were less affected by rapid changes in the osmolality of the growth medium when compared with the wild-type yeast cells, suggesting a role for Hsp 12 in the flexibility of the cell wall. This was also suggested by subjecting the yeast cells to rapid changes in barometric pressure where it was found that wild-type yeast cells were more resistant to cellular breakage.

NaOH was used to extract proteins from the cell walls of the yeast Saccharomyces cerevisiae. This treatment was shown not to disrupt yeast cells, as NaOH-extracted cells displayed a normal morphology upon electron microscopy. Moreover, extracted and untreated cells had qualitatively similar protein contents upon disruption. When yeast was grown in the presence of 1M mannitol, two proteins were found to be present at an elevated concentration in the cell wall. These were found to be the late-embryogenic-abundant-like protein heat-shock protein 12 and the glycolytic enzyme phosphoglycerate mutase. The presence of phosphoglycerate mutase in the cell wall was confirmed by immunocytochemical analysis. Not only was the phosphoglycerate mutase in the yeast cell wall found to be active, but whole yeast cells were also able to convert 3-phosphoglycerate in the medium into ethanol, provided that the necessary cofactors were present.

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.

The role of juxtamembrane stalk glycosylation in modulating stalk cleavage and shedding of membrane proteins remains unresolved, despite reports that proteins expressed in glycosylation-deficient cells undergo accelerated proteolysis. We have constructed stalk glycosylation mutants of angiotensin-converting enzyme (ACE), a type I ectoprotein that is vigorously shed when expressed in Chinese hamster ovary cells. Surprisingly, stalk glycosylation did not significantly inhibit release. Introduction of an N-linked glycan directly adjacent to the native stalk cleavage site resulted in a 13-residue, proximal displacement of the cleavage site, from the Arg-626/Ser-627 to the Phe-640/Leu-641 bond. Substitution of the wild-type stalk with a Ser-/Thr-rich sequence known to be heavily O-glycosylated produced a mutant (ACE-JGL) in which this chimeric stalk was partially O-glycosylated; incomplete glycosylation may have been due to membrane proximity. Relative to levels of cell-associated ACE-JGL, rates of basal, unstimulated release of ACE-JGL were enhanced compared with wild-type ACE. ACE-JGL was cleaved at an Ala/Thr bond, 14 residues from the membrane. Notably, phorbol ester stimulation and TAPI (a peptide hydroxamate) inhibition of release-universal characteristics of regulated ectodomain shedding-were significantly blunted for ACE-JGL, as was a formerly undescribed transient stimulation of ACE release by 3, 4-dichloroisocoumarin. These data indicate that (1) stalk glycosylation modulates but does not inhibit ectodomain shedding; and (2) a Ser-/Thr-rich, O-glycosylated stalk directs cleavage, at least in part, by an alternative shedding protease, which may resemble an activity recently described in TNF-alpha convertase null cells [Buxbaum, J. D., et al. (1998) J. Biol. Chem. 273, 27765-27767].

Polysaccharides, secondary metabolites and poly-phenolics are known to co-isolate with nucleic acids from plant tissues resulting in inhibition of molecular manipulations. RNA isolated from the polyphenolic-rich resurrection plant, Myrothamnus flabellifolius, was demonstrated to inhibit a standard polymerase chain reaction used as an assay despite the inclusion of the polyphenolic-binding compound poly(1-vinylpyrrolidone-2) (PVP) into the RNA isolation medium. This inhibition was, however, reversed by the addition of PVP into the PCR mixture itself. Confirmation of the inhibitory effect of polyphenolics on PCR was obtained by addition of green tea polyphenolics to the standard PCR assay. This inhibition was also reversed by the simultaneous inclusion of PVP.

Specialized proteases, referred to as sheddases, secretases, or membrane-protein-solubilizing proteases (MPSPs), solubilize the extracellular domains of diverse membrane proteins by catalyzing a specific cleavage in the juxtamembrane stalk regions of such proteins. A representative MPSP (tumor necrosis factor-alpha convertase) was cloned recently and shown to be a disintegrin metalloprotease that is inhibited by peptide hydroxamates including the compound TAPI. Substrate determinants that specify cleavage by MPSPs remain incompletely characterized, but may include the physicochemical properties of the stalk or unidentified recognition motifs in the stalk or the extracellular domain. We constructed a mutant angiotensin-converting enzyme (ACE) in which the stalk has been replaced with an epidermal growth factor (EGF)-like domain (ACE-JMEGF), to test the hypothesis that MPSP cleavage requires an open, comparatively unfolded or extended stalk. Wild-type ACE is a type I transmembrane (TM) ectoprotein that is efficiently solubilized by a typical MPSP activity. We found that ACE-JMEGF was solubilized inefficiently and accumulated in a cell-associated form on transfected Chinese hamster ovary (CHO) cells; cleavage was stimulated by phorbol ester and inhibited by TAPI, features typical of MPSP activity. Determination of the C-terminus of soluble ACE-JMEGF revealed that, surprisingly, cleavage occurred at a Gly-Phe bond between the fifth and sixth cysteines within the third disulfide loop of the EGF-like domain. Reduction of intact CHO cells with tributylphosphine resulted in the rapid release of ACE-JMEGF (but not wild-type ACE) into the medium, suggesting that a proportion of membrane-bound ACE-JMEGF is cleaved but remains cell-associated via disulfide tethering. The mechanism for the release of ACE-JMEGF in the absence of chemical reduction is unclear. We conclude that the presence of a compact, disulfide-bridged domain does not per se inhibit cleavage by an MPSP activity, but ectodomain release is prevented by disulfide tethering to the TM domain.

We have isolated a peptide from extracts of sinus glands of Jasus lalandii, a South African spiny lobster, by high-performance liquid chromatography (HPLC) and identified it as crustacean hyperglycaemic hormone (cHH) by (i) a conspecific bioassay measuring glucose elevation in the haemolymph and (ii) an immunoassay using an antiserum raised against cHH of the American lobster. The J. lalandii peptide has a free N-terminus as evidenced by sequencing the first 30 amino acid residues of the intact peptide. Further primary structural data were obtained from sequencing HPLC-purified tryptic and Asp-N proteolytic digests and by cyanogen bromide cleavage of the native, unreduced peptide. In this way, less than 400 sinus glands were used to provide the full sequence. Mass spectrometric analysis in conjunction with inferences based on interspecies sequence homology of cHH molecules unequivocally assigned the complete primary structure of cHH in a member of the crustacean infraorder Palinura for the first time. Our results show 51-76% homology with cHHs known from other decapod infraorders, the major difference being a free N-terminus and several amino acid substitutions interspersed in the non-conserved regions of the molecule. The J. lalandii cHH sequence presented here differs from a partial cHH sequence previously reported from allegedly the same species.

Diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE) or juxtamembrane (stalk) deletion or chimaera mutants. Determination of the C termini (i.e. the cleavage sites) of released, soluble wild-type and mutant ACE by MALDI-TOF mass spectrometry indicated that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane, but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, kinetic analyses of release rates indicated that a minimum distance from the membrane must be preserved. Interestingly, soluble full-length (anchor-plus) WT-ACE incubated with fractions of, or intact, CHO cells was not cleaved. In all cases, release was stimulated by a media change or by the addition of phorbol ester, with rate enhancements of 5- and 50-fold, respectively, for WT-ACE. The phorbol ester effect was abolished by staurosporine, a protein kinase C (PKC) inhibitor. We propose that the CHO cell MPSP that solubilizes ACE: (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain and (4) is regulated in part by a PKC-dependent mechanism.

Many structurally and functionally diverse membrane proteins are solubilized by a specific proteolytic cleavage in the stalk sequence adjacent to the membrane anchor, with release of the extracellular domain. Examples are the amyloid precursor protein, membrane-bound growth factors, and angiotensin-converting enzyme (ACE). The identities and characteristics of the responsible proteases remain elusive. We have studied this process in Chinese hamster ovary (CHO) cells stably expressing wild-type ACE (WT-ACE; human testis isozyme) or one of four juxtamembrane (stalk) mutants containing either deletions of 17, 24, and 47 residues (ACE-JM delta 17, -JM delta 24, and -JM delta 47, respectively) or a substitution of 26 stalk residues with a 20-residue sequence from the stalk of the low-density lipoprotein receptor (ACE-JMLDL). The C termini of released, soluble WT-ACE and ACE-JM delta 17 and -JMLDL were determined by MALDI-TOF mass spectrometry analyses of C-terminal peptides generated by CNBr cleavage. Observed masses of 4264 (WT-ACE) and 4269 (ACE-JM delta 17) are in good agreement with an expected mass of 4262 for the C-terminal CNBr peptide ending at Arg-627, indicating cleavage at the Arg-627/Ser-628 bond in both WT-ACE and ACE-JM delta 17, at distances of 24 and 10 residues from the membrane, respectively. Data for ACE-JM delta 24 are also consistent with cleavage at or near Arg-627. For ACE-JMLDL, in which the native cleavage site is absent, observed masses of 4372 and 4542 are in close agreement with expected masses of 4371 and 4542 for peptides ending at Ala-628 and Gly-630, respectively, indicating cleavages at 17 or 15 residues from the membrane. These data indicate that the membrane-protein-solubilizing protease (MPSP) in CHO cells is not constrained by a particular cleavage site motif or by a specific distance from the membrane but instead may position itself with respect to the putative proximal, folded extracellular domain adjacent to the stalk. Nevertheless, cleavage at a distance of 10 residues from the membrane is more favorable, as ACE-JM delta 17 is cleaved 12-fold faster than WT-ACE. In contrast, ACE-JM delta 24 is released 17-fold slower, suggesting that a minimum distance from the membrane must be preserved. This is supported by results with the ACE-JM delta 47 mutant, which is membrane-bound but not cleaved, likely because the entire stalk has been deleted. Finally, soluble full-length (anchor-plus) WT-ACE is not cleaved when incubated with various CHO cell fractions or intact CHO cells. On the basis of these and other data, we propose that the CHO cell MPSP that solubilizes ACE (1) only cleaves proteins embedded in a membrane; (2) requires an accessible stalk and cleaves at a minimum distance from both the membrane and proximal extracellular domain; (3) positions itself primarily with respect to the proximal extracellular domain; and (4) may have a weak preference for cleavage at Arg/Lys-X bonds.

The complete amino acid sequence of histone H3 from Saccharomyces cereviriae (var.) has been established. The protein molecule consists of 135 residues. Since mammalians and yeast diverged from a common ancestor 15 amino acid substitutions have occurred. These substitutions mainly occur in the C-terminal third of the polypeptide chain. The evolutionary significance of the large number of substitutions is discussed.

1. 1. The 0.5 M perchloric acid extract prepared from chromatin of wheat germ, Triticum aestivum, contains a group of histones formerly called plant histones. These can be resolved by gel filtration on Bio-Gel P-60 with subsequent CM-cellulose ion-exchange chromatography into five histone fractions containing families of histones H2A and H2B. 2. 2. The partial amino acid sequences of histone H2A variants H2A(1)Triticum, H2A(2)Triticum and H2A(3)Triticum are presented. Extensive sequence homology exists between calf thymus histone H2A and wheat embryo H2A histones. Differences are largely due to conservative amino acid substitutions and in two of the variants, viz. H2A(2) and H2A(3) to N-terminal extensions of the polypeptide chains.

1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids. 2. In the carboxy-terminal two thirds of the molecule there is a high degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions. 3. In the N-terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.

Sperm histone H2B(2)Parechinus consists of a single polypeptide chain of the following 143 amino acids: Pro-Arg-Ser-Pro-Ala-Lys-Thr-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Arg-Lys-Gly-Ser-Pro-Ser-Arg-Lys-Ala-Ser-Pro-Lys-Arg-Gly-Gly-Lys-Gly-Ala-Lys-Arg-Ala-Gly-Lys-Gly-Gly-Arg-Arg-Arg-Arg-Val-Val-Lys-Arg-Arg-Arg-Arg-Arg-Arg-Glu-Ser-Tyr-Gly-Ile-Tyr-Ile-Tyr-Lys-Val-Leu-Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Arg-Ala-Met-Ser-Val-Met-Asn-Ser-Phe-Val-Asn-Asp-Val-Phe-Glu-Arg-Ile-Ala-Gly-Glu-Ala-Ser-Arg-Leu-Thr-Ser-Ala-Asn-Arg-Arg-Ser-Thr-Val-Ser-Ser-Arg-Glu-Ile-Gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His-Ala-Val-Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr-Lys-Tyr-Thr-Thr-Ser-Arg. In a previously published partial sequence positions 25, 34, 40 and 41 were incorrectly assigned. The amino-terminal one third of sperm histone H2B(2)Parechinus is highly basic containing a repeating pentapeptide occurring over the first 24 residues. This repeating pentapeptide is different to that of sperm histone H2B(1)Parechinus. In this region there is very little amino acid sequence homology between H2B(1)Parechinus, H2B(2)Parechinus on the one side and H2Bcalf thymus on the other side, but all three proteins are highly basic in their N-terminal parts. The carboxyl-terminal two thirds of sperm histone H2B(2) is hydrophobic except for a short polar region of 20 amino acids. The carboxyl-terminal region shows a very high amino acid sequence homology between the three H2B histones. Much of the variability occurs in the short polar region whereas the hydrophobic regions are almost completely constant. The majority of the amino acid substitutions that occur in the carboxyl end are conservative. The three H2B protein sequences are compared and the functional and evolutionary significance of the changes is discussed.

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphylococcus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1) and bovine H1.

Two of the four electrophoretic histone H2B variants present in wheat embryos have been isolated. The complete primary structure of the H2B(2) variant has been deduced from sets of overlapping peptides generated by CNBr cleavage, Staphyloccocus aureus V8 protease, endoproteinase Arg-C, the post-proline cleaving enzyme, chymotrypsin and cleavage in dilute acid. A minimum of 17 peptides were required to establish the sequence. This variant has a blocked N terminus and comprises a total of 149 amino acids. The C-terminal two-thirds of the protein are highly homologous to vertebrate H2B. In contrast, the N-terminal third is entirely different and contains an N-terminal extension of 23 residues in which the sequence Ala-Glu-Lys or variants are repeated several times. This region is also highly homologous to the H2B from Tetrahymena pyriformis. It shows in addition similarities to wheat H2A(1), and bovine H1.

Two histones have been isolated and purified from sperm cells of the sea urchin Parechinus angulosus. They have been designated sperm histone H2B(1) Parechinus and sperm histone H2B(2) Parechinus on criteria of solubility and partial sequence homology with H2Bcalf. H2B(1) consists of a polypeptide chain of the following 144 amino acid residues: Pro-Ser-Gln-Lys-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Thr-Lys-Arg-Ser-Pro-Gln-Lys-Gly-Gly-Lys-Gly-Gly-Lys-Gly-Ala-Lys-Arg-Gly-Gly-Lys-Ala-Gly-Lys-Arg-Arg-Arg-Gly-Val-Gln-Val-Lys-Arg-Arg-Arg-Arg-Arg-Arg-Glu-Ser-Tyr-Gly-Ile-Tyr-Ile-Tyr-Lys-Val-Leu-Lys-Gln-Val-His-Pro-Asp-Thr-Gly-Ile-Ser-Ser-Arg-Ala-Met-Ser-Val-Met-Asn-Ser-Phe-Val-Asn-Asp-Val-Phe-Glu-Arg-Ile-Ala-Ala-Glu-Ala-Gly-Arg-Leu-Thr-Thr-Tyr-Asn-Arg-Arg-Ser-Thr-Val-Ser-Ser-Arg-Glu-Val-Gln-Thr-Ala-Val-Arg-Leu-Leu-Leu-Pro-Gly-Glu-Leu-Ala-Lys-His-Ala-Val-Ser-Glu-Gly-Thr-Lys-Ala-Val-Thr-Lys-Tyr-Thr-Thr-Ser-Arg. In a previously published partial sequence serine was incorrectly assigned to position 30. A comparison of the carboxyl-terminal two thirds of H2B(1) Parechinus and H2Bcalf reveals that 79% of the corresponding positions are occupied by identical amino acid residues. Much of the variability occurs in a short polar region of 20 amino acid residues. The remainder of C-terminal regions is hydrophobic and almost completely invariable. A comparison of the amino-terminal one third of H2B(1) Parechinus and H2Bcalf reveals that they are both highly basic with a considerable homology of the position of basic amino acids. Further, H2B(1) Parechinus possesses an amino terminal extension of 19 amino acids. These 19 amino acids can be recognized as a repeating pentapeptide.

Ten isohistones from the embryo of the sea urchin Parechinus angulosus have been isolated by preparative gel electrophoresis and characterised by electrophoretic mobility in two detergent systems, amino acid composition and partial sequences. This brings the total number of different histones identified which are synthesized at one or the other time during the life cycle of the sea urchin to a minimum of 24 structurally characterised polypeptides.

The primary structure of sperm histone H1Parechinus has been determined. H1Parechinus consists of a polypeptide chain of the following 248 amino acid residues: Pro-Gly-Ser-Pro-Gln-Lys-Arg-Ala-Ala-Ser-Pro-Arg-Lys-Ser-Pro-Arg-Lys-Ser-Pro-Lys-Lys-Ser-Pro-Arg-Lys-Ala-Ser-Ala-Ser-Pro-Arg-Arg-Lys-Ala-Lys-Arg-Ala-Arg-Ala-Ser-Thr-His-Pro-Pro-Val-Leu-Glu-Met-Val-Gln-Ala-Ala-Ile-Thr-Ala-Met-Lys-Glu-Arg-Lys-Gly-Ser-Ser-Ala-Ala-Lys-Ile-Lys-Ser-Tyr-Met-Ala-Ala-Asn-Tyr-Arg-Val-Asp-Met-Asn-Val-Leu-Ala-Pro-His-Val-Arg-Arg-Ala-Leu-Arg-Asn-Gly-Val-Ala-Ser-Gly-Ala-Leu-Lys-Gln-Val-Thr-Gly-Thr-Gly-Ala-Ser-Gly-Arg-Phe-Arg-Val-Gly-Ala-Val-Ala-Lys-Pro-Lys-Lys-Ala-Lys-Lys-Thr-Ser-Ala-Ala-Ala-Lys-Ala-Lys-Lys-Ala-Lys-Ala-Ala-Ala-Ala-Lys-Lys-Ala-Arg-Arg-leads to Lys-Ala-Lys-Ala-Ala-Ala-Lys-Arg-Lys-Ala-Ala-Leu-Ala-Lys-Lys-Lys-Ala-Ala-Ala-Ala-Lys-Arg-Lys-Ala-Ala-Ala-Lys-Ala-Lys-Lys-Ala-Lys-Lys-Pro-Lys-Lys-Lys-Ala-Ala-Ala-Lys-Lys-Ala-Lys-Lys-Pro-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Ala-Lys-Lys-Pro-Ala-Lys-Lys-Ser-Pro-Lys-Lys-Lys-Lys-Ala-Lys-Arg-Ser-Pro-Lys-Lys-Ala-Lys-Lys-Ala-Ala-Gly-Lys-Arg-Lys-Pro-Ala-Ala-Lys-Lys-Ala-Arg-Arg-Ser-Pro-Arg-Lys-Ala-Gly-Lys-Arg-Arg-Ser-Pro-Lys-Lys-Ala-Arg-Lys. The protein consists of three domains. Compared to other H1 and H5 histones, there is a very similar hydrophobic central domain and the carboxyl-terminal domain is very rich in lysine and alanine. H1Parechinus is similar to H5 histones in that the carboxyl-terminal domain also contains many arginine residues close to the carboxyl terminus. The carboxyl-terminal domain of H1Parechinus appears to have been constructed by a series of variable duplications. The amino-terminal domain of H1Parechinus is longer and quire different to that of other H1 and H5 histones and is characterized by a repeating tetrapeptide of the general type Ser-Pro-(basic)2. The known sequence of a histone H1 gene from Psammechinus miliaris [Schaffner, W. et al. (1978) Cell, 14, 655-671] is compared to the sequence of H1Parechinus. Again the central hydrophobic domains are similar whereas the amino terminal domains are very different. The functions of the various domains of sperm histone H1Parechinus are discussed.

The histone complement of chromatin from early gastrula, late gastrula and from fully differentiated gut cells of the sea urchin Parechinus angulosus has been fractionated by molecular sieve and ion-exchange chromatography. Several of the subfractions thus isolated have been characterized by amino acid composition and partial amino acid sequences as a series of variants of the histones H1, H2A and H2B. Specific histone variants are present in chromatin at specific stages of differentiation.

It has been observed that proline residues often initiate overlaps during sequenator analysis. The cause has been shown to be an abnormally slow cleavage reaction. The kinetics of the cleavage reaction has been studied and found to obey pseudo-first-order kinetics. There are considerable differences in reaction rates depending on the position of proline in the sequence, as demonstrated for the four prolines in the N-terminal section of the H2B histone from chicken.

Histones H2B have been isolated from the terminally differentiated diploid erythrocytes of three different classes, amphibia (Xenopus laevis), reptilia (Crocodilus niloticus) and aves (Gallus domesticus). Partial amino acid sequences revealed three regions of sequence variation, each variant involving a single amino acid substitution.

Two histone H1 variants have been isolated from wheat germ with M r in the region 23 000–27 000. Partial sequences from their globular regions are reported.