Fractionation by centrifugation of leaf proteins in press juices from Brassica and other species as a function of pH

Non-green leaf protein concentrates can be produced after separation of chlorophyll-associated proteins from chlorophyll-free proteins by sedimenting the chlorophyll-containing membranes suspended in press juice. To make the use of low speed centrifugation possible, the influence of pH on protein sedimentation rate was investigated, especially for Brassica Oleracea (17 cultivars). Press juices produced on a laboratory scale from greenhouse grown plant material were adjusted to the desired pH and centrifuges briefly at 5000 g or 15,000 g. The chlorophyll-associated protein from B. Oleraces sedimented rapidly at pH values both below and above 6.0(original pH of the press juice). When the pH of the press juice was adjusted to 7.5, all the chlorophyll-associated protein sedimented at 15,000 g, whereas about 45%of the original protein remained in the supernatant. An increase in sedimented chlorophyll-associated protein at higher pH was also observed for Brassica napus, Helianthus annuus and Vicia faba, but not for Beta vulgaris and Dactylis glomerata. Theoretically, the protein sedimentation pattern typical for B. oleracea might depend primarily on protein aggregation, but shrinking of thylakoids at pH values higher than 6 could contribute. The deviation from this pattern observed for B. vulgaris and D. glomerata might be caused by differences in composition of proteins or low molecular species. In practice, a pH above 7.0 seems to be useful for separation of chlorophyll-associated proteins from chlorophyll-free proteins by centrifugation of press juice from Brassica species without heat treatment.     

Molecular dynamics simulation of human interleukin-4: comparison with NMR data and effect of pH,counterions and force field on tertiary structure stability

The human protein interleukin-4 (IL-4) has been simulated at two different pH values 2 and 6, with different amounts of counterions present in the aqueous solution, and with two different force-field parameter sets using molecular dynamics simulation with the aim of validation of force field and simulation set-up by comparison to experimental nuclear magnetic resonance data, such as proton–proton nuclear Overhauser effect (NOE) distance bounds, ³ J(HN,HC_α) coupling constants and backbone N–H order parameters. Thirteen simulations varying in the length from 3 to 7 ns are compared.

At pH 6 both force-field parameter sets used do largely reproduce the NOE's and order parameters, the GROMOS 45A3 set slightly better than the GROMOS 53A6 set. ³ J values predicted from the simulation agree less well with experimental values. At pH 2 the protein unfolds, unless counterions are explicitly present in the system, but even then the agreement with experiment is worse than at pH 6. When simulating a highly charged protein, such as IL-4 at pH 2, the inclusion of counterions in the simulation seems mandatory.     

The Mouse p (pink‐eyed dilution) and Human P Genes,Oculocutaneous Albinism Type 2 (OCA2), and Melanosomal pH

Recessive mutations of the mouse p (pink‐eyed dilution) gene lead to hypopigmentation of the eyes, skin, and fur. Mice lacking a functional p protein have pink eyes and light gray fur (if non‐agouti) or cream‐colored fur (if agouti). The human orthologue is the P protein. Humans lacking a functional P protein have oculocutaneous albinism type 2 (OCA2). Melanocytes from p‐deficient mice or OCA2 individuals contain small, minimally pigmented melanosomes. The mouse and human proteins are predicted to have 12 membrane spanning domains and possess significant sequence homology to a number of membrane transport proteins, some of which are involved in the transport of anions. The p protein has been localized to the melanosome membrane. Recently, it has been shown that melanosomes from p protein‐deficient melanocytes have an abnormal pH. Melanosomes in cultured melanocytes derived from wild‐type mice are typically acidic, whereas melanosomes from p protein‐deficient mice are non‐acidic. Melanosomes and related endosome‐derived organelles (i.e., lysosomes) are thought to have an adenosine triphosphate (ATP)‐driven proton pump that helps to generate an acidic lumen. To compensate for the charge of these protons, anions must also be transported to the lumen of the melanosome. In light of these observations, a model of p protein function is presented in which the p protein, together with the ATP‐driven proton pump, regulates the pH of the melanosome.     

Evidence for a folded conformation of the cell wall protein fromAcetabularia (Polyphysa) cliftonii

Summary The cell wall protein fromAcetabularia has a non-random structure in aqueous solution at pH 5.3, as determined on the basis of intrinsic viscosity, sedimentation velocity and small angle X-ray scattering experiments. This non-random structure is stable in a pH range of 4.5–6.8, as observed on the basis of circular dichroism and viscosity measurements, supporting that the cell wall protein has a specific folded structure. All hydrodynamic measurements, including small angle X-ray scattering in solution, in this pH range are consistent with a prolate ellipsoid model for the shape of this protein, with overall dimensions ofc=86.0 Å,b=7.0 Å, anda=7.5 Å, and with a radius of gyration ofR=39.5 Å. The possibility of a coiled shape was investigated using a worm-like chain model, but it was inconsistent with the experimental data. Instead, a filled particle with uniform density which is equivalent in the scattering behavior is proposed. By a comparison of the observed radius of gyration, R_g=39.5 Å, and the radius of gyration of the cross section,R _c =7.5 Å, we were able to describe the cell wall protein in terms of a prolate ellipsoid of revolution. Comparisons of the experimental scattering curve, plotted as logl (h) versus logh, with the corresponding plots of normalized intensities, calculated for particles of particular shape and various axial ratios indicate a very asymmetric shape for the cell wall protein fromAcetabularia.This research was supported by a grant of the Deutsche Forschungsgemeinschaft.     

pH-Dependent Binding of Synthetic β-Amyloid Peptides to Glycosaminoglycans

The seinile plaques found within the cerebral cortex and hippocampus of the Alzheimer disease brain contain β-amyloid peptide (Aβ) fibrils that are associated with a variety of macromolecular species, including dermatan sulfate proteoglycan and heparan sulfate proteoglycan. The latter has been shown recently to bind tightly to both amyloid precursor protein and A/β, and this binding has been attributed largely to the interaction of the core protein of heparan sulfate proteoglycan with Aβ and its precursor. Here we have examined the ability of synthetic Aβ s to bind to and interact with the glycosaminoglycan moieties of proteoglycans. Aβ(1–28) associates with heparin, heparan sulfate, dermatan sulfate, and chondroitin sulfate. The interaction of these sulfated polysaccharides with the amyloid peptide results in the formation of large aggregates that are readily sedimented by centrifugation. The ability of both Aβ(1–28) and Aβ(1–40) to bind glycosaminoglycans is pH-dependent, with increasing interaction as the pH values fall below neutrality and very little binding at pH 8.0. The pH profile of heparin-induced aggregation of Aβ(1–28) has a midpoint pH of approximately 6.5, suggesting that one or more histidine residues must be protonated for binding to occur. Analysis of the Aβ sequence reveals a consensus heparin-binding domain at residues 12–17, and this motif contains histidines at positions 13 and 14 that may be involved in the interaction with glycosaminoglycans. This hypothesis is supported by the following observations: (a) Aβ(13–17) binds tightly to a heparin affinity column at pH 4.0, but not at pH 8.0; and (b) an Aβ(13–17) in which histidine residues 13 and 14 have been replaced with serines does not bind to a heparin column at either pH 8.0 or 4.0. Together, the data indicate that Aβ is capable of binding to the glycosaminoglycan chains of proteoglycans, and such an interaction may be relevant to the etiology and pathology of Alzheimer's disease.     

Characteristics of an ideotype acid tolerant pasture legume symbiosis in Mediterranean agriculture

A series of experiments has led to the following concept of eight characteristics being required in an acid-tolerant pasture legume symbiosis for use in ley-farming: In the bulk soil i -microsymbiont (preferably of the Bradyrhizobium genus) capable of maintaining high numbers into autumn, through processes which allow saprophytic function at low pH such as regulation of its internal pH, ii -microsymbiont with a carboxylated cell surface electrochemistry stable under the influence of ambient pH in its interactions with soil colloids, minerals and root surfaces. In the rhizosphere iii -microsymbiont capable of appreciable growth in response to substrate availability, iv icrosymbiont able to recognise root exudates allowing interaction with its nodD gene protein, v -microsymbiont whose nodABC gene products (nod metabolites) are pH stable and which induce cortical cell division and root-hair curling in the host, vi -microsymbiont whose surface polysaccharides and proteins are pH stable to allow attenuation of the cell at the root surface, At the root surface vii-host apoplast function unaffected by low pH such that it may (a) produce pH stable exudates capable of interacting with the rhizobial nodD gene protein and (b) receive rhizobial nod metabolites and respond physiologically, viii-root able to produce pH stable organic acids for linkage with rhizobial cell surface structures.These ideotype characteristics reflect our current understanding of the mechanisms of acid tolerance in the nodulation phase of species such as Medicago polymorpha and M. murex, and acid soil tolerance in rhizobial inoculant strains such as WSM540.     

THE LIMITED PROTEOLYSIS OF BOVINE NEUROPHYSINS BY CATHEPSIN D

Abstract— The limited proteolysis of the bovine neurophysins at acid pH has been studied and the enzyme responsible has been characterized. Only 15 per cent of the catheptic activity in 4-year-old acetone-dried posterior pituitary lobe powder is soluble at pH 4.0. Solubility increases as the age of the powder decreases and the cathepsin is completely soluble in the presence of 1% Triton X-100. Acid proteinase activity in the neurohypophysis is not thiol activated and is inhibited by 3-phenylpyruvic acid. Bovine serum albumin was degraded at only 1 per cent of the rate of haemoglobin but with the same pH optimum (3.7). On this basis the enzyme was identified as cathepsin D. Neurophysin-I is degraded in two stages by cathepsin D; the first product (neurophysin-I′) runs faster and the second product (neurophysin-I″) runs slower than the native protein on starch-gel electrophoresis at pH 8.1. Neurophysin-II is also degraded in two stages; the first product has a higher electrophoretic mobility than the native protein and is identical in mobility with the faster-running component of the so-called neurophysin-M of Hollenberg and Hope (1967b). Prolonged incubation with the cathepsin gives rise to a slower-running component. Neurophysin-C is not attacked by the acid proteinase. Neurophysin-I′ and I″ have been isolated by ion-exchange chromotography. They have the same N-terminal amino acid (alanine) and C-terminal sequence (Ala-Phe-Ser) as the native protein and both bind 8-argininevasopressin. Neurophysin-I′ is identical in amino acid composition with the native protein but neurophysin-I″ has lost one leucine and two aspartic acid residues. Reduction, ¹⁴C-alkylation and separation of the fragments by starch-gel electrophoresis shows that the structural and functional integrity of neurophysin-I″ is maintained by the disulphide bonds, even though a tripeptide has been split out of the interior of the molecule. The low molecular weight material produced by catheptic digestion of neurophysin-I has been purified and shown to have a composition of one leucine and two aspartic acid residues. It is suggested that extensive in vivo proteolysis of neurophysin by lysosomal cathepsin, with consequent abolition of hormone-binding ability, is unlikely.     

Environmental factors influencing the chaperone-like activity of α-crystallin

The effects of mild environmental changes (e.g. the addition of divalent cations or EDTA, as well as variations of buffer pH) on the heat stability and chaperone-like activity of native α-crystallin, and denatured–renatured α-crystallin in the native molar isoform ratio, have been investigated using circular dichroism (CD) spectropolarimetry and functional assays. The presence or absence of divalent cations has little or no effect on the secondary structure of renatured samples, although chaperone-like activity levels can vary widely; the only relevant spectral difference observed is a loss of some α-helical content in all the renatured samples relative to the native protein, but this change has no impact on function. The range of concentration over which the inhibitory Mg²⁺ effect is observed is 10-fold higher for dialyzed fresh protein than for protein renatured into buffers containing Mg²⁺, but for both sets of samples, the full effect is established below physiological Mg²⁺ concentrations. Renaturing into various pH buffers, in contrast, affects both heat stability and chaperone-like activity below pH 7.0, with essentially no functionality observed at pH 6.0. CD spectra of these samples indicate that acidic conditions lead to some degree of unfolding, and that this unfolding correlates directly with functionality. Similar results are obtained for fresh protein dialyzed against these pH levels. Overall, these results suggest that heat stability is a function of the protein's secondary structure and folding state, while chaperone-like activity is primarily a function of factors at the tertiary and quaternary levels of organization.     

Current two-dimensional electrophoresis technology for proteomics

Two-dimensional gel electrophoresis (2-DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of promising alternative or complementary technologies (e.g. multidimensional protein identification technology, stable isotope labelling, protein or antibody arrays) that have emerged recently, 2-DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2-DE enables the separaration of complex mixtures of proteins according to isoelectric point (pI), molecular mass (M_r), solubility, and relative abundance. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or post-translational modifications. This is in contrast to liquid chromatography-tandem mass spectrometry based methods, which perform analysis on peptides, where M_r and pI information is lost, and where stable isotope labelling is required for quantitative analysis. Today's 2-DE technology with IPGs (Görg et al., Electrophoresis 2000, 21, 1037–1053), has overcome the former limitations of carrier ampholyte based 2-DE (O'Farrell, J. Biol. Chem. 1975, 250, 4007–4021) with respect to reproducibility, handling, resolution, and separation of very acidic and/or basic proteins. The development of IPGs between pH 2.5–12 has enabled the analysis of very alkaline proteins and the construction of the corresponding databases. Narrow-overlapping IPGs provide increased resolution (δpI = 0.001) and, in combination with prefractionation methods, the detection of low abundance proteins. Depending on the gel size and pH gradient used, 2-DE can resolve more than 5000 proteins simultaneously (˜2000 proteins routinely), and detect and quantify < 1 ng of protein per spot. In this article we describe the current 2-DE/MS workflow including the following topics: sample preparation, protein solubilization, and prefractionation; protein separation by 2-DE with IPGs; protein detection and quantitation; computer assisted analysis of 2-DE patterns; protein identification and characterization by MS; two-dimensional protein databases.     

Thiosulfate sulfur transferases (Rhodaneses) ofChlorobium vibrioforme f.thiosulfatophilum

Two enzymes containing thiosulfate sulfur transferase activity were purified fromChlorobium vibrioforme f.thiosulfatophilum by ion exchange chromatography, gel filtration and isoelectrofocusing. Enzyme I is a basic protein with an isoelectric point at pH 9.2 and has a molecular weight of 39,000. TheK _m-values for thiosulfate and cyanide of the purified basic protein were 0.25 mM (thiosulfate) and 5 mM (cyanide). Enzyme II is an acidic protein. The enzyme has an isoelectric point at pH 4.6–4.7 and a molecular weight of 34,000. TheK _m-values of the acidic protein were found to be 5 mM for thiosulfate and 125 mM for cyanide.In addition to thiosulfate sulfur transferase activity, cellfree extracts ofChlorobium vibrioforme f.thiosulfatophilum also contained low thiosulfate oxidase activity and negligible thiosulfate reductase activity. The percent distribution of thiosulfate sulfur transferase and thiosulfate oxidase activities in the organism was independent of the offered sulfur compound (thiosulfate, sulfide or both) in the medium.Abbreviations C Chlorobium - SDS sodium dodecylsulfate Dedicated to Prof. Dr. Norbert Pfennig on the occasion of his 60th birthday     

The impact of pH and nutrient stress on the growth and survival of Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen that is able to colonise various host environments and is associated with both gastrointestinal and vaginal maternal carriage. Maternal vaginal carriage represents the major source for transmission of S. agalactiae to the foetus/neonate and thus is a significant risk factor for neonatal disease. In order to understand factors influencing maternal carriage we have investigated growth and long term survival of S. agalactiae under conditions of low pH and nutrient stress in vitro. Surprisingly, given that vaginal pH is normally <4.5, S. agalactiae was found to survive poorly at low pH and failed to grow at pH 4.3. However, biofilm growth, although also reduced at low pH, was shown to enhance survival of S. agalactiae. Proteomic analysis identified 26 proteins that were more abundant under nutrient stress conditions (extended stationary phase), including a RelE family protein, a universal stress protein family member and four proteins that belong to the Gls24 (PF03780) stress protein family. Cumulatively, these data indicate that novel mechanisms are likely to operate that allow S. agalactiae survival at low pH and under nutrient stress during maternal vaginal colonisation and/or that the bacteria may access a more favourable microenvironment at the vaginal mucosa. As current in vitro models for S. agalactiae growth appear unsatisfactory, novel methods need to be developed to study streptococcal colonisation under physiologically-relevant conditions.     

Cloning,functional expression and expression studies of the nitrate transporter gene from Chlorella sorokiniana (strain 211-8k)

The nitrate transporter from Chlorella sorokiniana (accession number AY026523) has been cloned by screening a cDNA library based on mRNA isolated after 30 min treatment of Chlorella with 5 mM nitrate and with a RT-PCR product (730 bp) as a probe. The Chlorella sequence has similarity to known nitrate transporters of the NRT2 family (high-affinity nitrate transporters). The cDNA clone was used for functional expression in Xenopus oocytes and a nitrate-dependent current was measured at pH 5.5 but not at pH 7.4. A second algal gene or a second gene product was not needed for functional expression in Xenopus. Inhibitor studies in Chlorella indicated that protein phosphorylation/dephosphorylation is involved in nitrate induction of ChNRT2.1. In addition to nitrate, ChNRT2.1 expression is induced by nitroprusside, a NO donor, and is affected by glucose.     

Studies of the conformational stability of invasion plasmid antigen B from Shigella

Shigella spp. are the causative agent of shigellosis, the second leading cause of diarrhea in children of ages 2–5. Despite many years of research, a protective vaccine has been elusive. We recently demonstrated that invasion plasmid antigens B and D (IpaB and IpaD) provide protection against S. flexneri and S. sonnei. These proteins, however, have very different properties which must be recognized and then managed during vaccine formulation. Herein, we employ spectroscopy to assess the stability of IpaB as well as IpgC (invasion protein gene), IpaB's cognate chaperone, and the IpaB/IpgC complex. The resulting data are mathematically summarized into a visual map illustrating the stability of the proteins and their complex as a function of pH and temperature. The IpaB/IpgC complex exhibits thermal stability at higher pH values but, though initially stable, quickly unfolds with increasing temperature when maintained at lower pH. In contrast, IpaB is a much more complex protein exhibiting increased stability at higher pH, but shows initial instability at lower pH values with pH 5 showing a distinct transition. IpgC precipitates at and below pH 5 and is stable above pH 7. Most strikingly, it is clear that complex formation results in stabilization of the two components. This work serves as a basis for the further development of IpaB as a vaccine candidate as well as extends our understanding of the structural stability of the Shigella type III secretion system.     

Thermal and chemical denaturation of Colocasia esculenta tuber agglutinin from α2β2 to unfolded state

The major tuber storage protein of Colocasia esculenta, is a monocot mannose-binding, widely used dietary lectin, containing two polypeptides of 12.0 and 12.4 kDa. By both gel filtration and dynamic light scattering at pH 7.2, the lectin has a α₂β₂ form of apparent molecular mass of 48.2 kDa and a hydrodynamic radius of 6.1 ± .2 nm; however, at pH 3, it migrates as αβ and has a reduced hydrodynamic radius of 4.6 ± .3 nm. Our circular dichroism spectroscopy studies show that the lectin retains approximately 100% of its secondary structure between pH 2–8, going down to ~90% for extreme acidic/alkaline conditions. The fluorescence emission maxima of 346 to 350 nm for pH 4 to 10 show that the tryptophan residues are relatively exposed. The unfolding is a simple two-state process, N₄ ? 4U, as seen in our denaturation scan profiles. These denaturation profiles, monitored separately by fluorescence, far-UV CD, and near-UV CD, are completely super imposable. Analyses of these profiles provide an estimate of several thermodynamic parameters at each guanidinium chloride concentration, including the melting temperature T_g, which is 346.9 K in 0 M, but lowers to 321.8 K in 3.6 M. Dimeric and tetrameric interfaces observed in the crystal structure for the same protein are used to rationalize solution data in some detail.     

Interaction of Melanin with Proteins – The Importance of an Acidic Intramelanosomal pH

Melanin is a highly irregular heteropolymer consisting of monomeric units derived from the enzymatic oxidation of the amino acid tyrosine. The process of melanin formation takes place in specialized acidic organelles (melanosomes) in melanocytes. The process of melanin polymerization requires an alkaline pH in vitro, and therefore, the purpose of an acidic environment in vivo remains a mystery. It is known that melanin is always bound to protein in vivo. It is also seen that polymerization in vitro at an acidic pH necessarily requires the presence of proteins. The effect of various model proteins on melanin synthesis and their interaction with melanin was studied. It was seen that many proteins could increase melanin synthesis at an acidic pH, and that different proteins resulted in the formation of different states of melanin, i.e., a precipitate or a soluble, protein‐bound form. We also present evidence to show that soluble protein‐bound melanin is present in vivo (in B16 cells as well as in B16 melanoma tissue). An acidic pH appeared to be necessary to ensure the formation of a uniform, very high molecular weight melano–protein complex. The interaction between melanin and proteins appears to be largely charge‐dependent as evidenced by zeta potential measurements, and this interaction is also increased in an acidic pH. Thus, it appears that an acidic intramelanosomal pH is essential to ensure maximum interaction between protein and melanin, and also to ensure that all the melanin formed is protein‐bound.     

Stimuli-responsive separation of proteins using immobilized liposome chromatography

The possibility of the stimuli-responsive separation of proteins was investigated using immobilized liposome chromatography (ILC) as novel aqueous two-phase systems. The specific capacity factor (k_s) of β-galactosidase, obtained by analysis of ILC, was varied by changing the pH of the solution and was maximized at the specific pH of 5 (k_s,max=5.57). The k_s values were found to correspond well with their local hydrophobicities, which can be determined by the aqueous two-phase partitioning method. The variation of k_s, therefore, indicates a change in the surface properties of a protein during conformational change under pH stimuli. A similar phenomenon is observed in the case of other proteins (α-glucosidase, k_s,max=11.3 at pH 4; carbonic anhydrase from bovine, k_s,max=6.53 at pH 4). The difference in the height and/or the position of the peaks of the k_s–pH curves of each protein suggests a difference in their pH denaturation in the ILC column. Based on these results, the mutual separation of the above proteins at pH 4 could be successfully performed by selecting their specific capacity factor as a design parameter.     

Optimal conditions for hepatitis B core antigen production in shaked flask fermentation

The effects of various environmental factors such as pH (5, 6, 7, 8 and 9), temperature (30, 37 and 40°C) and rotational speed (150, 200 and 250 rpm) on the growth and the hepatitis B core antigen (HBcAg) production ofEscherichia coli W3110IQ were examined in the present study. The highest growth rate is achieved at PH 7, 37°C and at a rotational speed of 250 rpm which is 0.927 h⁻¹. The effect of pH on cell growth is more substantial compared to other parameters; it recorded a 123% different between the highest growth rate (0.927 h⁻¹) at pH 7 and lowest growth at pH 5. The highest protein yield is achieved at pH 9, rotational speed of 250 rpm and 40°C. The yield of protein at pH 7 is 154% higher compared to the lowest yield achieved at pH 5. There is about 28% different of the protein yield for theE. coli cultivated at 250 rpm compared to that at 150 rpm which has the lowest HBcAg yield. The yield of protein at 40°C is 38% higher compared to the lowest yield achieved, at 30°C.     

Use of enzymatic cell wall degradation for improvement of protein extraction from Chondrus crispus,Gracilaria verrucosa and Palmaria palmata

The effect of polysaccharidases (κ-carrageenase, β-agarase, xylanase, cellulase) on the protein extraction from three rhodophytes has been studied. The kinetic parameters (apparent V _m, apparent K _m) and the optimum activity conditions (pH, temperature) of each enzyme were determined by using pure substrates. All the tested enzymes possess Michaelis Menten mechanism with estimated substrate saturating concentrations of 8 000 mg l⁻¹(carrageenan) for κ-carrageenase, 8 000 mg l⁻¹ (agar) for β-agarase, 5000 mg l⁻¹ (xylane) for β-xylanase and 6 000 mg l⁻¹ (carboxymethylcellulose) for cellulase. The optimum activity conditions are pH 6.5–6.8 at 45°C for carrageenase, pH 6–6.5 at 55°C for agarase, pH 5 at 55°C for xylanase and pH 3.8 at 50°C for cellulose. Different alga/enzymes couples (κ-carrageenase/Chondrus crispus, β-agarase/Gracilaria verrucosa, β-xylanase/Palmaria palmata) were tested under the optimum activity conditions. Alga/cellulase + specific enzyme (e.g. Chondrus crispus/carrageenase + cellulase) systems were also studied at the optimum activity conditions of a specific enzyme (e.g. carageenase). The use of the only cellulose was also tested on each alga. Except for Palmaria palmata, the highest protein yields were observed with the procedures using cellulase coupled with carrageenase or agarase for an incubation period limited to 2 h. The Chondrus crispus/carrageenase + cellulose and Gracilaria verrucosa/agarase + cellulase systems gave ten-fold and three-fold improvements, respectively, in protein extraction yield as compared to the enzyme-free blank procedure. The combined action of xylanase and cellulose on protein extraction from Palmaria palmata does not significantly improve protein yield. The best overall protein yield for P. palmata is for P. palmata/xylanase with a 14-h incubation time. This study shows the interest in the use of a polysaccharidase mixture for improving protein extractibility from certain rhodophytes. This biotechnology approach, adapted from procedures for protoplast production or enzymatic liquefaction of higher plants, could be tested as an alternative method to obtain proteins from seaweeds of nutritional interest.     

Biochemical Aspects of Parasitism by the Angiosperm Parasites: Host-parasite Interrelationship in Phosphatase Activity

Infection by Cuscuta and Orobanche causes significant losses in dry solids and protein content in host plant or plant part. Changes occur in phosphatase activity towards fructose-1,6-diphosphate at alkaline pH and β-glycerophosphate at acid pH, expressed per mg protein or g fresh tissue. The leaves of all hosts infected by Orobanche show an increase in the alkaline fructosediphosphatase activity, whereas as far as the infection by Cuscuta is concerned the general response is a decrease in the enzyme in the shoots. The alterations in the phosphatase activity towards β-glycerophosphate at acid pH in the shoots are not consistent. However, there is a marked increase in the acid phosphatase activity against β-glycerophosphatase in the roots of the infected hosts. The significance of these findings has been discussed in the light of host-parasite interrelationship.