Fusion and histocompatibility in Rhodophyta

Fully developed thalli of Chondrus crispus, Gracilaria chilensis, Gymnogongrus furcellatus and Mazzaella laminarioides were used to assess tissue compatibility. The effect of thallus polarity on grafting and regeneration was also evaluated. Fusion did occur between fragments of the same life history phase in C. crispus, G. chilensis, G. furcellatus and M. laminarioides. Fusion between sporophytic and gametophytic tissue occurred in C. crispus, G. chilensis and M. laminarioides. Intergeneric fusion was observed between C. crispus and M. laminarioides, but not between G. chilensis and G. furcellatus.Outer cell wall, cortex and medulla were continuous at the contact face in compatible combinations. Medullary cells in the attached fragments were thinner and longer than normal cells, forming an interwoven scar plate. Thallus polarity did not modify fusion and regeneration.     

ENDOPHYTIC ALGAE OF CHONDRUS CRISPUS (RHODOHYTA). II. ACROCHAETE HETEROCLADA SP. NOV., A. OPERCULATA SP. NOV., AND PHAEOPHILA DENDROIDES (CHLOROPHYTA)1

The occurrence of three endophytic green algae within Chondrus crispus Stackh. is reported. Two of them are new to science and are described as Acrochaete heteroclada Correa and Nielsen sp. nov. and A. operculata Correa and Nielsen sp. nov. The algae were studied in unialgal culture and in association with the host following infection of C. crispus under laboratory conditions. The experimental infection showed A. heteroclada to be initially epiphytic, with endophytic filaments growing into the cortex of the host during late infection. A. operculata is entirely endophytic when associated with the host. Phaeophila dendroides (Crouan frat.) Batters behaves as an epiphyte at 15 and 24°C but penetrates the host tissues at 20°C. For all three species, a close physical association with the host is established only when zoospores settle and germinate on the surface of C. crispus.     

Influence of the activation status and of ATP on phosphoribulokinase degradation

The light-regulated chloroplast enzyme phosphoribulokinase (EC 2.7.1. 19) exists in two forms. In darkness this enzyme is present in an oxidized form, which is inactive. It is activated in the light by a thioredoxin-mediated reduction. In extracts from young wheat leaves (Triticum aeestivum L.) phosphoribulokinase as well as some other thioredoxin-modulated enzymes can be activated by the artificial reductant dithiothreitol (DTT). The influence of the activation status and of the substrate ATP on phosphoribulokinase stability was investigated in the presence of endogenous endopeptidases from senescing wheat leaves. Similar experiments were performed with purified phosphoribulokinase from spinach in the presence of exogenous, purified endopeptidases (chymotrypsin and trypsin). Phosphoribulokinase stability was analysed by immunoblotting and activity measurements. Both systems led to similar conclusions. DTT (reductant and ATP (substrate) stabilized phosphoribulokinase in wheat leaf extracts as well as partially purified phosphoribulokinase from spinach. The combination of both effectors was far more protective than either effector alone. DTT had hardly any effect on the degradation of thioredoxin-independent chloroplast enzymes such as glutamate synthase and glutamine synthetase. These results suggest that the activation status and substrate concentrations are not only important for the activity of phosphoribulokinase, but are also relevant for the susceptibility of this enzyme to proteolysis.     

The production of antimicrobial compounds by British marine algae I. Antibiotic-producing marine algae

Using five species of bacteria as the test organisms, 151 species of British marine algae have been screened for the production of antibiotics. Of these, Asparagopsis armata, Bonnemaisonia asparagoides, Bonnemaisonia hamifera, Chondrus crispus, Dilsea carnosa, Gloiosiphonia capillaris, Sphondylothamnion multifidum, Desmarestia aculeata, Desmarestia ligulata, Laminaria digitata, Dictyopteris membranacea, Dictyota dichotoma, Halidrys siliquosa and most members of the family Rhodomelaceae appear to possess outstanding antibacterial properties. Although the production of antibiotics would appear to be a characteristic of several families, it has not been possible to establish any major correlation between taxonomy and antibiotic production. In the case of two closely related and morphologically similar species, Chondrus crispus and Gigartina stellata, the former possesses considerable degrees of antimicrobial activity whilst the latter exhibits no such activity. The results also indicate that the production of antibiotics by the algae is affected by the season of the year.     

The NADP-linked glyceraldehyde-3-phosphate dehydrogenases of Anabaena variabilis and Synechocystis PCC 6803, which lack one of the cysteines found in the higher plant enzyme,are not reductively activated

Light activation of NADP-linked glyceraldehyde-3-P dehydrogenase involves reductive cleavage of a disulfide bond. We have proposed that the inactivating disulfide locks the two domains of the enzyme, preventing catalysis, and we have tentatively identified the two critical cysteine residues in the chloroplast enzyme (D. Li, F.J. Stevens, M. Schiffer and L.E. Anderson (1994) Biophys J. 67: 29–35). We reasoned that if activation of this enzyme involves these cysteines that enzymes lacking one or both should be active in the dark and insensitive to reductants. One of these cysteines is present in the enzymes from Anabaena variabilis and Synechocystis PCC 6803 but the other is not. Consistent with the proposed mechanism, glyceraldehyde-3-P dehydrogenase is not affected by DTT-treatment in extracts of either of these cyanobacteria. Fructosebisphosphatase is DTT-activated in extracts of both of these cyanobacteria and glucose-6-P dehydrogenase is inactivated in Synechocystis, as in higher plant chloroplasts. Apparently reductive modulation is possible in these cyanobacteria but glyceraldehyde-3-P dehydrogenase is not light activated.     

Stress tolerance and reactive oxygen metabolism in the intertidal red seaweeds Mastocarpus stellatus and Chondrus crispus

Mastocarpus stellatus and Chondrus crispus are morphologically similar red seaweeds that co-occur on rocky intertidal seashores in the Northern Atlantic. Mastocarpus stellatus grows higher on the shore and is more tolerant of environmental stress, caused by factors such as freezing and desiccation, than C. crispus. Here we report a correlation between reactive oxygen metabolism and stress tolerance, which suggests that reactive oxygen metabolism may play a role in stress tolerance of intertidal red seaweeds. Mastocarpus stellatus scavenged added H₂O₂ slightly faster, and was more resistant to oxidative stress induced by addition of H₂O₂ and Rose Bengal, than C. crispus. These data were consistent with higher levels of ascorbate and β-carotene and higher activities of catalase and glutathione reductase, in M. stellatus. Tocopherol content and activities of superoxide dismutase and ascorbate peroxidase were similar in both species. Activities of reactive oxygen scavenging enzymes generally increased with tidal height in M. stellatus; this was, however, not a consistent trend in C. crispus.     

PHOSPHORUS AND NITROGEN NUTRITION IN CHONDRUS CRISPUS (RHODOPHYTA): EFFECTS ON TOTAL PHOSPHORUS AND NITROGEN CONTENT,CARRAGEENAN PRODUCTION,AND PHOTOSYNTHETIC PIGMENTS AND METABOLISM1

The existence of a phenomenon in phosphorus (P) nutrition comparable to the “Neish effect” in nitrogen (N) nutrition (an inverse relation between seawater N enrichment and carrageenan content) was investigated in the temperate red alga Chondrus crispus Stackhouse. Plants were preconditioned for 17 d and then cultured under varying enrichments of P (0, 3, 6, 10, 15 μM P·wk^?1) and a constant N enrichment (53.5 μM N·wk^?1) for 5 wk. Tissue total P, tissue total N, and carrageenan contents were then determined. Identical experiments were performed using C. crispus collected during the fall, winter, spring, and summer seasons. The procedure was repeated using material collected during the following fall season and cultured under constant P (6 μM P·wk^?1) and varying N enrichments (0, 3, 6, 10, 25 μM N·wk^?1). In the fall (P) experiment, carrageenan content was the highest [53.1 ± 0.3% DW (dry weight)], and tissue total P content was the lowest (1.71 ± 0.27 mg P·g DW^?1) in plants that received no P enrichment. Carrageenan content was stable (46.1 ± 1.8% DW) for plants given enrichments of 3 μM P·wk^?1 and greater. Thus, a decrease in carrageenan content, concomitant with an increase in tissue total P content, was observed, but only at tissue total P levels below 2 mg P·g DW^?1. As these levels were always higher than 2 mg P·g DW^?1 in the winter, spring, and summer experiments, carrageenan content remained constant within each season at 46.2 ± 1.3, 43.1 m 0.7, and 44.5 ± 0.6% DW, respectively. Nitrogen enrichment of plants collected in the fall did not affect carrageenan content, which was stable at 49.3 ± 0.9% DW. When these plants were compared with those of the previous fall experiment (6 μM P·wk^?1 and 53.5 μM N·wk^?1), a slight increase in carrageenan content was noted. Thus, at sufficiently high concentration, N also decreased carrageenan content in C. crispus. Phosphorus nutrition had no significant effect on photosynthesis versus irradiance parameters (P_max, α, R_d, I_c, and I_k), the contents of the photosynthetic pigments chlorophyll-a, phycoerythrin (PE), phycocyanin (PC), and allophycocyanin (APC), and the ratios PE:APC and PC:APC. In contrast, N nutrition affected both P_maxand the photosynthetic pigment contents. The data indicate that N limitation reduces the number of phycobilisomes but not their size. The greater reduction in phycobiliprotein than chlorophyll-acontent corroborates the natural bleaching phenomenon regularly observed in C. crispus populations during summer when N levels are generally low in seawater. These results suggest that C. crispus in the temperate waters of the Bay of Fundy may experience N limitation, but P limitation is unlikely.     

Sulfated Oligosaccharides Mediate the Interaction between a Marine Red Alga and Its Green Algal Pathogenic Endophyte.

The endophytic green alga Acrochaete operculata completely colonizes the sporophytes of the red alga Chondrus crispus; however, it does not penetrate beyond the outer cell layers of the gametophytes. Given that the life cycle phases of C. crispus differ in the sulfation pattern of their extracellular matrix carrageenans, we investigated whether carra-geenan fragments could modulate parasite virulence. lambda-Carrageenan oligosaccharides induced release of H(2)O(2), stimulated protein synthesis, increased carrageenolytic activity, and induced specific polypeptides in the pathogen, resulting in a marked increase in pathogenicity. In contrast, kappa-carrageenan oligosaccharides did not induce a marked release of H(2)O(2) from A. operculata but hindered amino acid uptake and enhanced their recognition by the host, resulting in a reduced virulence. Moreover, C. crispus life cycle phases were shown to behave differently in their response to challenge with cell-free extracts of A. operculata. Gametophytes exhibited a large burst of H(2)O(2), whereas only low levels were released from the sporophytes.     

Regulation of phosphoribulokinase and glyceraldehyde 3‐phosphate dehydrogenase in a freshwater diatom,Asterionella formosa1

The regulation of phosphoribulokinase (PRK) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) was investigated in a freshwater pennate diatom, Asterionella formosa Hassall, and compared to the well‐studied chlorophyte Chlamydomonas reinhardtii P. A. Dang. As has been reported for a marine centric diatom, in A. formosa, PRK was not regulated by reduction with dithiothreitol (DTT) apart from a weak induction in the presence of NADPH and DTT. However, NADPH‐GAPDH was strongly activated when reduced, in contrast to a previous report on a diatom. Surprisingly, it was inhibited by NADPH, unlike in C. reinhardtii, while NADH‐GAPDH was not affected. NADH‐GAPDH was also strongly activated by DTT in contrast to most other photosynthetic cells. In A. formosa, unlike C. reinhardtii, 1,3‐bisphosphoglycerate, the substrate of GAPDH, activated this enzyme, even in the absence of DTT, when using both NADH and NADPH as cofactors. Some of these kinetic behaviors are consistent with regulation by protein–protein interactions involving CP12, a small protein that links PRK and GAPDH in cyanobacteria, green algae, and higher plants. This conclusion was supported by immunodetection of CP12 in crude extracts of A. formosa, using antibodies raised against CP12 from C. reinhardtii. This is the first report of the existence of CP12 in a diatom, but CP12 may be a common feature of diatoms since a bioinformatic search suggested that it was also present in the Thalassiosira pseudonana Hasle et Heimdal genome v3.0. Despite the presence of CP12, this work provides further support for the differential regulation of Calvin cycle enzymes in diatoms compared to green algae.     

The amino acid sequence of a flavodoxin from the eukaryotic red alga Chondrus crispus.

The amino acid sequence of the constitutive flavodoxin from the red alga Chondrus crispus was determined from the analyses of peptide fragments derived by enzymic digestions of the carboxymethylated protein. This is the first sequence reported for a flavodoxin from a eukaryote. The protein is composed of 173 amino acid residues and is a member of the longer-chain group of flavodoxins. The extent of sequence homology to the three other flavodoxins in the group for which sequences are available is in the range 36-39%, with the most strongly conserved regions being those implicated in binding of the FMN, the redox-active prosthetic group. Nevertheless, Chondrus crispus flavodoxin stands apart in a number of respects, in particular the possession of an unusually high content of proline, with these residues distributed more or less regularly along the peptide chain.     

Apoplastic oxidation of L-asparagine is involved in the control of the green algal endophyte Acrochaete operculata Correa & Nielsen by the red seaweed Chondrus crispus Stackhouse

Gametophytes of the marine alga Chondrus crispus are more resistant than tetrasporophytes to infection by the filamentous endophytic alga Acrochaete operculata. It has been shown recently that carrageenan oligosaccharides from the resistant gametophytic generation of C. crispus stimulate the secretion of L-asparagine (L-Asn) by the endophyte and that the host generates hydrogen peroxide and 2-oxo-succinamic acid after contact with this amino acid. Here the response of C. crispus to L-Asn and its effect on the pathogen is investigated. Chondrus crispus released hydrogen peroxide, ammonium ions, and a carbonyl compound into the medium when exposed to L-Asn. This response was correlated with an increase in oxygen consumption. Inhibitor studies indicated the involvement of a flavoenzyme in the reaction, which was sensitive to high concentrations of the reaction product, ammonium, and to chlorpromazine, quinacrine, and cyanide, inhibitors of L-amino acid oxidase. Cell wall macerate of C. crispus also responded to L-Asn, while protoplasts were inactive. Uptake of L-Asn into the cell was not necessary for the response, suggesting that the involved L-amino acid oxidase is apoplastic. Acrochaete operculata was more sensitive to hydrogen peroxide than C. crispus and settlement of A. operculata zoospores on C. crispus was reduced by 86% in the presence of L-Asn. This reduced settlement could be prevented with catalase. Chondrus crispus thus features an apoplastic amino acid oxidase, which is involved in the control of its endophytic pathogen. The modulation of the amino acid secretion in A. operculata by carrageenan oligosaccharides is therefore a key issue in the etiology of the association.     

MOLECULAR DELINEATION OF SPECIES AND SPECIES RELATIONSHIPS IN THE RED ALGAL AGAROPHYTES GRACILARIOPSIS AND GRACILARIA (GRACILARIALES)1

Delineation of species in the economically important agarophyte genera Gracilaria and Gracilariopsis has proven extremely difficult using available morphological characteristics. In this study, we examine the usefulness of two transcribed spacers for molecular systematic studies of these genera. The polymerase chain reaction was used to amplify the internal transcribed spacers (ITSs) and the intervening 5.8S ribosomal DNA of the nuclear ribosomal repeat region. In addition, a plastid spacer region and flanking regions of coding genes were amplified from the RUBISCO operon. Both regions were sequenced for individuals and populations of Gracilariopsis lemaneiformis (Bory) Dawson, Acleto, et Foldvik to determine the usefulness of these spacers in delimiting populations. These studies reveal that there is as much variation among individuals of a population as there is between individuals of geographically separate populations. In addition, the ITS spacer regions were compared between different species of Gracilariopsis and Gracilaria. The nuclear ITS spacer region is conserved at a species level in both genera and provides phylogenetically informative characters that can be used to examine species interrelationships among relatively closely related taxa. However, because of the difficulties of aligning this entire region among species from the two genera, the ITS region is not useful for examining intergenera relationships. ITS interspecies sequence comparisons indicate that Gracilariopsis lemaneiformis from California is significantly different from G. lemaneiformis from China and that a species of Gracilariopsis from Peru is more closely related to G. lemaneiformis from North Carolina than it is to the other Gracilariopsis species examined. In addition, these studies indicate that Gracilaria chilensis Bird, McLachlan, et Oliveira from New Zealand and Gracilaria tenuistipitata Chang et Xia from southeast Asia are as closely related as are Gracilaria verrucosa (Hudson) Papenfuss, G. pacifica Abbott, and Gracilaria robusta Kylin. Phylogenetic analysis of aligned plastid spacer sequences from Gracilaria and Gracilariopsis taxa provide similar conclusions about species relationships.     

Agglutinins from marine macroalgae of the southeastern United States

Protein extracts from 22 species of marine macroalgae from Florida and North Carolina were compared for their abilities to agglutinate sheep and rabbit erythrocytes. Protein extracts from 21 algal species agglutinated rabbit erythrocytes compared to 19 for sheep erythrocytes. However, agglutination by brown algal extracts was variable. The agglutination produced by protein extracts from Dictyota dichotoma could be blocked by addition of polyvinylpyrrolidone. Protein extracts from North Carolina macroalgae were also tested against five bacterial species. Three of these agglutinated bacterial cells. Ulva curvata and Bryopsis plumosa agglutinated all five species. Protein extracts from five species of Florida algae were tested for their effects on mitogenesis in mouse splenocytes and human lymphocytes. Gracilaria tikvahiae HBOI Strain G-5, Ulva rigida and Gracilaria verrucosa HBOI Strain G-16S stimulated mitogenesis in mouse splenocytes, while Gracilaria tikvahiae HBOI Strain G-16stimulated mitogenesis in human lymphocytes.     

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.     

Photoinhibition of Photosynthesis and its Recovery in Red Algae

In different marine red algae (Chondrus crispus, Delesseria sanguinea, Membranoptera alata, Phycodrys rubens, Phyllophora truncata, Polyneura hilliae) photoinhibition of photosynthesis has been investigated by means of both fluorescence and oxygen measurements. Measurements of absolute oxygen production show that photoinhibition causes a decline in the initial slope and in the rate of bending of the fluence rate-response curve (i.e. the photosynthetic efficiency at non-saturating fluence rates), as well as a decline in the photosynthetic capacity (Pm) at saturating fluence rates. Fluorescence data (Fv/Fm) were consistent with the results of oxygen measurements. Under excessive light photoinhibition protects photosynthesis against photo-damage in red algae. However, an increase in the initial fluorescence (Fo) after photoinhibitory treatment indicates that it could not prevent photodamage entirely. Action spectra of photoinhibition demonstrate that the main photoinhibition site in Polyneura hiliae is PS II, because far red light absorbed by PS I was ineffective. The strong increase of Fo in the blue wavelength range and the slight and partial recovery in weak blue light indicate that blue light especially causes photodamage. Recovery of photosynthesis requires dim white light conditions. Experiments with monochromatic light also show a wavelength dependence of recovery. Moreover, the recovery of photosynthesis after a photoinhibitory treatment is strongly temperature dependent, indicating participation of enzymatic processes. The comparison of fluorescence and oxygen measurement of the recovery shows different results in some species. The rate of oxygen production in red control light increased immediately after photoinhibited algae were exposed to weak light conditions. Surprisingly, the ratio of variable to maximum fluorescence (Fv/Fm) of Phyllophora truncata and the maximum fluorescence (Fm) of Polyneura hilliae show first a delay of the recovery under weak light conditions. Thus, in recovery experiments fluorescence and oxygen data are not quite consistent.     

Gas exchange of two CAM species of the genus Cissus (vitaceae) differing in morphological features

NADP-malate dehydrogenase extracted from darkened leaves of the C₃ plants pea, barley, wheat and spinach was activated by reduced glutathione, a monothiol, as well as by dithiothreitol (DTT). However, in the C₄ plants maize and Flaveria trinervia, only dithiothreitol could effectively activate the enzyme. There was no activation of the maize enzyme and little or no activation of the F. trinervia enzyme by glutathione. The failure of glutathione to activate NADP-MDH in leaf extracts of maize and F. trinervia may indicate there is some difference in disulfide groups of the protein compared to the C₃ plant enzyme. Both DTT and glutathione could activate NADP-malate dehydrogenase in a partially purified enzyme preparation from pea leaves with or without addition of partially purified thioredoxin. However, the required concentration of reductant was lower with addition of thioredoxin than in its absence. In extracts of C₃ species and the partially purified pea enzyme the level of activation after 40 to 60 min under aerobic conditions was higher (up to twofold) with DTT than with glutathione. Under anaerobic conditions, the initial rate of activation was about twice as high with DTT as with glutathione, but the total activation after 40 to 60 min was similar. Ascorbate was totally ineffective as a reducing agent in activating NADP-MDH from C₃ or C₄ plants, possibly due to its more positive redox potential.Abbreviations Chl Chlorophyll - DTT Dithiothreitol - GSH Reduced Glutathione - NADP-MDH NADP-malate Dehydrogenase     

Inactivation of pea leaf phosphofructokinase by light and dithiothreitol

When intact pea (Pisum sativum L.) plants are illuminated, the glycolytic enzyme phosphofructokinase is inactivated. In crude extracts the enzyme is inhibited by dithiothreitol. It would seem that this cytoplasmic enzyme, like glucose-6-P dehydrogenase, is light-inactivated when the enzymes of photosynthetic carbon metabolism are light-activated.     

LIFE HISTORY PHASES AND THE BIOMECHANICAL PROPERTIES OF THE RED ALGA CHONDRUS CRISPUS (RHODOPHYTA)

Chondrus crispus Stackhouse alternates between two isomorphic life history phases that differ in cell-wall phycocolloid composition. It has been long hypothesized that the gametophyte, with strong-gelling kappa-type carrageenans, is mechanically superior to the tetrasporophyte, with nongelling lambda-type carrageenans, which could contribute to the observed gametophytic dominance in many wave-swept environments. Standard mechanical tests were performed on distal tissues of C. crispus sampled from a range of environments in Narragansett Bay, Rhode Island, using a tensometer equipped with a video extensometer. Life history phase was by far the most important determinant of mechanical properties, whereas environmental factors had only modest influence (vertical distribution) or no effect (exposure); gametophytic distal tissues were 43% stronger, 21% more extensible, and 21% stiffer than tetrasporophytic distal tissues. However, the superior strength of gametophytic tissues was not evident at the stipe/holdfast junction (where breakage typically occurs), and the two phases were equally susceptible to dislodgment by a given force. The primary ecophysiological role of carrageenans in C. crispus may not be the provision of a structure to resist wave action.     

Isolation and characterisation of an acid phosphatase interfering with phosphorylase determinations in crude extracts from yeast

In phosphorylase assays in crude yeast extracts with glucose-1-phosphate (G-1-P) as substrate, 25–30% of the P_i-liberating activity could not be inhibited by antibodies against yeast phosphorylase and were attributed to the action of phosphatases. During phosphorylase preparation from baker's yeast (Saccharomyces cerevisiae), a phosphatase, molecular weight 45000±5000, with high specificity for G-1-P, pH-optimum 5.6, was isolated which appeared to be responsible for the interference. It did not hydrolyze other glycolytic intermediates, pyrophosphate or adenylates. No activation by Mg²⁺ or inhibition by (+)-tartrate, and only 40% inhibition by 50 mM F^- were observed, 5,5 dithiobis-(nitrobenzoic acid) (10mM) inactivated the enzyme completely. Its affinity for G-1-P was very low (K _m=40 mM). Consequently, it mainly interfered with the phosphorylase assay in the amylose synthesizing reaction, in which high G-1-P-concentrations have to be used. For phosphorylase assays in crude extracts, measurement of the phosphorolytic activity is recommended, in which the concentration of G-1-P is kept sufficiently low.Abbreviations G-1-P Glucose-1-phosphate - (NbS)₂ 5,5 dithiobis-(2-nitrobenzoic acid) - SDS Sodium dodecylsulfate     

ENDOPHYTIC ALGAE OF CHONDRUS CRISPUS (RHODOPHYTA). III. HOST SPECIFICITY1

Susceptibility of rhodophycean macroalgae to infection by the green endophytes, Acrochaete operculata Correa & Nielsen and A. heteroclada Correct & Nielsen was studied. Cross-infection experiments showed that A. operculata is host specific and developed only in sporophytic fronds of Chondrus crispus Stackh. and Iridaea cordata (Turn.) Bory. Although A. operculata penetrated equally the multilamellar outer cell wall of sporophytic and gametophytic fronds of C. crispus, subsequent development was arrested in the gametophytic fronds. Susceptibility of the sporophytic phase of C. crispus was detected early in the development of the host, at a discoid stage that is structurally distinct from the adult fronds. The evidence strongly suggests that host specificity in A. operculata is determined by cell-wall composition of the hosts, likely the carrageenan fraction. In contrast, A. heteroclada was not host specific, infecting all offered hosts, including carrageenophytes and agarophytes. Germination occurred on the surface of the hosts and led to the development of an epiphytic stage. Subsequent penetration in many cases involved total displacement of cortical tissue in the infected frond.