PROPERTIES OF PHOSPHOENOLPYRUVATE CARBOXYKINASE FROM ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

Phosphoenolpyruvate carboxykinase was purified to electrophoretic homogeneity from the brown alga. Ascophyllum nodosum (L.) le Jol. Its molecular mass was 60 kDa as determined by gel filtration. The pH optimum of the carboxylating reaction was 7.9 and the apparent K_m for PEP⁴, ADP and HCO₃^- were 0.036, 0.0116 and 50 mol · m^-3, respectively. Rates of light and dark carbon fixation are also reported for A. nodosum apices and it is shown that rates of in vitro PEPCK activity would account for the observed rate of dark fixation. The physiological role of PEPCK is discussed in relation to light-independent carbon fixation.     

PHOSPHOENOLPYRUVATE CARBOXYKINASE ACTIVITY IN ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE)1

PEP-dependent⁴ CO₂-fixation by extracts of Ascophyllum nodosum (L.) Le Jol. is reported. The carboxylation of PEP is Mn²⁺ dependent and ATP is shown to be a product. IDP was found to be less efficient as a phosphate acceptor than ADP and 3-mercaptopicolinic acid inhibited the carboxylation reaction. Extracts decarboxylated OAA only in the presence of ATP and had high activities of MDH and GOT. This evidence, together with the probable absence of PEPC, PEPCTrP, and PC in A. nodosum extracts, favors the view that PEPCK is responsible for the light-independent CO₂-fixation observed in this alga.     

COLONIZATION AND GROWTH OF ASCOPHYLLUM NODOSUM (PHAEOPHYTA) IN MAINE1

Colonization and growth of Ascophyllum nodosum (L.) Le Jol. were investigated in two Maine estuaries from 1972 to 1978. Following denudation of intertidal rock, substrata were initially colonized by Fucus vesiculosus L.; eventually, Ascophyllum supplanted Fucus, and became dominant in terms of percentage cover. Ascophyllum settled first and most densely in the low intertidal zone, but its fastest growth occurred in the mid-intertidal zone. Some, but not all, Ascophyllum germlings produced a vesicle within one year of colonization. The mean annual growth of A. nodosum was variable among sites, zones and years.     

ROLE OF SURFACE WOUNDS AND BROWN ALGAL EPIPHYTES IN THE COLONIZATION OF ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE) FRONDS BY VERTEBRATA LANOSA (RHODOPHYTA)1

Ascophyllum nodosum (L.) Le Jol. forms extensive beds in wave‐sheltered, rocky intertidal habitats on the northwestern Atlantic coast. This fucoid seaweed is host to an obligate red algal epiphyte, Vertebrata lanosa (L.) T. A. Chr. [=Polysiphonia lanosa (L.) Tandy], and two facultative brown algal epiphytes, Elachista fucicola (Velley) Aresch. and Pylaiella littoralis (L.) Kjellm. Although V. lanosa can occur throughout most of the length of host fronds, it largely predominates in midfrond segments. The two brown algal epiphytes are restricted to distal segments. Through field experiments conducted in Nova Scotia, Canada, we tested the hypothesis that surface wounds are required for the colonization of distal segments of host fronds by V. lanosa. Distal tissues normally have a smooth surface because of their young age (A. nodosum fronds grow apically). By creating small wounds that mimicked grazing wounds distributed elsewhere on host fronds, we demonstrated that V. lanosa can colonize distal frond segments during the growth and reproductive season (summer and autumn). Approximately half of the artificial wounds were colonized by V. lanosa during this time. The experimental exclusion of both brown algal epiphytes from distal frond segments did not affect colonization by V. lanosa. Thus, we conclude that the absence of surface irregularities on distal segments of host fronds, specifically small wounds, is the main factor explaining the absence of V. lanosa there. We propose that further experimental work clarifying epiphyte distribution in host beds will enhance our ability to understand the functional role of epiphytes in intertidal ecosystems.     

PHYSIOLOGICAL AND POPULATION ECOLOGY OF INTERTIDAL AND SUBTIDAL ASCOPHYLLUM NODOSUM (PHAEOPHYTA)1

Morphological, demographic and physiological characteristics of Rhode Island intertidal and subtidal populations of Ascophyllum nodosum (L.) Le Jolis were compared in order to examine factors influencing vertical distribution. The two populations had distinctive morphologies: subtidal plants were narrower (more terete) and highly branched compared with intertidal plants. The subtidal population showed signs of necrosis and breakage, which was reflected in significantly shorter mean plant size. High survivorship and low recruitment of both population resulted in relatively constant densities, averaging 91 and 50 plants per m² in the intertidal and subtidal habitats, respectively. Intertidal plants had higher mean annual growth rates (25 cm.yr^?1) than subtidal plants (2 cm.yr^?1). In general, intertidal plants had higher photosynthetic capacity and nutrient (NO₃^?) uptake rates than the subtidal population but maintained lower light-harvesting pigment and tissue nitrogen concentrations. Although Ascophyllum nodosum is capable of survival and growth in subtidal as well as intertidal areas, results of this study suggest that different selective pressures affect persistence in each habitat. The scarcity of plants in the subtidal environment may be due to the lack of a critical balance between algal production, allocation of photosynthate, and the negative effects of grazers or competitors.     

EXPRESSION,PURIFICATION, AND CHARACTERIZATION OF COLD-ADAPTED INORGANIC PYROPHOSPHATASE FROM PSYCHROPHILIC Shewanella sp. AS-11

In the presence of divalent cations, inorganic pyrophosphatase is activated to hydrolyze inorganic pyrophosphate to inorganic phosphate. Here, we clone, express, purify, and characterize inorganic pyrophosphatase from the psychrophilic Shewanella sp. AS-11 (Sh-PPase). The recombinant Sh-PPase was expressed in Escherichia coli BL21 (DE3) at 20°C using pET16b as an expression vector and purified from the cell extracts by a combination of ammonium sulfate fractionation and anion-exchange chromatography. Sh-PPase was found to be a family II PPase with a subunit molecular mass of 34 kD that preferentially utilizes Mn²⁺ over Mg²⁺ ions for activity. The functional characteristics of Sh-PPase, such as activity, temperature dependency, and thermal inactivation, were greatly influenced by manganese ions. Manganese ion activation increased the enzyme's activity at low temperatures; therefore, it was required to gain the cold-adapted characteristics of Sh-PPase.     

A FIELD-COMPATIBLE METHOD FOR EXTRACTION OF FINGERPRINT-QUALITY DNA FROM MACROCYSTIS PYRIFERA (PHAEOPHYCEAE)1

A method for extracting DNA from laminarialean algae resulting in DNA of sufficient quantity and purity for DNA fingerprints is presented. The method both eliminates the use of liquid N₂ and delays the use of toxic chemicals in the initial extraction steps; thus, it is appropriate for use in remote field locations. The algal samples were ground in a solution of hexadecyltrimethylammonium bromide (CTAB) and polyvinylpyrrolidone (PVPP) at room temperature and then stored for 1 week in the CTAB-PVPP solution at room temperature before extraction with chloroform-isoamyl alcohol and purification by cesium chloride ultracentrifugation. No degradation of DNA was observed. Hybridization of RsaI-digested Macrocystis pyrifera (L.) C. Ag. DNA with the M13repeat probe yielded a DNA fingerprint with 12 discrete bands 4–19 kb in molecular size.     

SETTLEMENT AND SURVIVAL OF POLYSIPHONIA LANOSA (CERAMIALES) SPORES ON ASCOPHYLLUM NODOSUM AND FUCUS VESICULOSUS (FUCALES)1

The settlement patterns of spores of Polysiphonia lanosa (L.) Tandy on Ascophyllum nodosum (L.) Le Jolis and Fucus vesiculosus L. were studied using a flow tank. Settlement sites were defined as ‘sheltered’ or ‘exposed.’ Surface area calculations revealed non-random settlement on A. nodosum, with higher than expected spore frequencies on the thallus and lateral pits and lower than expected frequencies on the vesicles. Settlement of F. vesiculosus was random and significantly lower than on A. nodosum. On the shore, survival of sporelings from September (post-sporulation) to May (pre-sporulation) was highly non-random on both basiphytes. On A. nodosum, lateral pits ('sheltered') showed the highest survival frequency. Here the proportion of surviving sporelings increased over the study period, whereas the proportion on open thallus area ('exposed') decreased. On F. vesiculosus also preferential survival occurred on ‘sheltered’ sites such as vesicle/thallus interfaces and wounds. Between September and May, all P. lanosa sporelings were lost from ‘exposed’ areas (thallus surface and vesicles). Overall, frequencies of surviving sporelings were much greater on A. nodosum than on F. vesiculosus. These results are discussed with reference to basiphyte morphology, epiphyte removal mechanisms and the survival stratagy of P. lanosa.     

麻蝇幼虫肠道类胰蛋白酶的分离纯化及性质(英文)

麻蝇幼虫肠液经硫铵沉淀, ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５离子交换层析, ＳＢＢＩ－Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析,分离纯化出一种分子量为 １６ｋＤ的蛋白酶。底物及抑制剂的特异性表明,该酶为类胰蛋白酶。其能够强烈地降解蛋白酶非专一底物酪蛋白和 Ｈｉｄｅ ｐｏｗｄｅｒ ａｚｕｒｅ,以及类胰蛋白酶专一底物 Ｂｚ－Ｐｈｅ－Ｖａｌ－Ａｒｇ ＮＡ, Ｂｚ－Ｐｒｏ－Ｐｈｅ－Ａｒｇ ＮＡ和Ｂｚ－Ｖａｌ－Ｇｌｙ－Ａｒｇ ＮＡ．该酶又能被丝氨酸蛋白酶抑制剂ＰＭＳＦ,类胰蛋白酶抑制剂 ＳＢ－ＢＩ和Ｌｅｕｐｅｐｔｉｎ强烈地抑制。蛋白酶在酸性环境下极不稳定,在弱碱环境（ｐＨ８．５－９．５）中活性最高。     

PARTIAL PURIFICATION AND CHARACTERIZATION OF THE ORGANIC SOLVENT-TOLERANT LIPASE PRODUCED BY Pseudomonas fluorescens RB02-3 ISOLATED FROM MILK

Eighty-five putative Pseudomonas isolates were obtained from various raw milk and pasteurized milk samples using Pseudomonas CFC agar. Among them, 36 isolates were identified as Pseudomonas fluorescens, and one isolate was identified as Pseudomonas putida. Lipase activity of the strains was quantitatively measured by the spectrophotometric method using p-nitrophenyl palmitate (p-NPP) as substrate. Detected lipase activity of the strains was between 10.03 U/mL and 22.16 U/mL. Pseudomonas fluorescens RB02-3 possessed the highest lipase activity. The extracellular lipase of P. fluorescens RB02-3 strain was homogeneously purified using a combination of ammonium sulfate precipitation, dialysis, and gel filtration column chromatography. This purification procedure resulted in 2.97-fold purification with 20.3% recovery. The enzyme was characterized, and exhibited maximum activity at pH 7.0 and 50°C; after it was incubated for 1 h it was activated in the presence of hexane, ethyl acetate, isopropanol, and ethanol and remained stable after the incubation was extended for 2 hr. The lipase was slightly inhibited in the presence of Zn²⁺, Co²⁺, Cu²⁺, Ni²⁺ salts, and ethylenediamine tetraacetic acid (EDTA), whereas Cd²⁺, sodium dodecyl sulfate (SDS), and Tween-80 had no effect on its activity.     

甲基营养菌WB-1甲胺磷降解酶的产生、部分纯化及性质

甲基营养菌WB１菌株在以甲胺磷作碳氮源的无机盐培养基上生长时,产生甲胺磷降解酶情况较好,培养20 h为细胞收获的适宜时期。所得细胞经过超声波破碎、吐温20抽提、热处理(60℃,9min)\,DEAE\|纤维素32和CM\|纤维素32柱层析等步骤,得到的酶制品比活力为180U/mg,收率78.8%,纯化倍数22.8。纯化的酶制品经连续聚丙烯酰胺凝胶电泳,呈现一条主带,酶活力染色则呈现一条与之对应的活性谱带;该酶催化反应的适宜pH为90,底物专一性不强,Hg²⁺\,Mn²⁺\,Cu²⁺对酶有强烈的抑制作用。部分纯化酶制品在-20℃(0.1mol/L的磷酸盐溶液中)存放5d,酶活力丧失约60%。     

PURIFICATION AND PARTIAL CHARACTERIZATION OF A GUT TRYPSIN-LIKE PROTEASE FROM LARVAE OF FLESH FLY BOETTCHERISCA PEREGRIN A (DIPTERA: SARCOPHAGAE)

Abstract A 16kD protease was purified from the gut extract of larvae of Boettcherisca peregrina , after ammonium sulfate precipitation, DEAE-Sephadex A-25 ion-exchange chromatography and SBBI-Sepharose 4B affinity chromatography. The results of substrate and inhibitor specificity indicated that the protease behaved as a trypsin-like protease. It possesses high activity against non-specific substrate casein and Hide powder azure, and against trypsin-specific substrates Bz-Phe-Val-Arg NA, Bz-Pro-Phe-Arg NA and Bz-Val-Gly-Arg NA. It can be strongly inhibited by PMSF, phenymethysulfonyl fluoride (serine protease inhibitor), SBBI, soybean Bowman-Birk inhibitor and Leupeptin (trypsin-specific inhibitor). Activity of this protease was found to be maximal at the alkaline range of pH 8. 5–9. 5.     

A NEW RECORD AND ERADICATION OF THE NORTHERN ATLANTIC ALGA ASCOPHYLLUM NODOSUM (PHAEOPHYCEAE) FROM SAN FRANCISCO BAY,CALIFORNIA, USA1

A new record of the Northern Atlantic fucoid Ascophyllum nodosum (L.) Le Jolis (Knotted wrack) was discovered on a shoreline in San Francisco Bay, California during a survey of intertidal habitats in 2001–2002. The alga showed no signs of deterioration 2.5 months after its initial detection. The healthy condition, presence of receptacles with developing oogonia, potential for asexual reproduction, and ability to withstand environmental conditions, both inside the Bay and on the outer Pacific coast, prompted a multiagency eradication effort. Given the relatively small area of shoreline inhabited by the alga, in combination with its absence in 125 other surveyed locations, we decided that manual removal of the seaweed would be the most environmentally sensitive yet effective eradication approach. No A. nodosum has been detected at the site since December 2002, and the species is thought to have been locally eradicated. The site continues to be monitored to assess the success of the eradication efforts.     

GROWTH,YIELD AND MORPHOLOGY OF ASCOPHYLLUM NODOSUM (PHAEOPHYTA) UNDER CONTINUOUS AND INTERMITTENT SEAWATER SPRAY CULTURE REGIMENS1

Growth and yield of the brown alga Ascophyllum nodosum (Le Jolis) were measured under various regimens of seawater spray culture. One-hour periods of desiccation throughout the day and night and cessation of spray throughout the night did not affect growth relative to that in continuous spray. Desiccation for four hours during the day resulted in death. Changes in morphology from the normal intertidal growth form to that resulting from spray culture are described.     

EFFECTS OF EXTERNAL INORGANIC NITROGEN CONCENTRATION ON METABOLISM,GROWTH AND ACTIVITIES OF KEY CARBON AND NITROGEN ASSIMILATORY ENZYMES OF LAMINARIA SACCHARINA (PHAEOPHYCEAE) IN CULTURE1

The growth rate of Laminaria saccharina (L.) Lamour. is dependent on inorganic nitrogen in culture. Growth rates were saturated between 5 and 10 μmol · L^?1 nitrate. The activities of ribulose-1,5 bisphosphate carboxylase, phosphoenolpyruvate carboxykinase, mannitol-1-phosphate dehydrogenase, nitrate reductase and glutamine synthetase also varied with the concentration of inorganic nitrogen in the medium. All enzyme activities were lowest at 2.5 μmol · L^?1 nitrate (the lowest concentration used) increasing to a maximum activity between 10 and 30 μmol · L^?1 nitrate. Most enzyme activities followed a hyperbolic curve resembling those described by the Michaelis-Menten equation, with different half-saturation constants.     

PURIFICATION AND CHARACTERIZATION OF GLYCOLATE OXIDASE FROM THE BROWN ALGA SPATOGLOSSUM PACIFICISM (PHAEOPHYTA)1

Glycolate oxidase was purified to apparent homogeneity from the brown alga Spatoglossum pacificum Yendo. The 1326-fold purified glycolate oxidase enzyme exhibited a specific activity of 22. 4 micromoles glyoxylate formed ·min^?1·mg protein^?1. The molecular weight of the native enzyme was estimated to be 230,000 by gel filtration. The subunit molecular weight of the enzyme was determined to be 49,000 by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, suggesting that the native enzyme is a tetramer. There were two absorption peaks at 345 and 445 nm, indicating that glycolate oxidase is a flavoprotein. This enzyme had a high isoelectric point (pI 9.6) and a high pH optimum (pH 8.3). The K_m values for glycolate and l -lactate were 0.49 and 5.5 mM, respectively. This enzyme also had a broad specificity for other straight-chain α-hydroxy acids but not for β-hydroxyacids. Cyanide, azide, N-ethylmaleimide, and p-chloromercuribenzoic acid did not affect the enzyme, whereas 2-pyridylhydroxymethanesulfonic acid strongly inhibited it. These properties of glycolate oxidase from the brown alga S. pacificum are similar to the properties of the glycolate oxidasesfrom higher plants. Polyclonal antibodies raised against the polypeptide fragment of Spatoglossum glycolate oxidase could recognize glycolate oxidase from Spinacia oleracea L., although the cross-reactivity was weak. The N-terminal sequence of two internal polypeptide fragments of the enzyme from S. pacificum showed a high degree of similarity to that of glycolate oxidase from higher plants. These results suggest that glycolate oxidase from higher plants and brown algae share the same ancestral protein.     

ISOLATION AND CHARACTERIZATION OF GOLGI APPARATUS FROM EGGS OF FUCUS (PHAEOPHYCEAE)1

A golgi-rich cell-free fraction has been obtained from eggs of Fucus serratus L. and characterized by enzyme markers (IDP-ase and TPP-ase) and electron microscopy. The results are correlated with cytochemical localization of IDP-ase and TPP-ase in situ.     

常山凝集素的分离纯化及其性质研究

 本文采用分子筛层析和离子交换技术,从植物常山(Dichroa febrifuga Lour)的鲜叶中,分离纯化出一种新的凝集素,定名为常山凝集素(DFL)。并对其理化性质进行了鉴定:测得其亚基分子量为37,000道尔顿;等电点为4.2。DFL是一种糖蛋白,糖含量为2.8％。DNS-CI法测得其肽链的N-末端为L-缬氨酸。本文还对其糖专一性、不同动物红细胞的凝集专一性,以及对猪精子和某些肿瘤细胞的凝集活性等生物学性质进行了研究。     

ISOLATION AND CHARACTERIZATION OF A DNA VIRUS INFECTING FELDMANNIA SIMPLEX (PHAEOPHYCEAE)1

We report on the isolation and characterization of a virus that is formed in modified zoidangia of the marine brown alga Feldmannia simplex (Crouan) Hamel (Ectocarpales, Phaeophyceae). Isolated virus particles had a buoyant density of about 1.35 g·mL^?1 in CsCl equilibrium gradients. They contained one major polypeptide (MW = 55,000) and at least six additional polypeptides (MW = 15,000–120,000). Four of these proteins were glycosylated. The viral genome consisted of double-stranded DNA and formed two freely migrating fractions in pulsed-field-gel electrophoresis, namely linear DNA with a size of 220 kilobase pairs, and fragments of 10–60 kilobase pairs. However, electron microscopic examination revealed that a substantial fraction of the viral DNA occurred as closed circles. We suggest that the viral DNA in native particles is circular but tends to break at random sites during the preparation.     

EXTRACTION,ASSAY AND SOME PROPERTIES OF FLORIDOSIDE PHOSPHATE SYNTHASE FROM PORPHYRA PERFORATA (RHODOPHYTA)1

A suitable method for extraction of floridoside phosphate synthase (FPS, UDP-galactose: sn-3-glycerol phosphate: 1→2′α-D-galactosyl transferase)from Porphyra perforata J. Ag. was developed. Two assay methods for enzyme activity were utilized, one measuring the amount of floridoside formed by using gas-liquid chromatography, the other measuring the sn-3-glycerol phosphate-dependent formation of UDP; both assays gave similar results. FPS is a soluble protein, and FPS activity in the extract as determined by the amount of product formed in vitro compared well with the in vivo rate of floridoside synthesis (4–7 μMmol product formed·h^?1·g^?1 fresh wt). The rate of product formation in vitro was linear up to 45 min and proportional to protein concentration in the assay mixture. The temperature optimum was 30–35° C. FPS was active over a range of pH values from 7.0–8.5. It was stable in concentrated solutions in the presence of 0.3 M ammonium sulfate, but activity was lost in diluted solution (protein concentration below 0.2 mg·mL^?1) or below 0.2 M ion strength. The data suggest that FPS may be an oligomeric protein which occurs free in the cytoplasm or loosely bound to a membrane. It may also be a regulatory protein controlling the overall rate of synthesis of floridoside in vivo.