Separation and characterization of the soluble and insoluble components of insoluble laminaran

Insoluble laminaran, a (1→3)-β-D-glucan from Laminaria hyperborea (L. cloustoni), has been fractionated by differential solubility into soluble and insoluble fractions. These fractions were degraded with a purified exo-(1→3)-β-D-glucanase from Basidiomycete sp. QM806 giving, as primary hydrolysis products, D-glucose, gentiobiose, laminarabiose, and 1-O-β-laminarabiosylmannitol. Gentiobiose was obtained in only trace amounts from the insoluble fraction of laminaran, suggesting the absence of branching. Successive application of periodate oxidation, reduction, mild acid hydrolysis, and enzymic degradation indicated that the branch in the soluble fraction consists of a single β-(1→6)-linked D-glucosyl residue. The results indicate that “insoluble” laminaran is apparently an aggregate of three closely related polysaccharide species: a soluble, branched, reducing component (soluble laminarose); an insoluble, unbranched, reducing component (insoluble laminarose); and an unbranched, nonreducing component (laminaritol) that has a monosubstituted mannitol residue at the reducing terminal. Laminaritol was found to be about equally distributed between the soluble and insoluble fractions. The average d.p. of the laminaran components is 20–25 residues, as determined from the relative amounts of enzymic hydrolysis products and from periodate-oxidation data.     

Salt soluble without jelly-like component from the oviducal gland induces chorionic expansion in the ova of the Japanese common squid Todarodes pacificus

Abstract

In vitro fertilization of squid requires the jelly substance found in the female oviducal gland; yet, the active component of this substance that facilitates fertilization remains unknown. Here, we used biochemical methods to separate the jelly substance of Todarodes pacificus (Oegopsida) and Sepioteuthis lessoniana (Myopsida family) into four fractions; specifically, two water soluble fractions (Molecular weight > 10,000 and < 10,000), one salt soluble fraction, and one insoluble fraction. The salt soluble fraction of T. pacificus induced chorionic expansion (perivitelline space formation), which precedes the normal embryonic development of ova. In contrast, the salt soluble fraction of S. lessoniana elicited insufficient expansion of the ova, only producing embryos with high abnormality rates. These results suggest that the salt soluble component(s) (not the jelly-like substances) in the oviducal gland induce chorionic expansion and hatching in Oegopsida, and that these components may be similar in Myopsida.     

Trypanosoma gambiense: Blocking ability of parasite and macrophage homogenates on attachment during phagocytosis

This study deals with the ability of the parasite and macrophage homogenates, each fractionated into soluble and insoluble parts, to block the attachment of Trypanosoma gambiense on the macrophage in the process of phagocytosis. The soluble parasite fraction and the insoluble macrophage fraction both showed a high blocking ability. However, the ability of the parasite fraction was completely lost on trypsin digestion, while that of the macrophage was not changed at all. In addition, sonication of the macrophage did not change its blocking ability.     

Wheat, canola and grain legume access to soil phosphorus fractions differs in soils with contrasting phosphorus dynamics

Despite the high phosphorus (P) mobilizing capacity of many legumes, recent studies have found that, at least in calcareous soils, wheat is also able to access insoluble P fractions through yet unknown mechanism(s). We hypothesized that insoluble P fractions may be more available to non-legume plants in alkaline soils due to increased dissolution of the dominant calcium(Ca)-P pool into depleted labile P pools, whereas non-legumes may have limited access to insoluble P fractions in iron(Fe)- and aluminium(Al)-P dominated acid soils. Four crop species (faba bean, chickpea, wheat and canola) were grown on two acid and one alkaline soil under glasshouse conditions to examine rhizosphere processes and soil P fractions accessed. While all species generally depleted the H₂O-soluble inorganic P (water Pi) pool in all soils, there was no net depletion of the labile NaHCO₃-extractable inorganic P fraction (NaHCO₃ Pi) by any species in any soil. The NaOH-extractable P fraction (NaOH Pi) in the alkaline soil was the only non-labile Pi fraction depleted by all crops (particularly canola), possibly due to increases in rhizosphere pH. Chickpea mobilized the insoluble HCl Pi and residual P fractions; however, rhizosphere pH and carboxylate exudation could not fully explain all of the observed Pi depletion in each soil. All organic P fractions appeared highly recalcitrant, with the exception of some depletion of the NaHCO₃ Po fraction by faba bean in the acid soils. Chickpea and faba bean did not show a higher capacity than wheat or canola to mobilize insoluble P pools across all soil types, and the availability of various P fractions to legume and non-legume crops differed in soils with contrasting P dynamics.     

Enzymic activities involved in the oothecal sclerotization of the praying mantid,Tenodera aridifolia sinensis saussure

The enzyme(s) responsible for the sclerotization of mantid ootheca is secreted by the left colleterial gland. From an extract of the glands of Tenodera aridifolia sinensis, two soluble enzyme fractions of different activities were obtained. One fraction acted on N-acetyldopamine (NADA), a precursor of a representative sclerotizing agent, and produced NADA-quinone. The other did not act on NADA itself but converted the quinone to a highly reactive intermediate, such as quinone methide, which was able to react nonenzymically with nucleophilic compounds. Other insoluble enzyme preparations obtained from the silk and pupal cuticle of the Japanese giant silk moth, Dictyoploca japonica, also had these two activities.     

Immunological,isoelectric, hydrophobic and molecular weight differences between soluble and ionically membrane-bound fractions of choline-o-acetyltransferase prepared from mouse and rat brain

Choline-O-acetyltransferase (EC 2.3.1.6; ChAT) was prepared from synaptosomal fractions (P₂) of mouse and rat brain in the presence of proteolytic inhibitors by the method of Gray and Whittaker (1962) as modified by (Salehmoghaddam and Collier, 1976). The P₂ fraction was hypo-osmotically shocked with glass distilled water and centrifuged to separate the cytoplasmic (S₃) and vesicle-bound (P₃) fractions. Fraction S₃ was saved for ChAT assay and compared with the ChAT fraction eluted from the P₃ by salt at a pH 7.4 or by detergent (Benishin and Carroll, 1983). These three fractions of ChAT were then compared by molecular weights, isoelectric points, immunoblotting with monoclonal or polyclonal antibodies and hydrophobicity. The results show that the S₃ fraction of ChAT has a molecular weight of 66 K_d, whereas the ionically-bound fraction of ChAT has a molecular weight of 73–78 K_d. SDS-PAGE of these two ChAT fractions followed by immunoblotting revealed the presence of two immunoreactive bands at 28–29 K_d and 50–51 K_d for the ionically bound ChAT fraction. Conversely, none of these antibodies immunostained any protein bands for the S₃ ChAT fraction even though one monoclonal antibody had been prepared against this ChAT fraction and the S₃ ChAT fraction had a similar specific activity prior to SDS-PAGE as did the salt solubilized ChAT fraction. However, anti-ChAT monoclonal antibody MB16 binds the native S₃ ChAT fraction in the co-precipitation assay.The S₃ fraction of ChAT had only one isoelectric point at pH 7.8, whereas the ionically bound and detergent soluble ChAT fractions had two isoelectric points at pH 8.1–8.15 and 7.45–7.5. The S₃ ChAT fraction also differed in hydrophobicity from the other two ChAT fractions. These differences between the S₃ and salt soluble ChAT fractions were not obviated by addition of Triton X-100 and thus could not be attributed to the association of lipids with either of the fractions. We conclude that the water soluble fraction of ChAT in central nerve terminals differs in its physical properties and its subcellular location from that which ionically binds to membranes.     

Cadmium bioaccumulation and detoxification in the gill and digestive gland of the Antarctic bivalve Laternula elliptica

Exposure to a sublethal concentration of cadmium (Cd; 50 microg L(-1)) resulted in significantly increased Cd concentrations in the gill and digestive gland of the Antarctic bivalve Laternula elliptica. Continuous accumulation of Cd in the two organs during the 14-day exposure period was associated with sequestration of Cd to both the soluble cytosolic and insoluble particulate cell fractions. However, the contribution of each cell fraction to Cd sequestration differed between the two organs; in the gill, a larger portion of Cd was associated with the insoluble fraction, while in the digestive gland, both the soluble and insoluble fractions sequestered similar amounts of Cd. Metal-binding components in the insoluble cell fraction were not identified in this study. On the other hand, a metallothionein-like protein (MTLP) was the major Cd-detoxifying component in the soluble cell fraction of the gill and digestive gland. The amount of MTLP increased linearly with exposure time and the amount of Cd accumulated in the tissue, which suggests a potential utility of MTLP as a biomarker for exposure to Cd and possibly other metals.     

Synthesis of early heat shock proteins in young leaves of barley and sorghum

The in vivo synthesis of early heat-shock proteins in young leaves of barley (Hordeum vulgare L.) and sorghum (Sorghum bicolor L.) was studied by one- and two-dimensional electrophoresis. Analysis of whole leaf protein patterns demonstrated clearly the enhanced resolution of heat-shock proteins, especially those of low molecular weight, when separated by two-dimensional electrophoresis. Comparison between the two cereals showed that a greater number and diversity of heat-shock proteins were induced in the subtropical C₄ (sorghum) species compared to the temperate C₃ (barley) species. Fractionation of whole leaf proteins into soluble and membrane fractions showed the majority of heat-shock proteins to be associated with the soluble fraction in both sorghum and barley. However, several low molecular mass (17-24 kilodalton) heat-shock proteins were clearly identified in the membrane fractions, indicating a likely association with thylakoid membranes in vivo during the early stages of a heat-shock response in both species.     

A protein-bound form of thioacetamide in liver nucleoli

The cellular distribution of ³⁵S from ³⁵S- thioacetamide was determined in rabbit liver subcellular fractions following its $\text{in vivo}$ administration. Of the various fractions isolated, only the nucleolar fraction contained ³⁵S counts that were insoluble in 10% trichloroacetic acid but soluble in trichloroacetic acid if the fraction was treated with trypsin but not RNase or DNase. These results demonstrate that a protein bound form of thioacetamide is present in the nucleolus following $\text{in vivo}$ administration of this drug.     

A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA-DRB-genes

Electrophoretic mobility shift assays reveal that HeLa neuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)_n(ga)_m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA bindinlg capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into head sensitive, water soluble and temporary stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase 1 footpoint analyses yield four different protein binding regions only on the (gt)_n(ga)_m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5′ of the (gt)_n part. Hence at lealst two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)_n(ga)_m-containing strand intron 2 in HLA-DRB genes     

The seed proteins of chick pea: Comparative studies ofCicer arietinum,C. reticulatum andC. echinospermum (Leguminosae)

Seed proteins fromCicer arietinum L.,C. reticulatum Ladiz. andC. echinospermum Davis were extracted and separated into water soluble (albumin) and water insoluble (globulin) fractions. These were analysed using three polyacrylamide gel systems: uniform pore slab gels, gradient gels and SDS disc gels. For all three species, albumins constitute just over one-third of total protein. Minor differences in the composition of this fraction were observed. Within the globulin fraction, seven disulphide-linked polypeptides were found. Four of these resemble the major polypeptide of legumin, consisting of constant small subunit (21,000 daltons) linked to variable large subunit (46,000, 41,000, 39,000 or 36,000 daltons), forming polypeptides of 67,000 (I), 62,000 (II), 60,000 (III) and 57,000 (IV) daltons respectively. Polypeptide I was prominent in both wild species, but absent fromC. arietinum. Polypeptides II and III were equally prominent inC. arietinum andC. reticulatum. Polypeptide IV was more prominent inC. echinospermum, which was deficient in polypeptide III. Polypeptides V (45,000 daltons) and VI (43,000 daltons), apparently composed of two equal subunits, were present in trace amounts in both wild species, but well represented inC. arietinum Polypeptide VII of 45,000 daltons (31,000 + 14,000) was present in all three species.     

Induction of oviposition by injection of male-derived extracts in two Callosobruchus species

In some insect species, certain substances in the seminal fluid of males induce egg production and laying in females. We determined the effects of male-derived substances on female oviposition behaviour in two Callosobruchus species, C. chinensis and C. maculatus. Aqueous extracts of the accessory gland; testis; and seminal vesicle, including the ejaculatory duct, were prepared. The injection of these extracts into abdomen of females induced oviposition in both species. Oviposition was induced by the testis and seminal vesicle extracts in C. chinensis and by the accessory gland extracts in C. maculatus. The extracts were separated into three fractions by ultrafiltration: fractions I, molecular weight (MW) <3 kDa; fraction II, 3-14 kDa; and fraction III, >14 kDa. Fraction III induced oviposition in both species. These results suggest that in these two species, the substances that induce oviposition have similar MW but are present in different organs. Oviposition was induced by high-MW (>14 kDa) substances in the testis and seminal vesicle in C. chinensis, and by high-MW substances in accessory gland in C. maculatus. Here, we have discussed the relationship between oviposition and the abovementioned male-derived substances.     

Studies of Sulfate Utilization by Algae. 7. In vivo Metabolism of Thiosulfate by Chlorella

Chlorella pyrenoidosa Chick (Emerson strain 3) utilizes thiosulfate for growth as effectively as sulfate, and more effectively than a variety of organic sulfur compounds containing sulfur in various oxidation states. Thiosulfates, differentially labeled with ³⁵S in either the SH— or SO₃ — sulfur moieties, were used to follow the incorporation of thiosulfate-sulfur into constituents of the insoluble fraction and of the soluble pools. Labeled sulfate was also used for purposes of comparison. Label from both sulfur atoms of thiosulfate and from sulfate is incorporated into the cysteine, homocysteine, and glutathione of the soluble pools, and into the methionine and cystine of protein in the insoluble fraction. Label from SO₃-sulfur of thiosulfate is incorporated more slowly into protein methionine and cystine than label from the SH-sulfur. Moreover, the SO₃-sulfur of thiosulfate is recovered largely as sulfate in both the soluble pools and the insoluble fraction, while only a trace of SH-sulfur is recovered as sulfate in either case. Consistent with this, the metabolism of the SO₃-sulfur of thiosulfate more closely resembles the metabolism of sulfate. Thus it would appear that exogenous thiosulfate undergoes early dismutation in which the SO₃-sulfur is preferentially oxidized, and the SH-sulfur is preferentially incorporated in a reduced state. These results are discussed in relation to the conversion of sulfate to thiosulfate by cell-free extracts of Chlorella previously described.     

Quantitative measurements of fibroin messenger RNA synthesis in the posterior silk gland of normal and mutant Bombyx mori

The amount of newly synthesized and accumulated fibroin messenger RNA has been measured quantitatively at various stages of posterior silk gland development in Bombyx mori. The two-step method involves fractionation on a Bio-Gel column which excludes the large mRNA, followed by RNAase T₁ digestion, and fractionation of the oligonucleotides on DEAE-Sephadex. Larvae in the feeding stages of the third and fourth instar synthesize and accumulate fibroin mRNA to about 2% of cellular RNA; this corresponds to 0.2 and 2 μg per pair of posterior glands in the third and fourth instars, respectively. More than 70% of this mRNA is degraded in vivo during the third and fourth moulting stages. Fibroin mRNA synthesis resumes again within the first 24 hours of the fifth instar; the mRNA accumulates and predominates over other DNA-like RNAs as the stage proceeds until finally it comprises about 3.5% of cellular RNA in a mature larva (170 μg per pair of posterior glands). These results indicate that more than 99% of the fibroin mRNA detected in the fifth instar is synthesized during this stage.Four spontaneous mutants of B. mori which synthesize very low levels of fibroin have been analyzed for their RNA content in the middle fifth instar. The total cellular RNA of the posterior gland is reduced to 4 to 7% of normal. Fibroin mRNA is more severely reduced to 1% of normal. In three heterozygotes, which have mutant phenotypes with respect to fibroin production, only slight increases of total cellular RNA and fibroin mRNA were observed. Thus, the primary biochemical lesion in these mutants is still unknown.The presumed ancestor to B. mori, the wild silkworm B. mandarina, was also analyzed for its fibroin mRNA. The mRNA isolated from fifth instar larvae of B. mandarina is indistinguishable from that of B. mori with respect to its nucleotide sequence, molecular weight and fraction of total cellular RNA.     

Carbohydrate Metabolism During Ascospore Development in Yeast

Carbohydrate metabolism, under sporulation conditions, was compared in sporulating and non-sporulating diploids of Saccharomyces cerevisiae. Total carbohydrate was fractionated into trehalose, glycogen, mannan, and an alkali-insoluble fraction composed of glucan and insoluble glycogen. The behavior of three fractions was essentially the same in both sporulating and non-sporulating strains; trehalose, mannan, and the insoluble fraction were all synthesized to about the same extent regardless of a strain's ability to undergo meiosis or sporulation. In contrast, aspects of soluble glycogen metabolism depended on sporulation. Although glycogen synthesis took place in both sporulating and non-sporulating strains, only sporulating strains exhibited a period of glycogen degradation, which coincided with the final maturation of ascospores. We also determined the carbohydrate composition of spores isolated from mature asci. Spores contained all components present in vegetative cells, but in different proportions. In cells, the most abundant carbohydrate was mannan, followed by glycogen, then trehalose, and finally the alkali-insoluble fraction; in spores, trehalose was most abundant, followed by the alkali-insoluble fraction, glycogen, and mannan in that order.     

ANTIBIOTIC SUBSTANCES FROM A STRAIN OF BACILLUS SUBTILIS

SUMMARY: Strain B₇ of B. subtilis , isolated from soil collected from the gardens of the Bose Institute, Calcutta, was found to elaborate at least four different antibiotic substances. Fraction I was a brick red amorphous powder with certain resemblances to the bacillomycin group of antibiotics, soluble in alcohols, pyridine, etc., but insoluble in ether, water and acids. It had an ultraviolet absorption maximum at 276 mμ, melted with decomposition at 311°, and was a polypeptide positive to ninhydrin. On hydrolysis 12 amino acids were found. Fraction II was a white amorphous powder soluble in alcohols and ether but insoluble in water and petroleum ether. It melted at 158–162° and was a ninhydrin positive polypeptide composed of 7 amino acids. Fraction III was a yellow amorphous powder soluble in water and acetone, less soluble in ether and practically insoluble in chloroform. It was not a polypeptide. Fraction IV was a yellowish white amorphous powder with solubility characteristics similar to those of fractions I and II. It was a polypeptide composed of 10 amino acids.     

Acetate Metabolism by an Obligate Phototrophic Strain of Pandorina morum1

SYNOPSIS. Acetate metabolism was studied in 2 strains of the green alga Pandorina morum. Both strains were capable of mixotrophic growth in the light, but only one strain was capable of heterotrophic growth in the dark. ¹⁴C-2-acetate uptake by both strains was studied in the light and dark, in the presence and absence of CO₂ and 3(3,4-dichlorophenyl)-1,1-dimethylurea (10^?5M). The distribution of radioactivity incorporated into the insoluble, aqueous and chloroform soluble fractions of the cells was determined. The strain incapable of heterotrophic growth in the dark was found to incorporate very little acetate in the dark, and its ability to incorporate acetate into the insoluble fraction was severely limited under all conditions. Incorporation into the aqueous and chloroform-soluble fractions in the light was similar in both strains. The reduced incorporation into the insoluble fraction was almost totally the result of limited incorporation of acetate into polysaccharides by the obligate phototrophic strain.     

Purification and characterization of the major storage proteins of Phaseolus vulgaris seeds,and their intracellular and cotyledonary distribution

Several extraction and fractionation procedures have been employed to isolate the major storage proteins of mature seeds of Phaseolus vulgaris cv. “Seafarer”; three proteins which were soluble at pH 4,7, and one that was insoluble at that pH were identified. The characteristic subunits of the three pH 4.7 soluble proteins had MW's 50000 and 47000, 32000, and 23000 respectively; those of the pH 4.7 insoluble fractions had MW 60000 and 20000. Amino acid compositions, N-terminal amino acid residues and the presence of carbohydrate in these proteins have been determined. All these proteins occurred in the protein body fraction and their relative amounts were different in the outer and central parts of the cotyledons.     

Interchain heterogeneity in 2-hydroxyethylglutamine-l-valine random copolymers

Solid state circular dichroism (c.d.) and infrared (i.r.) studies of water soluble and insoluble fractions of poly(hydroxyethylglutamine-valine) random copolymers, prepared from parent γ-benzyl l-glutamate valine copolymers, show that interchain conformational heterogeneity with interchain compositional heterogeneity is present when the respective N-carboxyanhydrides are copolymerized in dioxan or benzene/methylene chloride. Use of previously determined reactivity ratios for the aforementioned copolymer systems permits the determination of the variation of the average copolymer composition, $\text{f}$_G, with conversion. The experimentally determined average copolymer composition.$\text{f}$_G for the use of the respective reactivity ratios and the copolymer hydroxyethylglutamine, valine are predicted by the use of the respective reactivity ratios and the copolymer composition equation. As the valine content of the copolymer chains in the fractions increases, the expected increase in β-sheet contribution is seen. Comparison of the experimentally determined solid state c.d. spectra with Greenfield and Fasman's computer generated c.d. spectra for varying amounts of α-helix, β-sheet and random structures, shows that the water insoluble fractions with their increased valine contents have a greater contribution of β-sheet structure than the respective soluble fractions.