Soluble and Insoluble Protein Patterns during Induction of Freezing Tolerance in Black Locust Seedlings

Protein metabolism plays a major role in the development of freezing tolerance in plants. Soluble and insoluble protein concentrations were followed during induction of freezing tolerance in black locust (Robinia pseudoacacia L.) stem tissues. Soluble proteins were fractionated using two-dimensional polyacrylamide gel electrophoresis and examined for the presence of glycoprotein fractions. Soluble protein concentration remained relatively constant during early stages of induction of freezing tolerance but increased significantly during later stages, while insoluble protein concentration remained relatively constant throughout induction. A new soluble protein component appeared during later stages of induction and was identified as a glycoprotein. Some glycoproteins are known to have a high water-binding capacity, which could play a role in intracellular resistance to ice formation during development of freezing tolerance.     

Quantitative and qualitative variations in the glycoproteins in the chick embryo brain

Sugar content was examined in soluble and insoluble glycoproteins extracted from the chick embryo brain at different developmental stages. The content of hexosamines and uronic acids in the soluble fraction is higher during the whole period examined. The difference between the two fractions reaches a maximum at the 15th day. The insoluble fraction shows the highest content of sialic acid and fucose in comparison with the soluble one, especially toward hatching. The sialic acid/fucose ratio shows a different pattern in the two fractions examined, particularly in the soluble glycoproteins. The patterns of sialic acid and fucose indicate that quantitative and qualitative developmental changes occur in the soluble and insoluble glycoproteins. All sugars examined show significant changes on the 15th day, suggesting that this stage may represent a critical period in the development of the chick embryo brain.     

Soluble invertase expression is an early target of drought stress during the critical,abortion-sensitive phase of young ovary development in maize

To distinguish their roles in early kernel development and stress, expression of soluble (Ivr2) and insoluble (Incw2) acid invertases was analyzed in young ovaries of maize (Zea mays) from 6 d before (-6 d) to 7 d after pollination (+7 d) and in response to perturbation by drought stress treatments. The Ivr2 soluble invertase mRNA was more abundant than the Incw2 mRNA throughout pre- and early post-pollination development (peaking at +3 d). In contrast, Incw2 mRNAs increased only after pollination. Drought repression of the Ivr2 soluble invertase also preceded changes in Incw2, with soluble activity responding before pollination (-4 d). Distinct profiles of Ivr2 and Incw2 mRNAs correlated with respective enzyme activities and indicated separate roles for these invertases during ovary development and stress. In addition, the drought-induced decrease and developmental changes of ovary hexose to sucrose ratio correlated with activity of soluble but not insoluble invertase. Ovary abscisic acid levels were increased by severe drought only at -6 d and did not appear to directly affect Ivr2 expression. In situ analysis showed localized activity and Ivr2 mRNA for soluble invertase at sites of phloem-unloading and expanding maternal tissues (greatest in terminal vascular zones and nearby cells of pericarp, pedicel, and basal nucellus). This early pattern of maternal invertase localization is clearly distinct from the well-characterized association of insoluble invertase with the basal endosperm later in development. This localization, the shifts in endogenous hexose to sucrose environment, and the distinct timing of soluble and insoluble invertase expression during development and stress collectively indicate a key role and critical sensitivity of the Ivr2 soluble invertase gene during the early, abortion-susceptible phase of development.     

Lymphocyte cytotoxicity in x-irradiation-induced rat small bowel adenocarcinoma. III. Blocking by 3 M KCL extract

Hypertonic salt extracts (3 M KCl) of x-irradiation-induced Holtzman rat small bowel adenocarcinomas blocked the in vitro destruction of allogeneic cultured cells of this malignancy by sensitized lymphoid cells obtained from tumor-bearing animals. The protective effect were mediated by a blocking action at both the effector and the target cell level. The extracts were separated into 50% ammonium sulfate soluble and insoluble fractions with the soluble fraction being more effective in blocking the cytotoxic responses through interaction with the lymphoid cells whereas the insoluble one had a greater effect upon tumor target cells. Associated with both fractions was the oncofetal glycoprotein previously identified with the cellular membrane of this x-ray-induced malignancy. Immunoglobulins were identified with insoluble fraction; some were able to bind the oncofetal protein, thus clasifying it as a fetal antigen. The protective effects of the soluble fraction and this neoantigen were found to be citric acid labile, whereas the effects due to the insoluble fraction were unchanged.     

Generation of the heterodimeric precursor GP3 of the Chlamydomonas cell wall

The cell wall of the unicellular green alga Chlamydomonas reinhardtii exclusively consists of hydroxyproline‐containing glycoproteins. Protein chemical analysis of its polypeptide constituents was hindered by their cross‐linking via peroxidase‐catalysed intermolecular isodityrosine formation and transaminase‐dependent processes. To overcome this problem, we have identified putative soluble precursors using polyclonal antibodies raised against deglycosylation products of the highly purified insoluble wall fraction and analysed their amino acid sequence. The occurrence of the corresponding polypeptide in the insoluble glycoprotein framework was finally probed by epitope mapping of the polyclonal antibodies using overlapping scan peptides which, together, cover the whole amino acid sequence of the putative precursor. As a control, peptide fragments released from the insoluble wall fraction by trypsin treatment were analysed by mass spectroscopy. By this approach, the heterodimeric, chaotrope‐soluble glycoprotein GP3 proved to be a constituent of the insoluble extracellular matrix of Chlamydomonas reinhardtii. Furthermore, we have shown that the polypeptide backbones of both GP3 subunits are encoded by the same gene and differ by a C‐terminal truncation in the case of GP3A.     

Fluctuation in free and conjugated polyamines in Scots pine seedlings after changes in temperature and daylength

Free, soluble and insoluble conjugated polyamines from the needles, roots and stem of five month old Scots pine (Pinus sylvestris L.) seedlings inoculated with Suillus variegatus (Fr.) O. Kuntze and seedlings without inoculation were analysed during decrease in daylength and temperature. Temporary changes in free, soluble and insoluble conjugated polyamine pools caused by a decrease in daylength or temperature were observed. Inoculation of pine seedlings affected significantly the polyamine levels of five month old pine seedlings. The roots of inoculated seedlings contained significantly higher levels of free and soluble conjugated purtrescine and free, soluble conjugated and insoluble conjugated spermidine than the roots of noninoculated seedlings. The needles of inoculated seedlings contained significatly higher concentrations of free putrescine and soluble conjugated spermidine but lower amount of free spermine than the needles of noninoculated seedlings. The stems of inoculated seedlings contained higher concentrations of free putrescine but lower amounts of insoluble conjugated spermine. Changes in polyamine levels in noninoculated seedlings were observed after shortening of the daylength, whereas in inoculated ones changes were induced mainly by the decrease in temperature. The possible role of polyamines in the initial stage of cold hardening process is discussed.     

The biosynthesis of nicotinamide adenine dinucleotide during early stages of frog embryonic development of haploid and diploid embryos.

1. Concentration of NAD during embryonic development of haploid and diploid embryos of frog was followed. NAD content in haploid embryonic forms is twice that in diploid embryos. 2. The variation of the NMN adenylyltransferase activity in the oocytes and during the first states of embryonic development as surveyed in the nuclear soluble fraction and the nuclear insoluble fraction (chromatin). 3. The enzyme activity in the soluble fraction is low during embryonic development and shows higher values in haploid embryos. 4. In the nonfertilized mature oocytes, the NMN adenylyltransferase activity is sixfold higher in the insoluble chromatin fraction than in the soluble fraction. 5. The evolution of the NMN adenylyltransferase in the insoluble chromatin fraction also shows higher values in haploid embryos, as compared with diploid forms.     

Peptic erosion of gastric mucus in the rat

1. The effect of pepsin on the loss of mucus glycoprotein from the gastric epithelial mucus layer was studied in the rat. 2. Pepsin was instilled into the gastric lumen, and luminal contents were subsequently assayed. 3. Glycoprotein loss increased with luminal pepsin, up to a concentration of 1 mg pepsin/ml. 4. Luminal glycoprotein had a molecular size distribution intermediate between subunit, and native mucus glycoprotein of the epithelial mucus layer. 5. Incubation of gastric epithelial scrapings with pepsin demonstrated that insoluble, native mucus glycoprotein was rapidly degraded to soluble glycoprotein of similar molecular size distribution to that found in vivo in the lumen.     

The extracellular domain of immunodeficiency virus gp41 protein: expression in Escherichia coli, purification, and crystallization.

The env gene of SIV and HIV-1 encodes a single glycoprotein gp 160, which is processed to give a noncovalent complex of the soluble glycoprotein gp120 and the transmembrane glycoprotein gp41. The extracellular region (ectodomain), minus the N-terminal fusion peptide, of gp41 from HIV-1 (residues 27-154) and SIV (residues 27-149) have been expressed in Escherichia coli. These insoluble proteins were solubilized and subjected to a simple purification and folding scheme, which results in high yields of soluble protein. Purified proteins have a trimeric subunit composition and high alpha-helical content, consistent with the predicted coil-coil structure. SIV gp41 containing a double cysteine mutation was crystallized. The crystals are suitable for X-ray structure determination and, preliminary analysis, together with additional biochemical evidence, indicates that the gp41 trimer is arranged as a parallel bundle with threefold symmetry.     

Soluble compound electron microscope (EM) autoradiography: a resolution source to test redistribution of soluble tritiated compounds during processing

The development of a resolution source that can be labeled with either a soluble or insoluble tritiated compound, and of a method for applying a dry, uniform monolayer of emulsion is reported. Influences due to redistribution of the soluble isotope during emulsion coating were measured by comparing the grain density distributions around the resolution source for soluble tritiated proline (3H-PRO) with that obtained for cross-linked tritiated bovine serum albumin (3H-BSA). The grain density distributions resulting from a standard method of emulsion application (partly gelled/loop method) are compared to that obtained from a dry stripping film. It was found that only the dry stripping film gave a grain distribution which was statistically not different for the soluble and insoluble specimens.     

The composition and protein metabolism in the immature rabbit intervertebral disc

The nucleus pulposus (NP) and annulus fibrosus (AF) of immature rabbit intervertebral discs (IVD) have been subjected to the dissociative extraction procedure of Sajdera and Hascall (1969). The soluble, insoluble and unextracted fractions so obtained were analysed for total nitrogen, collagen, tyrosine, uronic acid, hexosamine and sialic acid content. A high proportion of non-collagenous protein, hexose and sialic acid in the NP insoluble fraction suggests the presence of glycopeptides associated with collagen and/or proteoglycans. The levels of proteoglycan in the soluble NP and AF fraction are similar. Immature (soluble) collagen, however, resides largely in the AF region. The metabolism of rabbit IVD protein components was also investigated both chemically and by autoradiography. L-Tyrosine-3,5-H3 was administered intraperitoneally (3 mc/kg) to 4 week-old rabbits. Animals were sacrificed at various time intervals and the harvested tissues extracted as before and lumbar discs collected. The levels of L-Tyrosine-3,5-H3 in the NP and AF insoluble and soluble fractions were determined using a tritium scintillation counting procedure and localisation by autoradiography. Pronounced extracellular activity of proteoglycan and glycoprotein is not evident before 24 hours. Soluble collagen, however, is synthesized and dispersed within 4 hours of isotope administration.     

The effect of plant cytokinin hormones on the production of ethylene, nitric oxide, and protein nitrotyrosine in ageing tobacco leaves

Transgenic plants with genetically increased or decreased levels of cytokinins were used to investigate the effect of cytokinin level on the production of ethylene, a plant hormone with suggested role in senescence, and the production of nitric oxide, potentially important signalling and regulatory molecule. The production of these gases was followed during the course of leaf development and senescence. The production of ethylene and nitric oxide is under genetic control of genes other than those involved in regulation of senescence. The difference in basic ethylene and NO levels in different tobacco cultivars was higher than their changes in senescence. The results of this study did not indicate a direct link between ethylene production and cytokinin levels. However, there was a decreased production of NO in senescent leaves. Low cytokinins level was associated with increased NO production during leaf development. Protein nitrotyrosine proved to be a better indicator of the reactive nitrogen species than measuring of the NO production. Higher nitrotyrosine concentrations were found in insoluble proteins than in the soluble ones, pointing to membrane proteins as the primary targets of the reactive nitrogen species. In plants with elevated cytokinin levels the content of nitrated proteins decreased both in soluble and insoluble fractions. This finding indicates an antioxidative function of cytokinins against reactive nitrogen species.     

The effect of ascorbic acid on the nature and production of collagen and elastin by rat smooth-muscle cells.

1. The effects of various concentrations of ascorbic acid on the quality and quantity of the insoluble extracellular matrices produced by two strains of cultured rat smooth-muscle cells were studied. 2. Ascorbic acid was necessary for the appearance of insoluble collagen in the extracellular matrix. 3. Secretion of soluble collagen continued in the absence of ascorbic acid, but this soluble collagen was markedly underhydroxylated. 4. The amount of insoluble collagen present in the matrix was directly related to the ascorbic acid concentration. 5. The insoluble collagen that appeared in the matrix under conditions where ascorbic acid was limiting was no more than 7% underhydroxylated. 6. In contrast, the amount of insoluble elastin produced was inversely proportional to the ascorbic acid concentration. 7. The elastin produced in the absence of ascorbic acid had the expected amino acid composition, but hydroxyproline was absent. 8. The hydroxyproline content of elastin was also directly dependent on the ascorbic acid concentration. 9. Ascorbic acid had variable effects on the quantity of glycoprotein(s) present in the matrix. 10. The appearance of insoluble collagen in the extracellular matrices produced by cultured human fibroblasts and calf endothelial cells was also completely dependent on the presence of ascorbic acid.     

Properties of catalytic,linker and chitin-binding domains of insect chitinase

Manduca sexta (tobacco hornworm) chitinase is a glycoprotein that consists of an N-terminal catalytic domain, a Ser/Thr-rich linker region, and a C-terminal chitin-binding domain. To delineate the properties of these domains, we have generated truncated forms of chitinase, which were expressed in insect cells using baculovirus vectors. Three additional recombinant proteins composed of the catalytic domain fused with one or two insect or plant chitin-binding domains (CBDs) were also generated and characterized. The catalytic and chitin-binding activities are independent of each other because each activity is functional separately. When attached to the catalytic domain, the CBD enhanced activity toward the insoluble polymer but not the soluble chitin oligosaccharide primarily through an effect on the Km for the former substrate. The linker region, which connects the two domains, facilitates secretion from the cell and helps to stabilize the enzyme in the presence of gut proteolytic enzymes. The linker region is extensively modified by O-glycosylation and the catalytic domain is moderately N-glycosylated. Immunological studies indicated that the linker region, along with elements of the CBD, is a major immunogenic epitope. The results support the hypothesis that the domain structure of insect chitinase evolved for efficient degradation of the insoluble polysaccharide to soluble oligosaccharides during the molting process.     

Characterisation of a thermostable alpha-amylase from Bacillus brevis.

Biochemical characterization of a novel heat-stable alpha-amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevis alpha-amylase acted via endo-attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The alpha-amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.     

Changes in Cell Wall Polysaccharides of Green Bean Pods during Development

The changes in cell wall polysaccharides and selected cell wall-modifying enzymes were studied during the development of green bean (Phaseolus vulgaris L.) pods. An overall increase of cell wall material on a dry-weight basis was observed during pod development. Major changes were detected in the pectic polymers. Young, exponentially growing cell walls contained large amounts of neutral, sugar-rich pectic polymers (rhamnogalacturonan), which were water insoluble and relatively tightly connected to the cell wall. During elongation, more galactose-rich pectic polymers were deposited into the cell wall. In addition, the level of branched rhamnogalacturonan remained constant, while the level of linear homogalacturonan steadily increased. During maturation of the pods, galactose-rich pectic polymers were degraded, while the accumulation of soluble homogalacturonan continued. During senescence there was an increase in the amount of ionically complexed pectins, mainly at the expense of freely soluble pectins. The most abundant of the enzymes tested for was pectin methylesterase. Peroxidase, beta-galactosidase, and alpha-arabinosidase were also detected in appreciable amounts. Polygalacturonase was detected only in very small amounts throughout development. The relationship between endogenous enzyme levels and the properties of cell wall polymers is discussed with respect to cell wall synthesis and degradation.     

Alterations in trehalase solubility during development in the cellular slime mould Dictyostelium discoideum

Previous studies have indicated that during development in the slime mould Dictyostelium discoideum, compartmentation of the isoenzymes of trehalase (alpha, alpha'-trehalose 1-D-glucohydrolase, (EC 3.2.1.28) occurs between the extracellular and intracellular environments. The compartmentation of trehalase between soluble and particulate cell fractions was examined in this work. The trehalase present in crude homogenates prepared during the first 12 h of development was completely soluble. Starting at about the pseudoplasmodial stage (i.e. the 14th hour of development), trehalase activity became associated with insoluble cellular material and this increased to a maximal value in homogenates from mature sorocarps, where 50% of the activity was insoluble. Spore cells accounted for only 2 to 3% of the trehalase associated with mature sorocarps, with the remaining 97% being localized in stalk cell material. Although trehalase recovered from spores was completely soluble, more than half of that from the stalk was recovered in the buffer-insoluble pellet fraction.     

Quantitative determination of calcium oxalate and oxalate in developing seeds of soybean (Leguminosae)

Developing soybean seeds accumulate very large amounts of both soluble oxalate and insoluble crystalline calcium (Ca) oxalate. Use of two methods of detection for the determination of total, soluble, and insoluble oxalate revealed that at +16 d postfertilization, the seeds were 24% dry mass of oxalate, and three-fourths of this oxalate (18%) was bound Ca oxalate. During later seed development, the dry mass of oxalate decreased. Crystals were isolated from the seeds, and X-ray diffraction and polarizing microscopy identified them as Ca oxalate monohydrate. These crystals were a mixture of kinked and straight prismatics. Even though certain plant tissues are known to contain significant amounts of oxalate and Ca oxalate during certain periods of growth, the accumulation of oxalate during soybean seed development was surprising and raises interesting questions regarding its function.     

Structural basis of calcification inhibition by alpha 2-HS glycoprotein/fetuin-A. Formation of colloidal calciprotein particles

Genetic evidence from mutant mice suggests that alpha(2)-HS glycoprotein/fetuin-A (Ahsg) is a systemic inhibitor of precipitation of basic calcium phosphate preventing unwanted calcification. Using electron microscopy and dynamic light scattering, we demonstrate that precipitation inhibition by Ahsg is caused by the transient formation of soluble, colloidal spheres, containing Ahsg, calcium, and phosphate. These "calciprotein particles" of 30-150 nm in diameter are initially amorphous and soluble but turn progressively more crystalline and insoluble in a time- and temperature-dependent fashion. Solubilization in Ahsg-containing calciprotein particles provides a novel conceptual framework to explain how insoluble calcium precipitates may be transported and removed in the bodies of mammals. Mutational analysis showed that the basic calcium phosphate precipitation inhibition activity resides in the amino-terminal cystatin-like domain D1 of Ahsg. A structure-function analysis of wild type and mutant forms of cystatin-like domains from Ahsg, full-length fetuin-B, histidine-rich glycoprotein, and kininogen demonstrated that Ahsg domain D1 is most efficient in inhibiting basic calcium phosphate precipitation. The computer-modeled domain structures suggest that a dense array of acidic residues on an extended beta-sheet of the cystatin-like domain Ahsg-D1 mediates efficient inhibition.