Structural studies on the glycoproteins from bovine cervical mucus.

The depolymerization of bovine cervical glycoprotein resulting from cleavage of disulphide bonds. Pronase digestion and both procedures sequentially was assessed by using gel filtration. Cleavage of disulphide bonds followed by Pronase digestion produced more extensive depolymerization than did either treatment alone, and gel filtration of the products resulted in two major peaks of glycosylated material on Sepharose CL-2B and Sepharose 4B. The glycopolypeptides in both peaks had similar sugar and sulphate compositions, but they migrated to different extents on gel electrophoresis. Electrophoretic studies indicated that both glycopolypeptides were derived from the same glycoprotein molecule and not from a mixture of two similar glycoproteins. Pronase digestion of glycoproteins in which the disulphide bonds had been labelled with iodo-[1-14C]acetamide revealed that most of the cysteine residues were situated in regions susceptible to Pronase. The results show the presence of two types of structural regions in bovine cervical glycoprotein, namely 'naked' peptide or non-glycosylated regions and glycopolypeptide subunit regions in which glycopolypeptides of two different sizes predominate. Comparison of the cervical glycoproteins isolated from mucus secreted during oestrus and pregnancy, by the methods outlined above, did not reveal any structural differences in the glycoproteins to explain the different physical properties of the mucus secreted under these conditions.     

Changing distribution patterns of α-glucosidase isoenzymes in extracts of antennal segments and in the haemolymph of the cotton stainer Dysdercus intermedius dist.

• 1.1. The α-glucosidase (E.C. 3.2.1.20) activities in extracts of the 2nd and 4th antennal segments (AS) and in the haemolymph of the cotton stainer were quantitatively examined by use of trehalose, p-nitrophenyl-α-glucoside, sucrose, turanose, maltose, isomaltose and palatinose as substrates.
• 2.2. Three peaks of activity were isolated by gel filtration from the extracts of both AS, but only two peaks from the haemolymph.
• 3.3. The peaks III of both AS differ in their substrate specificities from those of the peaks I and II.
• 4.4. It was evident from experiments, which used fractional extraction and variation of pH in addition to gel filtration that more than a single enzyme contributed to the formation of each peak.
• 5.5. Results are discussed with reference to the hypothesized presence of sugar reception processes in insect gustatory cells.

     

Sucrose synthase in rice plants : growth-associated changes in tissue specific distributions

Different parts of the rice (Oryza sativa L.) plant at different growth stages were analyzed for sucrose synthase (SS) by enzyme activity assay and enzyme-linked immunosorbent assay directly on the extracts or on the eluates from a gel filtration column. On a dry matter basis, the amount of soluble protein and SS activity decreased significantly, but the amount of enzyme protein changed little in growing leaves. In the grain, the SS activity was the highest at the early ripening stage and decreased later, but the amount of SS protein increased with the increase in maturity. In the root, a low activity of SS was detectable only in the tillering but not in other stages. Immunoblotting of SS protein extracted from different parts of rice showed two bands. Elution patterns of crude extracts from a gel filtration column showed the presence of several types of SS protein. Among them, two to three types with larger elution volumes had the SS activity but others with smaller elution volumes (considered as the aggregated forms) had no activity. The SS purified from different parts of the plant showed similar but distinctly different electrophoretic mobilities in a native gel. It has been concluded that different isozymes are expressed in different tissues at different growth stages.     

Purification and characterization of a lectin from wild sunflower (Helianthus tuberosus L.) tubers

A lectin (HTTL) was isolated from Helianthus tuberosus L. (wild sunflower) tubers using ion-exchange chromatography, gel filtration, and affinity chromatography. The lectin agglutinated both untreated and trypsin-treated rabbit erythrocytes and did not agglutinate human blood cells of groups A, B, and O. The gel filtration showed the native molecular mass of 72 kDa and subunit molecular masses of 17 and 18.5 kDa on 12% SDS-PAGE. The lectin activity was inhibited by D-mannose. The tetrameric protein revealed a unique characteristic by forming a broad zone of protein in native PAGE at pH 8.3, which dissociated into seven subunits of varying e/m ratios on acid gel at pH 4.3. These seven bands revealed two polypeptide species of molecular masses 17 and 18.5 kDa on 12% SDS-PAGE, as in the case of the native protein. The result indicated that of the seven subunits, three were homotetramers of 17 kDa, one was a homotetramer of 18.5 kDa, and three were heterotetramers of 17 and 18.5 kDa. The lectin was thermostable with broad pH optima (pH 4-8) and had no requirement for divalent metal cations for its activity. The amino acid composition showed that the lectin contained higher amounts of glycine, alanine, and lysine, but no methionine. The sugar content was estimated to be 5.3% mannose equivalent. The HTTL was mitogenic to mouse spleen (total) cells at 25 microg/ml concentration. The lectin showed characteristics different from those of the earlier reported H. tuberosus tuber lectins and hence opens up a new avenue to investigate the structure-function relationship of lectin in Helianthus species.     

Characterization of solubilized insulin receptors from rat liver microsomes. Existence of two receptor species with different binding properties

Insulin receptors were solubilized from rat liver microsomes by the nonionic detergent Triton X-100. After gel filtration of the extract on Sepharose CL-6B, two insulin-binding species (peak I and peak II) were obtained. The structure and binding properties of both peaks were characterized. Gel filtration yielded Stokes radii of 9.2 nm (peak I) and 8.0 nm (peak II). Both peaks were glycoproteins. At 4 degrees C peak I showed optimal insulin binding at pH 8.0 and high ionic strength. In contrast, peak II had its binding optimum at pH 7.0 and low ionic strength, where peak I binding was minimal. For peak I the change in insulin binding under different conditions of pH and ionic strength was due to a change in receptor affinity only. For peak II an additional change in receptor number was found. Both peaks yielded non-linear Scatchard plots under most of the buffer conditions examined. At their binding optima at 4 degrees C the high affinity dissociation constants were 0.50 nM (peak I) and 0.55 nM (peak II). Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of peak I revealed five receptor bands with Mr 400 000, 365 000, 320 000, 290 000, and 245 000 under non-reducing conditions. For peak II two major receptor bands with Mr 210 000 and 115 000 were found. The peak II receptor bands were also obtained after mild reduction of peak I. After complete reduction both peaks showed one major receptor band with Mr 130 000. The reductive generation of the peak II receptor together with molecular mass estimations suggest that the peak I receptor is the disulfide-linked dimer of the peak II receptor. Thus, Triton extracts from rat liver microsomes contain two receptor species, which are related, but differ considerably in their size and insulin-binding properties.     

Adenosine deaminase activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages

Adenosine deaminase (ADA) undergoes changes in specific activity during in vitro culture of human peripheral blood monocytes and pulmonary alveolar macrophages. Monocyte adenosine deaminase activity increases during the first 3 days of culture; after 3 days the specific activity decreases below the levels observed for freshly isolated cells. In contrast, ADA activity of pulmonary alveolar macrophages increases throughout the 14-day culture period studied. Based on pH optima, starch gel electrophoresis and gel filtration column chromatography, the changes in adenosine deaminase activity in monocyte-macrophage cultures are related to changes in the molecular form of the enzyme. Freshly isolated monocytes contain mainly ES, while at day 14, starch gel and gel filtration experiments demonstrate the appearance of EI. Human pulmonary macrophages contain primarily EI or EL; following several days in culture, there is an increase in the amount of ES present.     

ISOLATION AND CHARACTERIZATION OF BOVINE BRAIN CATHEPSIN D

Bovine brain cathepsin D was purified 1774-fold with a 19% recovery by affinity chromatography on immobilized pepstatin. Approximately 2 mg of enzyme protein were isolated from 150 g (wet weight) of bovine brain. The enzyme eluted from gel filtration as a single peak with a molecular weight of 40,000–42,000. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the predominant band migrated with a molecular weight of 48,000: however, less distinct bands were also present in the molecular weight ranges of 31,000 and 13,000. The isolated enzyme had isoelectric points over a range of pH 5–7 with 3 major peaks occurring at pH 5.6, 6.1, and 6.6. The amino acid composition of brain cathepsin D showed substantial differences from that reported for cathepsin D isolated from bovine spleen. Amino-terminal sequence analysis revealed an Asp-Val-lle sequence by Edman degradation. With hemoglobin as the substrate the enzyme had an apparent K, of 60mM.     

Isolation of two glycolipid transfer proteins from bovine brain: reactivity toward gangliosides and neutral glycosphingolipids

Two glycolipid transfer proteins that catalyze the transfer of gangliosides and neutral glycosphingolipids from phosphatidylcholine vesicles to erythrocyte ghosts have been isolated from calf brain. Purification procedures included differential centrifugation, precipitation at pH 5.1, ammonium sulfate precipitation, and gel filtration on Sephadex G-50 and G-75. The final stage employed fast protein liquid chromatography (Mono S), producing two peaks of activity. Apparent purity of the major peak (TP I) was approximately 85-90%, as judged by sodium dodecyl sulfate/urea-polyacrylamide gel electrophoresis. That of the minor fraction (TP II) was less. The major band of both fractions had a molecular mass of approximately 20,000 daltons. Both proteins catalyzed the transfer of ganglioside GM1 as well as asialo-GM1, but transfer protein I was more effective with di- and trisialogangliosides. Transfer protein II appeared to be somewhat more specific for neutral glycolipids in that GA1 was transferred more rapidly than any of the gangliosides; however, lactosylceramide transfer was relatively slow. Neither protein catalyzed transfer of phosphatidylcholine.     

The Characterization of Equine Prealbumin (Pr) Proteins by Antigen-Antibody Crossed Electrophoresis

The characterization of equine prealbumin (Pr) proteins by antigen-antibody crossed electrophoresis. Acta vet. scand. 1979, 20, 180–190. — Selected equine Pr phenotypes from a total of 55 horses of mixed breeds were investigated. The horse sera were subjected to acid starch gel electrophoresis at pH 4.8, followed by right angle electrophoresis in agarose gels containing rabbit-produced anti-Pr protein. This technique gives peaks in the agarose gels corresponding to the Pr zones in acid gels. The investigation revealed patterns of the Pr protein which were more complex than those seen when using ordinary acid starch gel electrophoresis. The phenotypes FF, II and LL showed a total of eight peaks, each with three main peaks in the front. Ahead of these, the Pr II and Pr LL phenotypes each had a fourth small peak. The basic fast pattern for these two phenotypes therefore consisted of four bands. The Pr WW and Pr SS showed a similar picture as regards the fast moving peaks. The Pr NN type appeared with two peaks in the front, one small and one large and with two slow moving ones. The Pr UU type had four peaks, but only in the area of the main Pr U band in acid gels. Four heterozygous Pr phenotypes appeared as a combination of the corresponding homozygous phenotypes, the number and height of the peaks depending on positions and overlappings of these in the respective homozygotes. Thus the Pr FW phenotype showed a total of 10 peaks. The effect of variations in pH of the starch gel buffer was studied. The Pr NN and Pr FF phenotypes were run at pH 4.8, 5.0, 5.2 and 5.4. With increasing pH, the slow moving peaks weakened and moved closer to the fast ones. At pH 5.4 only one large fast moving peak remained.     

Pepsinogen B: the zymogen of pepsin B

1. Pepsinogen B, the precursor of pepsin B, has been isolated by ion-exchange chromatography and gel filtration from neutral extracts of pig gastric mucosa. The material possesses potential activity against acetyl-l-phenylalanyl-l-di-iodotyrosine and against gelatin but has little, if any, potential activity against haemoglobin. 2. The material appears homogeneous in the ultracentrifuge, but on gel filtration and on electrophoresis in starch gel it is shown to be contaminated with a small amount of material having potential activity against haemoglobin. On electrophoresis in starch gel also the material is shown to contain about equal amounts of two major components, both of which have potential activity against the synthetic substrate. Pepsin B has also been shown to contain two active components by electrophoresis under the same conditions. 3. The zymogen is similar to pepsinogen and pepsinogen C in its molecular weight and general physico-chemical properties, but differs from these zymogens in the nature of its N-terminal residues. It is possible that one of the components contains 1 mole of bound phosphate/mole. 4. The material is activated rapidly at pH2 and more slowly at pH4. At both pH values the kinetics of the activation reaction are complex.     

Studies on the fractionation and isolation of the neurotoxins in Echis carinatus venom

Summary The venom of the saw-scaled viper. Echis carinatus venom was fractionated into twelve fractions by gel filtration on Sephadex G-100. The electrophoretic pattern of the whole venom and of the toxic fraction, F₁ separated by gel filtration was studied at pH 8.6 and pH 4.2. Two different neurotoxins were isolated by paper strip electrophoresis. One produced only paralysis in the hind limbs while the other produced both paralysis in the hind limbs of the animal as well as general un-co-ordinated movements.     

Improved preparation of the integral membrane proteins of human red cells, with special reference to the glucose transporter

Human red cell membranes were isolated and partially stripped of peripheral proteins by gel filtration of hemolysates on a Sepharose CL-4B column at pH 8 connected in tandem to a Sepharose CL-6B column at pH 10.5. The eluted material was washed by centrifugations, once at pH 10.5 and twice at pH 12. In this way, water-soluble proteins and peripheral membrane proteins were thoroughly removed, and 0.2 g of integral membrane proteins could be prepared within 10 h from 0.2 litre of red cells. The exposure to high pH did not lower the D-glucose transport activity, and electrophoretically pure glucose transport protein could be isolated from this preparation. Gel filtration in sodium dodecyl sulfate separated the integral membrane components into four fractions, one of them containing 4.5-material; gel electrophoresis showed about 14 zones and two-dimensional electrophoresis resolved up to 100 mostly minor components, among which the glucose transporter focused around pH 7. However, purified glucose transporter focused around pH 8. Glucose and nucleoside transport proteins were co-purified in active form on DEAE-cellulose and a fraction isolated by adsorption to Mono Q was used for immunization of mice and production of monoclonal antibodies. One hybridoma produced antibodies that reacted with material in the 4.5-region, possibly the glucose transport protein, and not with band 3-material. Upon two-dimensional electrophoresis of integral membrane components that had been solubilized with octyl glucoside the immunoreactive and the silver-stained 4.5-material focused in a broad range from pH 6 to pH 9. A possible explanation for this heterogeneity might be interaction between the glucose and nucleoside transport proteins and negatively charged lipids.     

Expression of N-linked sialyl Le(x) determinants and O-glycans in the carbohydrate moiety of human amniotic fluid transferrin during pregnancy

Transferrin, a glycoprotein involved in iron transport in body fluids, was isolated from amniotic fluid of a hydramniospatient by sequential anion-exchange chromatography and gel filtration. The N-glycans of human amniotic fluid transferrin (hAFT) were enzymatically liberated by PNGase-F digestion, isolated by gel filtration and fractionated by (high-pH) anion-exchange chromatography. After alkaline borohydride treatment of native hAFT, the released O-glycans were isolated by gel filtration and fractionated by anion-exchange chroma-tography. Structure elucidation of 14 N- and 2 O-glycans was performed by 500 or 600 MHz1H-NMR spectroscopy. Besides conventional N-glycans established earlier for human serum transferrin (hST), new (alpha1-3)-fucosylated N- glycans were found, representing sialyl Le(x) elements. Furthermore, as compared to hST, a higher degree of (alpha1-6)-fucosylation and an increase in branching from di- to triantennary compounds has been detected. The presence of O-glycans is demonstrated for the first time in transferrin.      

Plant nucleases. I. Separation and purification of two ribonucleases and one nuclease from corn

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.     

Fractionation of individual, biologically active factor VIII multimers

We have designed an electrophoretic system for the fractionation of individual, biologically active multimers of factor VIII. Human factor VIII, purified by gel filtration on Sepharose CL-2B from plasma cryoprecipitate, was submitted to electrophoresis without SDS on 2.0% polyacrylamide gels in 0.04 M Tris/0.06 M Tes buffer, pH 7.5. Staining with Coomassie blue revealed a series of protein bands. Measurement of electrophoretic mobility showed constant size intervals between adjacent bands. Electrophoresis in a second dimension, in the presence of SDS, resulted in an identical order of mobilities, suggesting that the different migration rates of factor VIII proteins in the first electrophoretic system were size- and not charge-dependent. After electrophoresis in the absence of SDS both factor VIII coagulant and ristocetin cofactor activities as well as factor VIII-related antigen were recovered by elution from gel slices. The distribution of activity peaks resembled that of Coomassie-stained factor VIII proteins found in control gels. We thus demonstrate that an electrophoretic fractionation of factor VIII multimers is possible even at neutral pH where factor VIII activities are retained.     

Isolation and immunochemical characterization of fractions from membranes of Aspergillus fumigatus with protease activity

Two fractions exhibiting acid protease activity (AFPI and AFPII) were isolated by extraction of membrane vesicles of Aspergillus fumigatus with Triton X-100. These two fractions produced single bands in both polyacrylamide and sodium dodecyl sulfate polyacrylamide gel electrophoresis and showed apparent molecular weights of 73,000 and 43,000, respectively. Molecular weights determined by gel filtration in the absence and presence of Triton X-100 and sedimentation velocities in analytical ultracentrifugation indicated hydrophobic characteristics, since both fractions readily aggregated and complexed with Triton X-100; both exhibited elevated enzyme activities in the presence of Triton X-100. Carbohydrate content was 93% for AFPI and 85% for AFPII. The enzymatic fractions demonstrated different pH optima in the acid range as well as different temperature stabilities. Both protease fractions cross reacted in double immunodiffusion, while in crossed immunoelectrophoresis both demonstrated five precipitin peaks, each with similar patterns. AFPI demonstrated two additional precipitin peaks in crossed immunoelectrophoresis. As determined by crossed immunoaffinoelectrophoresis, the protease fractions demonstrated galactose and mannose residues. In biotin-avidin enzyme-linked immunosorbent assay both fractions reacted with allergic bronchopulmonary aspergillosis and aspergilloma sera. It can be concluded that two fractions with protease activity of A. fumigatus reported here may be of significance in Aspergillus-induced diseases.     

Partial purification and some properties of an alkaline cellulase from an alkalophilic Bacillus sp.

A newly isolated Bacillus species, which grew optimally at 30°C and pH 10, produced a carboxymethylcellulase in a medium containing 10 g CM-cellulose/l. The enzyme, when partially purified by gel filtration, had a mass of about 29 kDa as determined by both SDS-PAGE and gel filtration chromatography. It was optimally active at pH 9.5 and 40°C, and was stable from pH 7 to 11 at 4°C for 24 h. The enzyme was stimulated by Ca²⁺ (1mm) but was completely inhibited by Hg²⁺ (1mm). Neither EDTA nor EGTA (10mm) affected the activity.The author is with the Department of Biological Sciences, University of Jordan. PO Box 2686, Amman 11181, Jordan     

Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits

The subunits of phosphorylase kinase are separated and isolated in high yield by gel filtration chromatography in pH 3.3 phosphate buffer containing 8 M urea. Three protein peaks are obtained: the alpha and beta subunits coelute in the first, whereas the gamma and delta subunits are separate peaks. Upon dilution of the denaturant, catalytic activity reappears, associated only with the gamma subunit. As has been previously observed (Kee, S.M., and Graves, D.J. (1986) J. Biol. Chem. 261, 4732-4737), addition of calmodulin dramatically stimulates the reactivation of gamma. Inclusion of increasing amounts of the alpha/beta subunit mixture in the renaturation progressively decreases the activity of the renatured gamma or gamma-calmodulin. This inhibition by alpha/beta is likely due to specific interactions with the gamma subunit because the inhibition is less at pH 8.2 than at pH 6.8 and less when equivalent amounts of phosphorylated alpha/beta subunits are used (both alkaline pH and phosphorylation are known to stimulate the activity of the holoenzyme). These results suggest that the role of either the alpha or beta subunits, or perhaps both, in the nonactivated (alpha 2 beta 2 gamma 2 delta 2)2 complex of phosphorylase kinase is to suppress the activity of the gamma subunit and that activation of the enzyme, by phosphorylation for instance, is due to deinhibition caused by release of this quaternary constraint by alpha and/or beta upon gamma.     

Plant Nucleases. II. Properties of Corn Ribonucleases I and II and Corn Nuclease I

Three enzymes with ribonuclease activity, one of which also had deoxyribonuclease activity, have been isolated and partially purified from corn seeds and seedlings. The purification of Ribonuclease I from mature seed was previously reported. This enzyme has a pH optimum near 5.0, is loosely adsorbed to carboxymethyl-cellulose, and has a molecular weight of 23,000, determined by gel filtration.

Ribonuclease II was isolated from the microsomes of corn roots, and was partially purified by gel filtration. It has a pH optimum plateau from 5.4 to 7.0, and molecular weight of 17,000.

Nuclease I hydrolyzes both RNA and DNA. It was isolated from the large particles of a corn root homogenate and was partially purified on a carboxymethyl-cellulose column. It has a pH optimum at 6.2 and a molecular weight of 31,000.

The relative activities of the 3 enzymes for deoxyribonuclease and at pH 5 and pH 6.2 for ribonuclease may be used to characterize them during purification operations. Assays on homogenates of corn roots, and especially of the root tips, suggested that a fourth enzyme, which possesses deoxyribonuclease activity, is also present.

     

Amino acid activation in mammalian brain. Purification and characterization of tryptophan-activating enzyme from buffalo brain

l-Tryptophan-activating enzyme [l-tryptophan-tRNA ligase (AMP), EC 6.1.1.2] of water-buffalo brain was purified to near homogeneity by heat and pH treatments, ammonium sulphate fractionation, column chromatography on DEAE-cellulose, hydroxyapatite and Amberlite CG-50, and gel filtration on Sephadex G-200. The purified enzyme catalyses tryptophanyl-tRNA formation with yeast tRNA, but not with Escherichia coli tRNA. The enzyme exhibits multiple peaks of activity in Sephadex gel filtration with molecular weights corresponding to 155000, 105000 and 50000. However, only one peak of activity with molecular weight of 155000 can be detected when the enzyme is subjected to gel filtration at high concentration. Disc gel electrophoresis in the presence of sodium dodecyl sulphate reveals a single band with molecular weight of 55000. The activity of the enzyme is concentration dependent. Different K(m) and V(max.) values are obtained at different enzyme concentrations. These data suggest that this enzyme may exist in different quaternary structures, each with its own kinetic constants. The enzyme activity is inhibited by p-chloromercuribenzoate, and is not protected by the presence of the substrates, l-tryptophan, Mg(2+), ATP, in any combination.