DEAE-Sephacel Anion Exchange Chromatography Research Articles

Indole-3-acetic acid UDP-glucosyltransferase from immature seeds of pea is involved in modification of glycoproteins

The glycosylation of auxin is one of mechanisms contributing to hormonal homeostasis. The enzyme UDPG: indole-3-ylacetyl-β-d-glucosyltransferase (IAA glucosyltransferase, IAGlc synthase) catalyzes the reversible reaction: IAA+UDPG↔1-O-IA-glucose+UDP, which is the first step in the biosynthesis of IAA-ester conjugates in monocotyledonous plants. In this study, we report IAA-glucosyltransferase isolated using a biochemical approach from immature seed of pea (Pisum sativum). The enzyme was purified by PEG fractionation, DEAE-Sephacel anion-exchange chromatography and preparative PAGE. LC–MS/MS analysis of tryptic peptides of the enzyme revealed the high identity with maize IAGlc synthase, but lack of homology with other IAA-glucosyltransferases from dicots. Biochemical characterization showed that of several acyl acceptors tested, the enzyme had the highest activity on IAA as the glucosyl acceptor (Km=0.52mM, Vmax=161nmolmin−1, kcat/Km=4.36mMs−1) and lower activity on indole-3-propionic acid and 1-naphthalene acetic acid. Whereas indole-3-butyric acid and indole-3-propionic acid were competitive inhibitors of IAGlc synthase, d-gluconic acid lactone, an inhibitor of β-glucosidase activity, potentiated the enzyme activity at the optimal concentration of 0.3mM. Moreover, we demonstrated that the 1-O-IA-glucose synthesized by IAGlc synthase is the substrate for IAA labeling of glycoproteins from pea seeds indicating a possible role of this enzyme in the covalent modification of a class of proteins by a plant hormone.

Purification and characterization of potato ribonuclease P

Ribonuclease P (RNase P), a ribonucleo-protein endoribonuclease, responsible for 5′ maturation of precursor tDNA, is well characterized in bacteria, yeast and human, but not in plant. Attempt has been made to partially purify and characterize nuclear RNase P from potato. cDNAs encoding two putative protein subunits of potato ribonuclease P (RNase P), StPop5 and StRpp25, were picked from potato EST library based on homology with respective human RNase P protein subunits. Both the cDNAs, 435 bp long StPop5 and 765 bp long StRpp25, were RT-PCR amplified, cloned and sequenced. StPop5 exhibited 46 % nucleotide sequence similarity to the hPop5 sequence. The deduced amino acid sequence of StPop5 had 23 % identity and 35 % similarity to hPop5. Both hRpp25 and StRpp25 had 46 % nucleotide sequence homology, and 17 % identity and 27 % similarity at a length of 271 amino acids. The molecular masses of purified 6× His-tagged recombinant StPop5 and StRpp25 proteins were 18 kDa and 33 kDa respectively. Potato nuclear RNase P was partially purified from leaves employing DEAE-Sephacel anion-exchange chromatography, and from floral buds employing DEAE-Sephacel and HiTrap Q anion-exchange chromatography, ammonium sulfate precipitation and gel filtration chromatography. Immunoprecipitation with polyclonal antisera, raised against recombinant StPop5 and StRpp25, demonstrated association of these two proteins with floral bud RNase P activity but not with leaf RNase P activity.

Isolation and characterization of a new phycoerythrin from the cyanobacterium Synechococcus sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)6 subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.

Purification and Biochemical Characterization of Indole-3-acetyl-aspartic Acid Synthetase from Immature Seeds of Pea (Pisum sativum)

Indole-3-acetic acid (IAA) amidosynthetases catalyzing the ATP-dependent conjugation of IAA and amino acids play an important role in the maintenance of auxin homeostasis in plant cells. A new amidosynthetase, indole-3-acetic acid:l-aspartic acid ligase (IAA-Asp synthetase) involved in IAA-amino acid biosynthesis, was isolated via a biochemical approach from immature seeds of the pea (Pisum sativum L). The enzyme was purified to homogeneity by a three-step procedure, involving PEG 6000 fractionation, DEAE-Sephacel anion-exchange chromatography, and preparative PAGE, and characterized as a 70-kDa monomeric protein by analytical gel filtration and SDS-PAGE. Rabbit antiserum against recombinant AtGH3.5 cross-reacted with the pea IAA-Asp synthetase, and a single immunoreactive polypeptide band was observed at 70 kDa. The purified enzyme had an apparent isoelectric point at pH 4.7, the highest activity at pH 8.2, preferred Mg2+ as a cofactor, and was strongly activated by reducing agents. Similar to known recombinant GH3 enzymes, an IAA-Asp synthetase from pea catalyzes the conjugation of phytohormone acyl substrates to amino acids. The enzyme had the highest synthesizing activity on IAA, followed by 1-NAA, SA, 2,4-D, and IBA, whereas activities on l-Trp, IPA, PAA, (±)JA, and 2-NAA were not significant or not detected. Of 14 amino acids tested, the enzyme had the highest activity on Asp and lower activity on Ala and Lys. Glutamate was found to be a very poor substrate and no conjugating activity was observed on the rest of the amino acids. Steady-state kinetic analysis indicated that IAA and aspartate were preferred substrates for the pea IAA-Asp synthetase. The enzyme exhibited both higher affinities for IAA and Asp (K m = 0.2 and 2.5 mM, respectively) and catalytic efficiencies (k cat/K m = 682,608.7 and 5080 s−1 M−1, respectively) compared with other auxins and amino acids examined. This study describes the first amidosynthetase isolated and purified from plant tissue and provides the foundation for future genetic approaches to explain the role of IAA-Asp in Pisum sativum physiology.

CHARACTERIZATION OF PHYTOCHELATIN-LIKE COMPLEXES FROM FLAX (LINUM USITATISSIMUM) SEED

Proteins were extracted from dehulled and delipidated powder of flaxseed (cv. NorMan), and fractionated by DEAE-Sephacel anion exchange chromatography. Ultraviolet absorbance scans of ion exchange fractions eluting in buffers at pH 8.6 containing 0. 45 M and 0.50M NaCl revealed A 255 >A 280 . Furthermore, the A 255 was reduced upon acidification and restored by adjusting to the original pH, confirming spectral characteristics typical of metal-thiolate complexes. Characteristic circular dichroism spectra and reversible aggregation-dissociation behavior under low and high salt conditions observed for these fractions are also typical of metal binding phytochelatins reported for other plants, while the presence of aromatic amino acids indicated by fluorescence spectra suggests some similarity to nonmetallothionein proteins. Further work is underway to investigate the prevalence of these and other complexes as well as the distribution of metals in different protein fractions of flaxseed, as a function of cultivar and geographical location.

Differential glutathione S-transferase isozyme activities in rice and early watergrass seedlings

Glutathione S-transferase (GST) isozymes were investigated in one-leaf-stage rice (Oryza sativa L. cv. Nipponbare) and early watergrass (Echinochloa oryzicola Vasing) shoots after being induced by treatment with a combination of pretilachlor [2-chloro-2′,6′-diethyl-N-(2-propoxyethyl)acetanilide] and fenclorim (4,6-dichloro-2-phenylpyrimidine) using DEAE-Sephacel anion exchange chromatography. Non-treated plants contained GST isozymes that had activity to the following substrates: three isozymes for l-chloro-2,4-dinitrobenzene (CDNB), six isozymes for pretilachlor and three isozymes for fenclorim in rice shoots; and four isozymes for CDNB, three or four isozymes for pretilachlor and two or three isozymes for fenclorim in early watergrass shoots. Glutathione S-transferase isozyme activities of non-treated plants were higher in rice than in early watergrass, especially in the case of GST activity with fenclorim as a substrate. Pretreatment of rice roots with a combination of pretilachlor and fenclorim increased the activity of the constitutively expressed isozymes that exhibited activity with CDNB, pretilachlor and fenclorim. This pretreatment also caused the appearance of one new GST(fenclorim) isozyme. Pretreatment of early watergrass roots with a combination of pretilachlor and fenclorim produced almost no increase in activity of some constitutively expressed isozymes that exhibited activity to CDNB and fenclorim, although it partly increased the peaks to corresponding to pretilachlor. The induction of GST was higher in rice than that in early watergrass. These results indicated that the isozyme pattern and substrate specificity of GST isozymes in rice were different from those in early watergrass. Furthermore, the selectivity of pretilachlor between rice and early watergrass may be related to different constitutively expressed GST(pretilachlor) isozyme activities and the induction of GST(pretilachlor) isozyme activities in the combination treatment.

Benign hyperplasia of the human prostate is associated with tissue enrichment in chondroitin sulphate of wide size distribution.

Benign prostatic hyperplasia (BPH) involves qualitative and quantitative alterations in extracellular matrix (ECM) components affecting stromal-epithelial interactions. Glycosaminoglycans (GAGs) are polysaccharide components of the ECM whose role in the development of BPH is under investigation. GAGs were extracted from human prostates of normal and BPH origin and were subsequently fractionated through DEAE-sephacel anion exchange chromatography. The isolated GAG fractions were identified through electrophoresis on cellulose acetate membranes and treatment with GAG-degrading enzymes of known specificity. Their size distribution was determined through gradient polyacrylamide gel electrophoresis. Isolated prostatic GAGs included hyaluronic acid (HA), heparan sulphate (HS), and a mixture of dermatan sulphate (DS) and chondroitin sulphate (CS). The CS/DS ratio was significantly higher in hyperplastic as compared to normal prostates. A difference was also observed with respect to the apparent molecular mass of the DS-CS mixture, which reflects the CS enrichment in BPH. GAGs isolated from hyperplastic prostates were more diverse in size as compared to the corresponding glycans from normal prostates. The apparent increase in CS and decrease in DS content in prostates of patients with BPH is in good agreement with the pathological manifestation of increased cell proliferation in hyperplastic prostate tissue, since these glycan molecules have been reported to increase and decrease cell proliferation, respectively. Identification of the responsible enzymes involved in the homeostasis of CS and DS may provide alternative targets for pharmacological intervention.

Partial Purification and Characterization of Two Distinct Types of Caspases from Human Epidermis

Recent observations demonstrated that interleukin-1beta converting enzyme family proteases, now referred to as caspase family, play central roles in apoptosis, or programmed cell death. In this study, we tried to isolate and characterize epidermal caspases. By DEAE-Sephacel anion-exchange chromatography, human cornified cell extract showed two caspase-like fractions (F-I and F-II) with different substrate specificities. These were further purified by Sephacryl S-200, Mono Q ion exchange and Superose 6 gel chromatography. F-I showed a molecular weight of 30 kDa and specifically hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, a fluorogenic substrate for caspase-3 (CPP32) with a Km value of 13.8 microM. F-I generated a characteristic 85 kDa fragment from poly(ADP-ribose) polymerase. Inhibitor susceptibility of F-I was very similar to that of caspase-3, further confirming the caspase-3-like properties of F-I. In contrast, the molecular weight of F-II was estimated to be 110 kDa, which was much higher than the other caspases. F-II equally hydrolyzed acetyl-Asp-Glu-Val-Asp-methylcoumarinamide, and acetyl-Tyr-Val-Ala-Asp-methylcoumarinamide, caspase-1 (interleukin-1beta converting enzyme)-specific substrate, and was inhibited by acetyl-Tyr-Val-Ala-Asp-aldehyde and acetyl-Tyr-Val-Ala-Asp-aldehyde. Affinity labeling using biotinylated YVAD-cmk demonstrated several positive bands ranging from 25 to 35 kDa, supporting the hypothesis that F-II is a complex of multiple caspases. Reverse transcriptase-polymerase chain reaction analysis demonstrated that among known caspases tested, caspase-1, -2, -3, -4, and -7 were expressed in cultured human keratinocytes. These results suggest that multiple caspases are synthesized in human keratinocytes and are involved in terminal differentiation.

Induction of Glutathione S-Transferase Isozymes in Rice Shoots Treated with a Combination of Pretilachlor and Fenclorim.

DEAE Sephacel anion exchange chromatography was used to separate glutathione S-transferase (GST) isozymes in non-treated etiolated rice shoots (4 day-old) and those treated with a combination of pretilachlor [2-chloro-2′, 6′-diethyl-N-(2-propoxyethyl)-acetanilide] and fenclorim (4, 6-dichloro-2-phenylpyrimidine).Non-treated rice shoots contained isozymes which were active with the following substrates; CDNB: 1-chloro-2, 4-dinitrobenzene (2 isozymes), fenclorim (2 isozymes) and pretilachlor (4 isozymes). Pretreatment of the shoots with a combination of pretilachlor and fenclorim increased the activity of the constitutively-expressed isozymes that exhibited activity with CDNB, fenclorim and pretilachlor. The treatment also induced two new GST(fen) isozymes and one new GST(pret) isozyme.The results are suggestive of the diversity of GST isozymes in etiolated rice shoots, the selective enhancement of GST isozymes by herbicides and safeners, and safeners conjugated with glutathione, resulting in selective safening action in rice.

Phosphodiesterase isoenzymes in human ureteral smooth muscle: identification, characterization, and functional effects of various phosphodiesterase inhibitors in vitro.

Phosphodiesterases (PDE) are key enzymes regulating intracellular cyclic nucleotide metabolism and, thus, contraction and relaxation of the muscle. At present, five different families of isoenzymes of PDE exist that show a distinct species-specific and organ-specific distribution. The aim of the present study was to analyze the PDE isoenzymes present in the human ureter and to evaluate the functional effects of isoenzyme-specific inhibitors in this tissue. Normal ureteral tissue was obtained during radical nephrectomies, homogenized, centrifuged, and the supernatant fraction was separated using DEAE-Sephacel anion-exchange chromatography. PDE assay was then performed and the isoenzymes characterized on the basis of their kinetic characteristics and their sensitivity to allosteric modulators and inhibitors. In vitro, longitudinal ureteral strips as well as ureteral rings were precontracted, and different selective and nonselective PDE inhibitors were added incrementally. Three different PDE isoenzymes were identified: PDE I (Ca/calmodulin-stimulated), PDE II (cyclic guanosine monophosphate-stimulated), and PDE IV (cyclic adenosine monophosphate-specific). All PDE inhibitors relaxed the strips dose-dependently with an EC50 of 30 microM for papaverine, 40 microM for zaprinast, 25 microM for quazinone, and 0.1 microM for rolipram. The existence of three different PDE isoenzymes was shown in this study. The ureter-relaxing effect of the PDE IV inhibitor at low concentrations, combined with its low effect on the systemic circulatory parameters, may open a possibility of using selective PDE IV inhibitors in the treatment of ureteral colics or ureteral stones.

Separable growth and migration factors for large-cell lymphoma cells secreted by microvascular endothelial cells derived from target organs for metastasis.

Metastatic variant sublines of the murine large-cell lymphoma cell line RAW117 were tested for their growth and migration properties in vitro in medium conditioned by soluble factors released from syngeneic mouse liver-, lung-, and brain-derived microvessel endothelial cells. Medium conditioned with hepatic sinusoidal endothelial cells stimulated the growth of highly liver-colonising (RAW117-H10) and highly liver- and lung-colonising (RAW117-L17) sublines at higher rates than the poorly metastatic parental line (RAW117-P) (H10 greater than L17 greater than P). Medium conditioned with lung microvessel endothelial cells selectively stimulated the growth of the lung-colonising RAW117-L17 subline. Medium conditioned with brain microvessel endothelial cells showed no growth selectivity, and equivalently stimulated the growth of various RAW117 cell sublines. Medium conditioned with hepatic sinusoidal endothelial cells preferentially promoted the migration of the liver-colonising H10 and L17 sublines, and medium conditioned with lung endothelial cells differentially stimulated the migration of the lung-colonising L17 subline; whereas medium conditioned with brain endothelial cells only slightly stimulated the migration of L17, but not H10 or P cells. Fractionation of medium conditioned with hepatic sinusoidal endothelial cells by DEAE Sephacel anion exchange chromatography revealed that the growth-stimulating activities were clearly separable from migration-stimulating activities. The growth- and migration-stimulating activities released from organ microvessel endothelial cells may be important in determining the ability of RAW117 cells to selectively form metastatic colonies in particular organs.

Purification and properties of neuraminidase from the culture medium of Bacteroides loescheii

Neuraminidase was purified from the culture medium of Bacteroides loescheii ATCC 15930 by ultrafiltration followed by a DEAE-Sephacel anion exchange chromatography, fast-protein liquid chromatography using Mono P column and high-performance liquid chromatography using a Shim-pack Diol-300 column. The molecular weight of the purified enzyme was measured to be approximately 87 kDa and the optimal pH was at 4.8. Although the enzyme was able to hydrolyze the substrates with alpha,2-3, alpha,2-6, and alpha,2-8 linkages of N-acetylneuraminic acid, the rate of hydrolysis of N-acetylneuraminosyl-alpha,2-3-lactose was greater than that of the alpha,2-6-isomer.

Immunochemical characterization of two surface polysaccharides of Bacteroides fragilis

Immunochemical analysis of the capsular polysaccharide from Bacteroides fragilis NCTC 9343 revealed a novel structure composed of two distinct polysaccharides. Immunoelectrophoresis of an extract of purified surface polysaccharide from fermenter-grown organisms showed a complex precipitin profile with varying anodal mobility. DEAE-Sephacel anion-exchange chromatography of the polysaccharide extract failed to separate the majority of this aggregate. Disaggregation of this complex was accomplished by very mild acid treatment; purification was achieved by DEAE-Sephacel anion-exchange chromatography. Polysaccharide A had a neutral charge at pH 7.3, a net negative charge at pH 8.6, and an average Mr = 110,000; chemical analysis showed it to contain galactose, galactosamine, and an unidentified amino sugar. Polysaccharide B eluted from the anion-exchange column with increased salt concentration; it had a net negative charge and an average Mr = 200,000, and contained fucose, galactose, quinovosamine, galacturonic acid, and glucosamine. Neither of these polysaccharides contained detectable 3-deoxy-D-manno-octolusonic acid, and both were recognized as distinct antigens on the basis of their reactivity with monoclonal antibodies CE3 and F10, which reacted with the complex before acid treatment. These data indicate that the capsule of B. fragilis NCTC 9343 comprises two discrete, surface-exposed polysaccharides with differing physiochemical properties that are distinct from the lipopolysaccharide of this organism. The finding of two surface polysaccharides has not been described for other bacteria pathogenic to humans.

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS)

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO 2 and CH 4 at a specific rate of 50 nmol·min −1·mg −1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridiwn pasteurianum. The apparent K m for ferredoxin from M. barkeri was determined to be 2.5 μM. A ferredoxin dependence was also found for the formation of CO 2, H 2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H 2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.

Modulation of neurotoxic esterase activity in vitro by phospholipids

Neurotoxic esterase (NTE), the proposed molecular site for the initiation of organophosphorus-induced delayed neuropathy, is a membrane-associated enzyme. NTE activity was solubilized from chicken brain microsomal membranes with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and partially separated from other solubilized hen brain esterases by DEAE-Sephacel anionexchange chromatography using stepwise increases in salt concentration; however, there was poor recovery of NTE activity and only a slight increase in NTE specific activity. NTE activity in the “high salt” fraction (i.e., the NTE-enriched fraction) was markedly activated by a heatstable factor(s) present in other fractions eluted from the column. This activating factor was extracted with organic solvents, suggesting that it may be lipid. In a related study, purified phosphatidylcholine, phosphatidylethanolamine, and phosphatidylserine were also found to activate the partially separated NTE activity in a concentration-dependent manner while phosphatidylinositol was found to inhibit the same partially separated NTE fraction in a concentration-dependent manner. The results suggest that lipids may modulate NTE activity and that the loss of lipid cofactors during chromatographic separations may underlie some of the difficulties encountered in isolation of active NTE.

Purification and characterisation of ovine C4: Evidence for two molecular forms in ovine plasma

A relatively rapid procedure is described for the isolation of the fourth component of complement (C4) from ovine plasma. The method, which recovers approximately 30% C4, is based upon DEAE Sephacel anion exchange chromatography of PEG precipitated plasminogen depleted plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration. SDS-PAGE of purified ovine C4 under reducing conditions revealed a complex pattern of bands which was interpreted on the basis of a three polypeptide chain structure for each of two distinct species, or isotypes, of C4 molecule herein termed C4A and C4B. Each isotype differs in the mol. wt of the α chain—108 and 95 K respectively. Nucleophilic substitution of immunoprecipitated ovine C4 with radiolabelled methylamine revealed that both C4 species contained a reactive thiol ester site and that each could be cleaved into an activated form (presumably C4b) characterised by a truncated α′ chain some 8 K lower in mol. wt. A comparison of the isotype composition of purified C4 with that of immunoprecipitated C4 from the same animal indicated that the purification procedure favoured isolation of the C4B isotype. The mol. wts of both the α and β chains were lowered following digestion of ovine C4 with neuraminidase.

Purification and characterisation of the fourth component of bovine complement.

A relatively rapid method for the isolation of complement protein C4 from bovine plasma is described. The method consists of DEAE Sephacel anion exchange chromatography of plasminogen-depleted bovine plasma followed by cation exchange chromatography on CM Sepharose and finally gel filtration on a TSK G3000 SW column. A yield of approximately 20% was obtained. Conventional SDS-PAGE of purified bovine C4 showed the presence of alpha, beta and gamma polypeptide chains, the molecular weights of which were determined from Ferguson plots to be 95,000 +/- 2,500, 80,500 +/- 2,000 and 30,000 +/- 500 daltons, respectively. SDS-PAGE of C4 immunoprecipitated from the plasma of individual cattle in gels with a reduced proportion of crosslinker showed size polymorphism of the alpha chain. The presence of dual alpha chains was confirmed by radiolabelling their reactive thiol ester moiety with 14C methylamine. The difference in size of the two bovine alpha chains is approximately 1,800 daltons. On activation of bovine C4 both alpha chains were cleaved into alpha' chains (87,000 and 85,000 daltons) characteristic of C4b.

Isolation, chemical and biological characterization of sulphated glycoproteins synthesized by rat buccal and palatal minor salivary glands in vivo and in vitro

35S-labelled sulphated glycoproteins (SGP) were isolated from these glands after the incorporation of radiosulphate in vivo and in vitro by fractionation of tissue and medium extracts on Sepharose 4B and partial purification by DEAE-Sephacel anion exchange chromatography. Fractions were assessed for purity by SDS-PAGE and by cellulose-acetate electrophoresis. Molecular weights ranged from 34,000 to 5 × 10 6. It was notable that the molecular size of SGP from the in vitro media was generally lower than from the corresponding tissue fractions, particularly for the palatal samples. The fractions were heterogeneous and contained no sulphated glycosaminoglycans; they had high levels of aspartic acid, glutamic acid, threonine and serine, but there was no major difference in amino-acid composition between them. Carbohydrate analysis indicated typical components associated with sulphated glycoproteins, including fucose, galactose, glucose, mannose, N-acetylgalactosamine, N-acetylglucosamine and N-acetylneuraminic acid. Protein:carbohydrate ratios ranged from 0.1:1.0–3.5:1.0 and ester sulphate from 0.8 to 16.2 per cent. All fractions exhibited blood-group A reactivity and aggregated Streptococcus sanguis NCTC 7864; several fractions interacted similarly with Streptococcus mutans OMZ61.

A solid-phase immunosorbent assay to determine the proteinase binding capacity of α2-macroglobulin using 125I-trypsin as indicator proteinase

Hyperimmune sera against human α 2macroglobulin were raised in rabbits following immunization with ‘s’ α 2-macroglobulin ∗∗ ∗∗ Nomenclature according to Barrett et al. [18]. over half a year. Immunoglobulins were prepared by DEAE-Sephacel anion exchange chromatography. The immunoglobulin preparations showed a remarkably high and equal titer for ‘s’ and ‘f’ α 2-macroglobulin (plasma α 2-macroglobulin fully saturated with pig pancreas trypsin), which amounted to 6.4·10 −6 as revealed by passive hemagglutination. Immunoimmobilization experiments revealed that at equilibrium, ‘s’ α 2-macroglobulin and both ‘f’ α 2-macroglobulins (27 and 82% saturation of ‘s’ α 2-macroglobulin with trypsin) had been bound to the same degree from the fluid phase to the monospecific antibodies that had been adsorbed to polystyrene tubes. Comparison of quantitative gel scans for disappearance of the intact α 2-macroglobulin subunit ( M r 182 000) with 125I-labeled trypsin binding capacity of immunoimmobilized α 2-macroglobulin-trypsin complexes showed conspicuous agreement. Rocket immunoelectrophoresis did not give significant differences between ‘s’ α 2-macroglobulin and ‘f’ α 2-macroglobulin. In the fluid phase, a binding ratio of 2.4 mol trypsin/mol α 2-macroglobulin was observed. Saturation of solid phase immunoimmobilized ‘s’ α 2-macroglobulin with trypsin could be accomplished by incubation with a 100–200-fold molar excess of enzyme for 10 min. The solid-phase experiments showed a binding ratio of 2.0 mol trypsin/mol α 2-macroglobulin. The high molar excess of trypsin needed to saturate solid-phase immunoimmobilized α 2-macroglobulin, which binds 20% less trypsin than in the liquid phase, is partially explained by an enhancement of the negative cooperativity of trypsin binding to α 2-macroglobulin found in the liquid-phase system. Assessment of the trypsin-binding capacity of α 2-macroglobulin immunoadsorbed from synovial fluids ( n = 19) of patients with seropositive rheumatoid arthritis yielded an inactive α 2-macroglobulin of 0–53% when compared to the trypsin-binding capacity of normal plasma α 2-macroglobulin.

Partial chemical characterization and biological activities of sulphated glycoproteins isolated from in-vivo pilocarpine-stimulated secretions of rat minor salivary glands

Following the incorporation of [ 35S]-sulphate into rats, 35S-labelled sulphated glycoproteins were isolated from the minor salivary gland secretions of pilocarpine-stimulated, immobilized animals. The secretory products were initially resolved on Sepharose 4B into two major radioactive fractions, one of which appeared at the void volume and represented only 7 per cent by weight of the applied fraction. The bulk of the products were of lower molecular weight and were further resolved by DEAE-Sephacel anion-exchange chromatography. Four distinct radioactive fractions were obtained all of which possessed blood-group A reactivity and aggregated Streptococcus sanguis NCTC 7864 but not Streptococcus mutans OMZ61. The radioactive fractions possessed protein/carbohydrate ratios in the range 1.6:1–5.8:1. Ester-sulphate contents ranged from 2.5 to 5.3 per cent with a higher value of 14.0 per cent being obtained for the most anionic fraction. Sialic acid was in the range 5.1 to 21.6 per cent. None of the fractions tested contained sulphated glycosaminoglycans.