In vitro biosynthesis of homogalacturonan by a membrane-bound galacturonosyltransferase from epicotyls of azuki bean.

A membrane preparation of 7-d-old seedlings from azuki bean (Vigna angularis) contained galacturonosyltransferase (GalAT) capable of transferring galacturonic acid (GalA) from UDP-GalA into polygalacturonic acid (PGA) as an exogenous acceptor. The enzyme was maximally active at pH 6.8-7.8 and 25-35 degrees C in the presence of 5 mM Mn2+ and 0.5% (w/v) Triton X-100. Acid-soluble low-Mr (average Mr 10,000) PGA was a more efficient acceptor substrate than acid-insoluble polymer (Mr 70,000). The apparent Michaelis constants for UDP-GalA and low-Mr PGA were 0.14 mM and 0.02 mg/ml, respectively. Various pectins with different degrees of methyl-esterification (DE) were poor acceptors, and the enzyme activity tended to decrease with decreasing DE of the pectins. The transfer products from incubation of the enzyme with UDP-14C-GalA and the low-Mr PGA yielded 14C-GalA2 as the major product upon digestion with an endopolygalacturonase (EPGase), confirming the incorporation of GalA into PGA through contiguous alpha-1,4-linkages.

A xylosyltransferase that synthesizes beta-(1-->4)-xylans in wheat (Triticum aestivum L.) seedlings.

A particulate preparation from 6-day-old seedlings of wheat (Triticum aestivum L.) was found to contain a xylosyltransferase (XylTase) which incorporated xylose (Xyl) from UDP-xylose into exogenous beta-(1-->4)-xylooligosaccharides with 2-aminopyridine-derivatized reducing end groups. High-performance liquid chromatographic analysis showed that the chain elongation of pyridylaminated beta-(1-->4)-xylotriose (Xyl3-PA) occurred by attachment of a series of one, two, or three xylosyl residues, depending on substrate concentrations and reaction times. Methylation analysis and beta-xylosidase digestion of the newly synthesized Xyl4-PA confirmed that the xylosyl residues were incorporated through beta-(1-->4)-linkages. The enzyme was maximally active at pH 6.8 and 20 degrees C, and required Triton X-100, which enhanced activity 5-fold at a concentration of 0.05-2%. Divalent ions, including Mn2+ and Mg2+, did not affect activity. Enzyme activity increased with increasing polymerization of xylosyl residues of the acceptor substrates: for instance, Xyl5-PA was almost 7 times as efficient as Xyl2-PA. The apparent Michaelis constants of the enzyme for Xyl3-PA and UDP-xylose were 13.5 and 7.9 mM, respectively. The enzyme also catalyzed incorporation of radioactive sugars (Xyl together with a small portion of L-arabinose) from UDP-[14C]xylose into higher beta-(1-->4)-xylooligosaccharides (degree of polymerization > 7) with or without (4-O-methyl-)glucuronosyl side chains at activities comparable to those observed for pyridylaminated xylooligosaccharides, and into several heteroxylans but with much lower efficiency. Enzymatic hydrolysis of the product with a beta-xylanase degraded it into mainly xylobiose, providing further evidence that the xylosyl residues are incorporated through beta-(1-->4)-linkages.

Purification and characterization of a beta-glucuronidase from Aspergillus niger.

A beta-glucuronidase from Pectinex Ultra SP-L, a commercial pectolytic enzyme preparation from Aspergillus niger, was purified 170-fold by ion-exchange chromatography and gel filtration. Apparent M(r) of the purified enzyme, estimated by denaturing gel electrophoresis and size-exclusion chromatography, were 68,000 and 71,000, respectively, indicating that the enzyme is a monomeric protein. It released uronic acids not only from p-nitrophenyl beta-glucosiduronic acid (PNP-GlcA) but also from acidic galactooligosaccharides carrying either beta-D-glucosyluronic or 4-O-methyl-beta-D-glucosyluronic residues at the nonreducing termini through beta-(1-->6)-glycosidic linkages. The enzyme exhibited a maximal activity toward these substrates at pH 3.0. A regioisomer, 3-O-beta-glucosyluronic acid-galactose, was unsusceptible to the enzyme. The enzyme did act on a polymer substrate, releasing uronic acid from the carbohydrate portion of a radish arabinogalactan-protein modified by treatment with fungal alpha-L-arabinofuranosidase. The enzyme produced acidic oligosaccharides by transglycosylation, catalyzing the transfer of uronic acid residues of PNP-GlcA and 6-O-beta-glucosyluronic acid-galactose to certain exogenous acceptor sugars such as Gal, N-acetylgalactosamine, Glc, and xylose.

Characterization of pectin methyltransferase from soybean hypocotyls.

Pectin methyltransferase (PMT) catalyzing the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the C-6 carboxyl group of galactosyluronic acid residues in pectin was found in a membrane preparation of etiolated hypocotyls from 6-d-old soybean (Glycine max Merr.). The enzyme was maximally active at pH 6.8 and 35-40 degrees C, and required 0.5% (w/v) Triton X-100. The incorporation of the methyl group was significantly enhanced by addition of a pectin with a low (22%) degree of methyl-esterification (DE) as exogenous acceptor substrate. The apparent Michaelis constants for SAM and the pectin (DE22) were 0.23 mM and 66 microg x ml(-1), respectively. Attachment of the methyl group to the carboxyl group of the pectin via ester linkage was confirmed by analyzing radiolabeled product from incubation of the enzyme with [14C]methyl SAM and the acceptor pectin. Size-exclusion chromatography showed that both enzymatic hydrolysis with a pectin methylesterase and a mild alkali treatment (saponification) led to the release of radioactive methanol from the product. Enzymatic hydrolysis of the product with an endopolygalacturonase degraded it into small pectic fragments with low relative molecular mass, which also supports the idea that the methyl group is incorporated into the pectin. The soybean hypocotyls were fractionated into their cell wall components by successive extraction with water, EDTA, and alkali treatment. Among the resulting polysaccharide fractions, high PMT activity was observed when a de-esterified polysaccharide derived from the EDTA-soluble fraction (the pectic fraction) was added as an alternative acceptor substrate, indicating that the enzyme may be responsible for producing methyl-esterified pectin in vivo.

Characterization of antigenic epitopes in anti-ulcer pectic polysaccharides from Bupleurum falcatum L. using several carbohydrases.

A polyclonal antibody (anti-bupleuran 2IIc/PG-1-IgG) against the "ramified" region (PG-1) of an anti-ulcer pectic polysaccharide was prepared and its antigenic epitopes were analyzed by using several carbohydrases. Enzymatic removal of arabinosyl residues from PG-1 by endo-(1-->5)-alpha-L-arabinanase (from Aspergillus niger) did not reduce the binding ability of anti-bupleuran 2IIc/PG-1-IgG to PG-1. When the endo-(1-->5)-alpha-L-arabinanase-resistant fraction (EA-1) was digested with rhamnogalacturonase A (rRGase A from A. aculeatus), a high-molecular-mass fragment fraction (RA-1) and an oligosaccharide fraction (RA-3) were obtained. RA-3 contained at least four kinds of oligosaccharides liberated from the rhamnogalacturonan core. This partial removal of the rhamnogalacturonan core in EA-1 also did not reduce the binding of the antibody to the polysaccharide. Further digestion of RA-1 with exo-(1-->3)-beta-D-galactanase (from Irpex lacteus), gave a high-molecular-mass fragment (EXG-1) and a trace of oligosaccharides (EXG-3). Methylation and FABMS analyses indicated that EXG-3 contained mono- and di-galactosyl oligosaccharides possessing terminal GlcA or GlcA4Me. Removal of the EXG-3 fraction from RA-1 by exo-(1-->3)-beta-D-galactanase significantly reduced the ability of the binding of the antibody to the polysaccharide. When PG-1 was digested with endo-(1-->6)-beta-D-galactanase (from Trichoderma viride) or beta-D-glucuronidase (from A. niger), the reactivities of both enzyme-resistant fractions to the antibody were decreased in comparison with that of PG-1. Both radish arabinogalactan (containing GlcA4Me) and beta-D-GlcpA-(1-->6)-beta-D-Galp-(1-->6)-D-Galp were shown to inhibit the reactivity of PG-1 to the antibody by competitive ELISA. These results suggest that 6-linked galactosyl chains containing terminal GlcA or GlcA4Me attached to (1-->3)-beta-D-galactosyl chains, are important sugar residues in the antigenic epitopes of the "ramified" region of bupleuran 2IIc.

alpha-L-fucosyltransferases from radish primary roots.

A novel alpha-L-fucosyltransferase capable of transferring L-fucose (L-Fuc) from GDP-L-Fuc to the O-2 of alpha-L-arabinofuranosyl residue (GDP-L-Fuc:alpha-L-arabinofuranoside 2-alpha-L-fucosyltransferase) has been found in the microsomal fraction of primary roots from 6-d-old radish (Raphanus sativus L.) seedlings. Enzyme activity was measured fluorometrically at 25 degrees C using a pyridylaminated trisaccharide, L-arabinofuranosylf alpha(1-->3)D-galactopyranosyl beta(1-->6)D-galactose (AraGalGal-PA) as the acceptor. This enzyme found in the microsomal fraction is maximally active at pH 6.8 and requires 0.1% (w/v) Zwittergent 3-16 and 5 mM Mn2+. Chemical and enzymatic analyses of fucosylated AraGalGal-PA confirmed the attachment of L-Fuc to the L-arabinofuranosyl (L-Araf) residue at O-2 by alpha-glycosidic linkage. Radiolabeling was used to assay L-Fuc transfer to L-Araf-containing galacto-oligomers and tamarind xyloglucan. The enzyme specific for the L-Araf residue undergoes development- and organ-specific expression in root tissue, whereas the L-Fuc transfer to tamarind xyloglucan can be detected in microsomal fractions from various organs in developing radish plants. Enzyme assays of membranes fractionated from microsomal fractions revealed that two distinct alpha-L-fucosyltransferases with different acceptor specificity are associated with Golgi membranes from primary roots, whereas hypocotyl Golgi membranes completely lack the enzyme specific for the L-Araf residue.

Purification and characterization of intracellular proteinases in Pleurotus ostreatus fruiting bodies.

A serine proteinase (ProA, EC 3.4.22.9) and two metalloendopeptidases (ProB, EC 3.4.99.32 and ProC, 3.4.24.4), have been purified to homogeneity from the fruiting bodies of Pleurotus ostreatus. ProA is a serine proteinase with a mass of 30 kDa, which has amidolytic and esterolytic activities besides proteolysis and catalyzes preferential cleavage of the peptide bonds involving the carboxyl groups of hydrophobic amino acid residues in oxidized bovine insulin B chain. The N-terminal amino acid sequence was VTQTNAPWGLSRL. ProB is a zinc-enzyme with a mass of 18 kDa, which is devoid of lysine, and its N-terminal sequence was ATFVGCSATRQ. The enzyme is inactivated completely by EDTA and 1,10-phenanthroline, and Zn(2+)-depleted ProB can regain the activity with Zn2+, Co2+, or Mn2+. Specific cleavage of Pro29-Lys30 in oxidized bovine insulin B chain, preferential generation of lysylpeptides from proteins, and a high susceptibility of polylysine suggest that ProB splits specifically the peptide bonds involving the alpha-amino group of lysyl residues. ProC is a metalloendopeptidase of a mass of 42.5 kDa, and Zn2+ was the most effective divalent metal ion to activate the EDTA-inactivated enzyme.

Characterization of a thermostable lysine-specific metalloendopeptidase from the fruiting bodies of a basidiomycete, Grifola frondosa.

A zinc-metalloendopeptidase, MEP, capable of catalyzing specific cleavage of acyl-lysine bonds (-X-Lys-) in polypeptides has been purified 212-fold in a yield of 24.7% from the fruiting bodies of Grifola frondosa, which is a popular edible mushroom called "MAITA-KE" in Japan. The purified enzyme consists of a single polypeptide chain with an apparent molecular mass of 20 kDa and a pI value of 7.46, contains 1 atom of zinc/molecule and can be inactivated with EDTA or 1,10-phenanthroline. Treatment of MEP with EDTA affords an apoenzyme, whose activity can be fully restored by the addition of Mn2+, Zn2+, Ca2+, or Co2+. Prominent features of MEP are its remarkable heat stability and its high affinity for beta-D-glucans and chitin. It hydrolyzes proteins maximally at pH 9-10, liberating only lysylpeptides. Polylysine and lysine copolymers with alanine, phenylalanine, or glutamic acid can serve as good substrates. Lysylalanine was liberated from bovine insulin and its oxidized B chain by the action of MEP. Mass spectrometric analysis by Frit-FAB MS of the fragments generated from horse heart cytochrome c presented unambiguous evidence to corroborate the specificity of MEP for acyl-lysine bonds.

The complete amino acid sequences of two serine proteinase inhibitors from the fruiting bodies of a basidiomycete, Pleurotus ostreatus.

The complete amino acid sequences of two isomeric endogenous inhibitors, IA-1 and IA-2, both of which specifically inhibit an intracellular serine proteinase (proteinase A) purified from the fruiting bodies of a higher basidiomycete, Pleurotus ostreatus, were determined. Both inhibitors are acidic polypeptides with respective molecular masses of 8307 and 8244 Da, as determined by plasma desorption mass spectral analyses, and their N-terminal serine residue is blocked by acetylation. The fragments generated from the inhibitors by proteolytic and chemical cleavages were subjected to amino acid composition, sequence, and mass spectral analyses. The sequence and molecular mass information for the peptides established that the inhibitors both consisted of 76 amino acid residues and differed from each other in that aspartic acid and glutamic acid residues at residues 12 and 15 of IA-1 were replaced by glycine and aspartic acid in IA-2, respectively. The molecular masses of IA-1 and IA-2 were calculated to be 8309 and 8237, based on the sequence data. The action of carboxypeptidase Y on IA-1 resulted in a complete loss of the inhibitory activity along with successive release of glutamine and threonine from the C-terminus. Cyanogen bromide cleavage of Met38-Pro39 and Met41-Lys42 in IA-1 and hydroxylamine degradation of IA-2 completely abolished their inhibitory activity. These results suggest that the whole molecules of both inhibitors are essential to their inhibitory activities. Their structural resemblance to propeptides of subtilisin family proteinases revealed their mechanism of action.

Inhibition of high-molecular-weight-(1-->3)-beta-D-glucan-dependent activation of a limulus coagulation factor G by laminaran oligosaccharides and curdlan degradation products.

Extensive surveys for the effects of various beta-D-glucans on the coagulation cascade in horseshoe crab amebocyte lysates showed that low-mol-wt-(1-->3)-beta-D-glucans and laminaran oligosaccharides inhibit the activation of a limulus coagulation factor G by high-mol-wt-(1-->3)-beta-D-glucans. The inhibitory properties are exclusively dependent upon their number-average mol wt (Mn) in a range of 342-58,100, which correspond to a degree of polymerization (dp) range of 2-359. The most effective is a laminaran dextrin of Mn 5800 (dp of 35-36), which causes 50% inhibition of factor G activation at a concentration of 3.16 ng/mL. The inhibition of the activation of factor G proportional to the concentration of the inhibitor, and the adsorption of factor G by inhibitory beta-D-glucan-conjugated cellulose suggested a high affinity of the inhibitory saccharides for the activator-recognition site of factor G. Branched (1-->6), (1-->3)-beta-D-glucans, laminarans, mixed linkage (1-->3), (1-->4)-beta-D-glucans, and partially substituted curdlan and laminaran were found to be inhibitory, possibly owing to clusters of consecutive (1-->3)-beta-D-glucopyranosyl residues as intrachain units. The inhibition appears to be related to the inability of the inhibitory (1-->3)-beta-D-glucans to form ordered conformations and to their tendency to take a random-coil structure in aqueous solution.

Production and characterization of antibodies to the beta-(1-->6)-galactotetraosyl group and their interaction with arabinogalactan-proteins.

Rabbit antisera were raised against beta-(1-->6)-galactotetraose coupled to bovine serum albumin (Gal4-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of alpha-L-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, beta-(1-->4)-galactan, and beta-(1-->3)-galactan, indicating their high specificity toward the consecutive beta-(1-->6)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immunobilized beta-(1-->6)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (1-->6)-linked beta-galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal4-BSA as antigen, which revealed that beta-(1-->6)-galactotriose and -tetraose were potent inhibitors, while beta-(1-->3)- or beta-(1-->4)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or beta-(1-->6)-galactotetraose.

alpha-l-Arabinofuranosidase from Radish (Raphanus sativus L.) Seeds.

An alpha-l-arabinofuranosidase has been purified 1043-fold from radish (Raphanus sativus L.) seeds. The purified enzyme was a homogeneous glycoprotein consisting of a single polypeptide with an apparent molecular weight of 64,000 and an isoelectric point value of 4.7, as evidenced by denaturing gel electrophoresis and reversed-phase or size-exclusion high-performance liquid chromatography and isoelectric focusing. The enzyme characteristically catalyzes the hydrolysis of p-nitrophenyl alpha-l-arabinofuranoside and p-nitrophenyl beta-d-xylopyranoside in a constant ratio (3:1) of the initial velocities at pH 4.5, whereas the corresponding alpha-l-arabinopyranoside and beta-d-xylofuranoside are unsusceptible. The following evidence was provided to support that a single enzyme with one catalytic site was responsible for the specificity: (a) high purity of the enzyme preparation, (b) an invariable ratio of the activities toward the two substrates throughout the purification steps, (c) a parallelism of the activities in activation with bovine serum albumin and in heat inactivation of the enzyme as well as in the inhibition with heavy metal ions and sugars such as Hg(2+), Ag(+), l-arabino-(1-->4)-lactone, and d-xylose, and (d) results of the mixed substrate kinetic analysis using the two substrates. The enzyme was shown to split off alpha-l-arabinofuranosyl residues in sugar beet arabinan, soybean arabinan-4-galactan, and radish seed and leaf arabinogalactan proteins. Arabinose and xylose were released by the action of the enzyme on oat-spelt xylan. Synergistic action of alpha-l-arabinofuranosidase and beta-d-galactosidase on radish seed arabinogalactan protein resulted in the extensive degradation of the carbohydrate moiety.

Substrate-induced activation of maltose phosphorylase: interaction with the anomeric hydroxyl group of alpha-maltose and alpha-D-glucose controls the enzyme's glucosyltransferase activity.

Maltose phosphorylase, long considered strictly specific for beta-D-glucopyranosyl phosphate (beta-D-glucose 1-P), was found to catalyze the reaction beta-D-glucosyl fluoride + alpha-D-glucose----alpha-maltose + HF, at a rapid rate, V = 11.2 +/- 1.2 mumol/(min.mg), and K = 13.1 +/- 4.4 mM with alpha-D-glucose saturating, at 0 degrees C. This reaction is analogous to the synthesis of maltose from beta-D-glucose 1-P + D-glucose (the reverse of maltose phosphorolysis). In acting upon beta-D-glucosyl fluoride, maltose phosphorylase was found to use alpha-D-glucose as a cosubstrate but not beta-D-glucose or other close analogs (e.g., alpha-D-glucosyl fluoride) lacking an axial 1-OH group. Similarly, the enzyme was shown to use alpha-maltose as a substrate but not beta-maltose or close analogs (e.g., alpha-maltosyl fluoride) lacking an axial 1-OH group. These results indicate that interaction of the axial 1-OH group of the disaccharide donor or sugar acceptor with a particular protein group near the reaction center is required for effective catalysis. This interaction appears to be the means that leads maltose phosphorylase to promote a narrowly defined set of glucosyl transfer reactions with little hydrolysis, in contrast to other glycosylases that catalyze both hydrolytic and nonhydrolytic reactions.

Purification of an exo-beta-(1----3)-D-galactanase of Irpex lacteus (Polyporus tulipiferae) and its action on arabinogalactan-proteins.

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.

[An experimental study on healing process of replanted mature permanent teeth. Changes of periodontal vascular network].

This study was conducted to clarify both changes of periodontal vascular architecture and concomitant remodelling phenomena associated with hard tissues in the healing process of replanted teeth of first premolar. Utilizing both vascular corrosive resin casts method, scanning electron microscopy, and histological examinations, 45 matured mongrel dogs were used for this study. The results were as follows: 1. 4 days after operation: Newly formed vascular networks with a exceedingly irregular course were observed on lower two thirds of the alveolar wall. They derived from comparatively less damaged periodontal vascular components. No vascular networks were observed surrounding in the crevicular area of the replanted tooth where the periodontal membrane tissue was thoroughly damaged when tooth was extracted. 2. 1 week after operation: Newly formed periodontal vascular networks with a slightly irregular course were observed over the entire alveolar bone surface. 3. 2 weeks after operation: Formation of Sharpey's fibers occurred. The surrounding alveolar bone was remodelled and rearrangement of periodontal vascular architecture was observed. Also, several Howship's lacunae were observed on the root surface where characteristic capillary loops with glomerular-like appearance penetrated into these lacunae. 4. 3 weeks after operation: Root resorption was advanced and capillary loops with glomerular-like appearance were extensively distributed in association with each lacunae. On the other hand, the less space where periodontal membrane vasculature occupied, the more space was occupied by osteoid tissue. 5. 4 weeks after operation: Blood vessels within the periodontal space were reduced in number and the osteoid tissue showed bony fusion adjacent to the extensively resorbing surface of dentin. 6. 12 weeks after operation: Functional arrangement of Sharpey's fibers was completed. Restoration of Howship's lacunae on the root surface and two layered arrangements of vascular network within the periodontal space were observed. Newly formed periodontal vascular architecture showed a fine meshwork pattern, which was somewhat different from that of the control (noreplantation) group. 7. 20 weeks after operation: Increased number of capillary loops was observed with leakage of methacrylate resin through the weakened endothelial linings of capillaries in one case. It is supposed that this leakage through capillaries is correlated with the inflammatory root resorption that occurs clinically in marginal periodontitis. Also in some cases, periodontal capillary network showed secondary occlusal traumatic changes. Above results indicated that periodontal vascular architecture varied depending upon the reactions of periodontium following tooth replantation and the prognosis of replanted tooth was deeply associated with repair of periodontal vascular network.

[A scanning electron microscopic study on the arteriovenous anastomoses of rabbit ear using corrosive resin casts].

Arteriovenous anastomoses (AVAs) partake in the regulation of blood flow, and although it has been speculated that AVAs in the rabbit ear play a major role in the regulation of the body temperature, no documented work is available on the structural characteristics of AVAs in the rabbit ear. Accordingly, the purpose of this investigation was to determine the frequency of AVAs and their structural characteristics by using the vascular corrosive resin casts and further examination under a scanning electron microscope (SEM). This method permitted not only the visualization of a three-dimensional view of AVAs, but also depicted a clear differentiation between AVAs and arterioles or venules. Within the vascular architecture of rabbit ear, three distinctive vascular layers could be identified. The frontal and dorsal epithelial vascular layers are distributed underneath the epithelium and formed with nearly all capillaries, and the intermediate vascular layer which is established with main arteries, veins and their branches. The AVAs were observed in all three vascular layers, although various venous valves were concentrated primarily in the intermediate vascular layer. In tabulating the frequency of appearance of AVAs, the mean number was 81.5 per centimeter square. The mean number of AVAs in each of the three vascular layers was 31.1 in the frontal layer, 17.1 in the intermediate layer and 33.1 in dorsal layer, respectively. The vasculature of rabbit ear was further anatomically divided into tip, middle and root portions. The mean number of AVAs in each portion, from the tip to the root was 112.9, 49.4 and 84.4, respectively. Several essential types of AVAs were observed, such as the S-shaped and similar forms which were the predominate type. Others observed were U-shaped, straight, Y-shaped and tri-branching types. Most AVAs within the intermediate vascular layer, consisting of a few small vessels which were mainly capillaries and venules, connected to the anastomosing portion of AVA. The high occurrence and strategic location of AVAs in the rabbit ear strongly suggest that AVAs in the rabbit ear partake in thermal regulation.

A beta-Galactosidase from Radish (Raphanus sativus L.) Seeds.

A basic beta-galactosidase (beta-Galase) has been purified 281-fold from imbibed radish (Raphanus sativus L.) seeds by conventional purification procedures. The purified enzyme is an electrophoretically homogeneous protein consisting of a single polypeptide with an apparent molecular mass of 45 kilodaltons and pl values of 8.6 to 8.8. The enzyme was maximally active at pH 4.0 on p-nitrophenyl beta-d-galactoside and beta-1,3-linked galactobiose. The enzyme activity was inhibited strongly by Hg(2+) and 4-chloromercuribenzoate. d-Galactono-(1-->4)-lactone and d-galactal acted as potent competitive inhibitors. Using galactooligosaccharides differing in the types of linkage as the substrates, it was demonstrated that radish seed beta-Galase specifically split off beta-1,3- and beta-1,6-linked d-galactosyl residues from the nonreducing ends, and their rates of hydrolysis increased with increasing chain lengths. Radish seed and leaf arabino-3,6-galactan-proteins were resistant to the beta-galase alone but could be partially degraded by the enzyme after the treatment with a fungal alpha-l-arabinofuranosidase leaving some oligosaccharides consisting of d-galactose, uronic acid, l-arabinose, and other minor sugar components besides d-galactose as the main product.

Arabinogalactan-Proteins from Primary and Mature Roots of Radish (Raphanus sativus L.).

Organ-specific variations in blood group H-like activity were observed in developing radish plants. A temporary increase in serological activity was found to occur in the roots at the earlier stages of development. Arabinogalactan-proteins (AGPs) were isolated from primary and mature roots, and investigated for changes in their physicochemical properties, structure, and serological activities. These root AGPs were composed mainly of l-arabinose and d-galactose but were distinguishable from each other in their contents of l-fucose as well as of protein and hydroxyproline. The structures of the carbohydrate moieties of the root AGPs were essentially similar to those of AGPs isolated from seeds and mature leaves in that they consisted of consecutive (1-->3)-linked beta-d-galactosyl backbone chains having side chains of (1-->6)-linked beta-d-galactosyl residues, to which alpha-l-arabinofuranosyl residues were attached in the outer regions. One prominent feature of the primary root AGPs was that they contained appreciable amounts of l-fucose, which was presumably responsible for expression of the serological activity. In their immunological reactions with rabbit anti-radish leaf AGP antibody, the root AGPs were shown to share common antigenic determinant(s) with those of seed and leaf AGPs.

Catalytic versatility of Bacillus pumilus beta-xylosidase: glycosyl transfer and hydrolysis promoted with alpha- and beta-D-xylosyl fluoride.

Bacillus pumilus beta-xylosidase, an enzyme considered restricted to hydrolyzing a narrow range of beta-D-xylosidic substrates with inversion of configuration, was found to catalyze different stereochemical, essentially irreversible, glycosylation reactions with alpha- and beta-D-xylopyranosyl fluoride. The enzyme promoted the hydrolysis of beta-D-xylopyranosyl fluoride at a high rate, V = 6.25 mumol min-1 mg-1 at 0 degrees C, in a reaction that obeyed Michaelis-Menten kinetics. In contrast, its action upon alpha-D-xylopyranosyl fluoride was slow and characterized by an unusual relation between the rate of fluoride release and the substrate concentration, suggesting the possible need for two substrate molecules to be bound at the active center in order for reaction to occur. Moreover, 1H NMR spectra of a digest of alpha-D-xylosyl fluoride showed the substrate to be specifically converted to alpha-D-xylose by the enzyme. The observed retention of configuration is not consistent with direct hydrolysis by this "inverting" enzyme but is strongly indicative of the occurrence of two successive inverting reactions: xylosyl transfer from alpha-D-xylosyl fluoride to form a beta-D-xylosidic product, followed by hydrolysis of the latter to produce alpha-D-xylose. The transient intermediate product formed enzymically from alpha-D-xylosyl fluoride in the presence of [14C]xylose was isolated and shown by its specific radioactivity and 1H NMR spectrum as well as by methylation and enzymic analyses to be 4-O-beta-D-xylopyranosyl-D-xylopyranose containing one [14C]xylose residue.(ABSTRACT TRUNCATED AT 250 WORDS)