Shin-ichi Ishii

Frontal affinity chromatography: theory for its application to studies on specific interactions of biomolecules.

Affinity chromatography is very useful in the investigation and characterization of specific interaction between biomolecules. Frontal analysis in affinity chromatography is advantageous from both theoretical and experimental viewpoints. The theory is very simple because we can describe this system by means of a simple equilibrium problem.Chromatographic data can be related easily to the amount of interacting molecules and the equilibrium constant. Useful equations analogous to those of enzyme kinetics can also be derived easily. Thus, frontal affinity chromatography provides information almost identical to that obtainable by enzyme kinetic studies. In addition, this method is more general because it does not depend on enzymatic activity. Experiment is very easy and does not require any special equipment. It is a powerful tool, especially for complicated systems where it has been difficult to find an appropriate method.

A new feature of angiotensin-converting enzyme in the brain: Hydrolysis of substance P

Highly purified rat brain angiotensin-converting enzyme hydrolyzes substance P which contains a C-terminal amino acid with an amidated carboxyl group. The hydrolysis of substance P verified by amino-group fluorometry and by high-performance liquid chromatography is inhibited by captopril, but not by phosphoramidon. The presence of sodium chloride is essential for the hydrolysis. The analyses of cleavage products indicate that the enzyme hydrolyzes substance P between Phe7-Phe8 and Phe8-Gly9 by an endopeptidase action, followed by successive release of dipeptides by a dipeptidyl carboxypeptidase action.

Legumain: asparaginyl endopeptidase

Publisher Summary This chapter describes asparaginyl endopeptidase from jack bean ( C. ensiformis ). During the characterization and purification of legumain from jack bean seeds, DNP-Pro-GIu-Ala-Asn-Val-Ile-Arg-NH 2 (DNP, dinitrophenyl) and DNP-Pro-GIu-Ala-Asn-NH 2 , which contain a sequence similar to that around one of the processing sites in proconcanavalin A, are used as substrates. The presence of the DNP group with absorption near 370 nm allows easy monitoring of separation between an enzymatic reaction product, DNP-Pro-Glu-Ala-Asn, and either of the substrates by high-performance liquid chromatography (HPLC), even when the enzyme in a crude extract of seeds is assayed. The addition of 2-mercaptoethanol to the incubation mixture is essential to yield the full activity. The final preparation of jack bean legumain obtained after the second gel-permeation chromatography on G3000SW gives a single peak in HPLC on a reversed-phase column and a single protein band with mobility corresponding to a molecular mass of about 37,000 Da on sodium dodecyl sulfate (SDS)-polyacrylamide gel after electrophoresis under reducing conditions. The inhibition profile of jack bean legumain suggests that the enzyme is a cysteine-type endopeptidase.

Evidence for the participation of two sperm proteases, spermosin and acrosin, in fertilization of the ascidian, Halocynthia roretzi: Inhibitory effects of leupeptin analogs on enzyme activities and fertilization☆

Ten kinds of argininal-containing compounds were examined for their inhibitory effects on the fertilization of the solitary ascidian and on the activities of acrosin and spermosin, trypsin-like proteases isolated from spermatozoa of this animal. Benzyloxycarbonyl-Val-Pro-argininal (I) and benzyloxycarbonyl-Phe-Leu-argininal (II) showed the strongest inhibition on the fertilization. Leupeptin (acetyl-Leu-Leu-argininal, III) was ranked next (I, II greater than III). The activity of ascidian acrosin was susceptible to most of the compounds, among which II was the best inhibitor and followed with I and III (II greater than I, III). Spermosin suffered significant inhibition only with I and II (I greater than II). These results suggest that not only acrosin but also spermosin is involved in fertilization of the ascidian.

Multiple forms in the subunit structure of concanavalin A

Abstract Concanavalin A, a phytohemagglutinin from jack beans, has at least two forms in the subunit structure. One of them is made up by a single subunit species of molecular weight 27·103 (probably a dimer at pH 5). The other may consist of two kinds of subunits (tetramer), both of which have molecular weight of about a half of the above-mentioned value. The presence of the third structure containing all the three subunits cannot be excluded. Chemical studies on the isolated subunit proteins suggest that the two smaller subunits have a close structural relation to the two fragments, respectively, arising from a polypeptide of the larger subunit when divided into nearly equal sizes.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi: Isolation and characterization

Abstract The lectin from the hemolymph of solitary ascidian, Halocynthia roretzi, has been isolated in an electrophoretically homogeneous form by affinity chromatography on a column of acid-treated Sepharose. It is a large protein with s20,w=24 S, composed of subunits with a molecular weight of 41,000. D-Galactose and various disaccharides containing D-galactose inhibit the hemagglutinating activity of the lectin. Thus, H. roretzi lectin is a D-galactose-specific lectin.

Identification of NDRG1 as an early inducible gene during in vitro maturation of cultured mast cells

Coculture of mouse bone marrow-derived mast cells (BMMC) with fibroblasts in the presence of stem cell factor (SCF) facilitates morphological and functional maturation toward a connective tissue mast cell (CTMC)-like phenotype. By means of cDNA subtraction, we identified several inducible genes during this mast cell maturation process. Of approximately 100 sequenced clones induced, nearly 50% were chromosome 14-associated serine proteases. Approximately 14% encoded NDRG1, a 43-kDa cytosolic protein that has been implicated in cell differentiation. NDRG1 was distributed in the cytosol of cultured mast cells and CTMC in rat skin. Overexpression of NDRG1 in RBL-2H3 cells resulted in enhanced degranulation in response to various stimuli. Thus, NDRG1 may be a mast cell maturation-associated inducible protein that allows the cells to be susceptible to extracellular stimuli leading to degranulation. Additionally, several unique maturation-associated inducible genes were identified, molecular and functional characterization of which will provide new insights into mast cell biology.

A novel lipopolysaccharide-binding hemagglutinin isolated from hemocytes of the solitary ascidian, Halocynthia roretzi: It can agglutinate bacteria

A hemagglutinin was isolated from hemocytes of the ascidian, Halocynthia roretzi, by a procedure including extraction and ion-exchange chromatography on CM-cellulose. The molecular weight of the hemagglutinin was estimated to be 120,000 by gel filtration. It was resistant to acid treatment but sensitive to alkali or heat treatment. The hemagglutinating activity was inhibited by heparin, chondroitin sulfate, and lipopolysaccharide (LPS), but not by mono- and disaccharides such as N-acetyl-galactosamine, galactose, and melibiose. The hemagglutinin showed binding ability to heparin and LPS, as demonstrated by heparin-Sepharose chromatography and centrifugation experiments, respectively. It was also found that the hemagglutinin can bind to various bacteria such as Escherichia coli, Bacillus subtilis, Vibrio anguillarum, Pseudomonas perfectomarinus, Achromobacter aquamarinus, and Alteromonas putrefaciens, and can agglutinate all of them.

Sodium dodecyl sulfate-induced conformational and enzymatic changes of multicatalytic proteinase.

Chymotrypsin-like activity of the multicatalytic proteinase (MCP) purified from eggs of the ascidian Halocynthia roretzi was activated by the addition of SDS. Complete activation was achieved simultaneously at the time of SDS addition, and this activity decreased as a function of time. Autonomous fluorescence of MCP also increased rapidly at the time of SDS addition and then decreased at a rate that depended on the SDS concentration. The decrease of autonomous fluorescence induced by SDS preceded that of the activity. These results suggest that a rapid conformational change of MCP induced by SDS results in the enhancement of chymotrypsin-like activity, followed by the decrease of this activity because of the lability of the activated conformation.

Galactose-specific lectin in the hemolymph of solitary ascidian, Halocynthia roretzi. Molecular, binding and functional properties

Galactose-specific lectin isolated from the hemolymph of solitary ascidian, Halocynthia roretzi, has been further characterized. The hemagglutinating activity of the lectin is Ca2+-dependent. The lectin has a large molecular form as revealed by gel-permeation chromatography, sedimentation equilibrium and velocity measurement, and electron microscopic observation. The lectin is adsorbed to columns of blue-Sepharose and phenyl-Sepharose, and eluted with ethylene glycol, not with lactose or high concentration of NaCl. The lectin shows a stimulatory effect on the superoxide anion production by guinea-pig polymorphonuclear leukocytes, and the effect is inhibited, among various sugars, most strongly by melibiose.