Takuji Sasaki

CHARACTERIZATION OF MAJOR PROTEINS IN SWEET POTATO TUBEROUS ROOTS

The tuberous roots, but not other organs, of sweet potato contained large quantities of two proteins which accounted for more than 80% of the total proteins. The two proteins, tentatively named sporamins A and B, were monomeric forms with similar M,s (25 000). They were separated from each other by electrophoresis on polyacrylamide gels in a non-denaturing buffer or a buffer containing sodium dodecyl sulphate without being reduced by dithiothreitol. They were very similar to each other with respect to amino acid composition, peptide map and immunological properties. These proteins decreased in preference to other proteins during sprouting. The amino acid sequencing of the amino terminal part of sporamin A suggested that it consists of at least two molecular species with different combinations of a few amino acids.

Purification and Some Characteristics of Extracellular Lipase from Fusarium oxysporum f. sp. lini.

An extracellular lipase was isolated from a culture filtrate of Fusarium oxysporum f. sp. lini SUF 402 by hydrophobic chromatography. Purity of the preparation was 38-fold and recovery yield was 32%. The molecular mass of the isolated enzyme was 30 kDa. The lipase had a sugar chain, and the N-terminal amino acid was modified. The optimun pH at 37°C was 7.0. The enzyme had higher activity toward p-nitrophenyl esters with long and middle chain fatty acids (C8-C18) than with a short chain fatty acid (C4). The enzyme was not inhibited by phenylmethylsulfonyl fluoride or 2-mercaptoethanol. In the case of the lipase, both the hydrolysis rate of tristearin and final concentration of fatty acid content were higher than those of triolein. The lipase was examined with respect to its ability of concentrating the n-3 polyunsaturated fatty acid (n-3 PUFA) content of partially hydrolyzed glycerides (TG and DG) obtained from two kinds of fish oil (cod liver oil and refined sardine oil). The lipase gave increases in n-3 PUFA contents as the hydrolysis progressed. The lipase concentrated only docosahexaenoic acid with little increase in icosapentaenoic acid. Maximal total n-3 PUFA contents were about 30% in both fish oils.

Purification and characterization of proteases responsible for vitellin degradation of the silkworm, Bombyx mori

Hydrolytic activity of benzoyl-dl-arginine-p-nitroanilide (BApNA) by crude egg extracts exhibited two peaks during the latter period of embryogenesis of the silkworm, Bombyx mori. The first peak appeared at the time of marked degradation of vitellin, and consisted of two proteases. The two proteases were purified from day 8 eggs to homogeneity by DEAE-cellulose, Sephadex G-75 and native-gel electrophoresis. One enzyme had a molecular mass of 30,000 Da and the other 24,000 Da. The NH2-terminal amino acid sequences were different from one another and only the 30 k-protease showed the sequence common to the trypsin family. Both enzymes showed similar properties in Km values and optimum pH, but different temperature dependence. Inhibition spectrum and substrate specificity suggest that these enzymes belong to the trypsin-like serine protease family. The purified proteases cleaved two yolk proteins, vitellin and egg-specific protein, into small peptides through limited hydrolysis, but could not attack the 30 kDa proteins which are the second major yolk proteins of silkworms. Degradation of vitellin was characterized by the preferential hydrolysis of a 178 kDa large subunit. From the biochemical properties and developmental changes, these two proteases appear to be responsible for the degradation of vitellin in eggs undergoing embryogenesis.

Structure, heterologous expression, and properties of rice (Oryza sativa L.) family 19 chitinases.

We identified four new family 19 chitinases in Oryza sativa L. cv. Nipponbare: one class I (OsChia1d), two class II (OsChia2a and OsChia2b), and one class IV (OsChia4a). OsChia2a resembled (about 60% identity) the catalytic domains of class I chitinases, but OsChia2b was almost identical (95% identity) to that of the class IV enzyme. OsChia1c, OsChia1cΔCBD (a deletion of OsChia1c lacking a chitin-binding domain, CBD), and OsChia2b were separately expressed and purified in Pichia pastoris. OsChia1c inhibited fungal growth significantly more than OsChia1cΔCBD or OsChia2b. The activities of these enzymes on chitin polymers were similar, but they acted differently on N-acetylchitooligosaccharides, (GlcNAC)n. OsChia1c slowly hydrolyzed (GlcNAC)6 and very poorly hydrolyzed (GlcNAC)4 and (GlcNAC)5. In contrast, OsChia2b efficiently hydrolyzed these oligosaccharides. The high antifungal activity and low hydrolytic activity of the class I enzyme towards (GlcNAC)n imply that it participates in the generation of N-acetylchitooligosaccharide elicitors from the cell walls of infecting fungi.

Amino acid sequence of a novel Kunitz-type chymotrypsin inhibitor from hemolymph of silkworm larvae, Bombyx mori

The primary structure of the chymotrypsin inhibitor (SCI‐III), from larvae of the silkworm, Bombyx mori, has been determined. This inhibitor seems to be a Kunitz‐type inhibitor judging from the sequence homology with the bovine basic pancreatic trypsin inhibitor, but has one more amino acid in the half‐cystine frame nearest the N‐terminus. The amino acid at the deduced reactive site (P1) is phenylalanine and this inhibitor is the first example of the Kunitz type that is assigned phenylalanine to its reactive site.

The occurrence of a premature form of egg-specific protein in vitellogenic follicles ofBombyx mori

SummaryAnalysis of yolk proteins of the silkworm,Bombyx mori, by SDS-polyacrylamide gel electrophoresis and immunoblotting showed that there was a developmental change in subunit composition of egg-specific protein; egg-specific protein consisting of 72 kDa subunits alone (premature form) was found in vitellogenic follicles, whereas the protein in mature eggs was composed of 72 kDa and 64 kDa subunits (mature form). The premature form of egg-specific protein was purified from young ovaries to homogeneity using a high performance liquid chromatography system. The purified protein had an apparent molecular mass of 225 kDa which could not be distinguished from that of the mature form. By circular dichroism analysis, both egg-specific proteins were estimated to have about 30% α-helix and 20% β-sheet, but the mature form showed a relatively rigid conformation in the aromatic region. The premature egg-specific protein purified from vitellogenic ovaries, consisted of three 72 kDa subunits, whereas mature egg-specific protein was composed of two 72 kDa subunits and one 64 kDa subunit. All of these subunits showed the same immunoreactivity towards antiserum raised against the mature form. An identical NH2-terminal amino acid sequence was found in both 72 kDa polypeptides and 64 kDa polypeptide for the initial 10 amino acids.

Amino acid sequence of alkaliphilic serine protease from silkworm, Bombyx mori, larval digestive juice

Alkaliphilic protease, P‐IIc, from silkworm, Bombyx mori, larval midgut digestive juice consists of 232 amino acids. It has a catalytic triad, Asp‐His‐Ser, invariably found in a serine protease. A shift of optimal pH value towards the alkaline side diminished at μ=1.0. This suggests the existence of an electrostatic interaction that affects the proteolytic activity. The higher Arg content may be responsible for this phenomenon. Two cysteine residues probably exist unpaired in a novel position among serine proteases.

The reactive site of silkworm hemolymph antichymotrypsin is located at the COOH-terminal region of the molecule

The complex of silkworm larval hemolymph antichymotrypsin (Mr = 43,000) and C-chain of bovine alpha-chymotrypsin was obtained. This complex showed two NH2-terminal amino acid sequences identical to those of intact silkworm antichymotrypsin and C-chain of alpha-chymotrypsin, respectively. Alkali treatment of the complex brought about its dissociation and the separated inhibitor component (Mr = 36,000) had an NH2-terminal amino acid sequence identical to that of intact silkworm antichymotrypsin. These results suggest that the reactive site of this inhibitor is located at the COOH-terminal region of the molecule and that the nature of association of this inhibitor and alpha-chymotrypsin is an acyl-bond.

Structure of fatty acid synthetase from the harderian gland of guinea pig: Proteolytic dissection and electron microscopic studies

Limited proteolysis and electron microscopic observation of fatty acid synthetase from the Harderian gland of guinea pig was performed to elucidate the higher-order structures of this multifunctional protein. Staphylococcus aureus V8 protease dissected the 250,000 Mr subunit of fatty acid synthetase into 120,000, 70,000, 35,000 and 30,000 Mr fragments, which were aligned in this order from the NH2 terminus. Some of the protease-resistant fragments produced with elastase, trypsin and lysyl endopeptidase were purified and fragment-specific antibodies (A40L, A33E and A25T) were prepared. A25T and A33F specifically bound the 35,000 and 30,000 Mr fragments, and A40L recognized the region between the 120,000 and 70,000 Mr fragments. Electron microscopic studies employing rotary shadowing, unidirectional shadowing and negative staining revealed that the overall dimension of the enzyme was 22 nm x 15 nm x 7 nm, and that two elongated subunits mainly composed of three subregions were in contact with each other at a few, three at most, points with two holes between them. The outer two attachment sites were often not in contact, indicating a certain flexibility of subunits at their ends. Immunocomplexes composed of fatty acid synthetase and fragment-specific antibodies were isolated and observed under the electron microscope. The attachment sites of A40L and A33E were located at the end of the minor and the major axes of the ellipsoidal contour of the molecule, respectively. Based on these results, the three-dimensional structure of animal fatty acid synthetase is discussed.

Purification and Properties of the Endo-1,4-β-Glucanase from Ruminococcus albus and Its Gene Product in Escherichia coli

Abstract Endoglucanases, EGI and EgI, were produced from the same Ruminococcus albus gene in R. albus and recombinant Escherichia coli , respectively. EGI was purified from R. albus culture supernatant and EgI was extracted from the transformant E. coli (JM101/pURA1) and purified. The purified enzymes EGI and EgI revealed maximum endoglucanase activity at a same pH of 6.8 and a temperature of 37°C. Both enzymes were stable at temperatures below 30°C. In addition, about 10% of their original activities were conserved even after boiling for 10 min. Amino acid sequences of both enzymes at the N-terminal (Ala-Ala-Asp-Glu-Ser-Glu-Thr-Glu-Asn-Val-Pro-Val-Ser-Gln-Thr-His--) were consistent with each other. The antiserum against EgI reacted with both EgI and EGI, indicating that both their protein moieties were the same immunologically. However, the molecular size of EGI (43,000) was larger than that of EgI (39,000) due to the presence of sugar moiety. The specific activity (54 units/mg) of EGI was almost double that (27 units/mg) of EgI. EGI was immunologically different from the endoglucanase purified in the previous paper [Ohmiya et al. : Carbohydrate Res., 166, 145–155 (1987)].