Activation of prephenoloxidase. 3. Release of a peptide from prephenoloxidase by the activating enzyme

Activation reaction of prephenoloxidase (pre-enzyme) was analyzed using a system of homogeneous pre-enzyme and highly purified prephenoloxidase-activating enzyme (PPAE) from the silkworm $\text{Bombyx mori}$. When pre-enzyme was activated by PPAE, release of a peptide was demonstrated. Results of polyacrylamide gel electrophoresis revealed that only a single peptide is liberated from pre-enzyme. Several lines of evidence indicated that the released peptide is inhibitory on PPAE.     

Evidence for the Existence of Isozymes of Glucose Isomerase from Streptomyces phaeochromogenes

Glucose isomerase from Streptomyces phaeochromogenes was purified from a commerical preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5 m, and hydroxyapatite column chromatographies. It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite. They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties. The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide. The results suggest that glucose isomerase from S. phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.     

Evidence for the existence of isozymes of glucose isomerase from Streptomyces phaeochromogenes.

Glucose isomerase from Streptomyces phaeochromogenes was purified from a commercial preparation, Swetase, by DEAE-cellulose, Bio-Gel A-0.5 m, and hydroxyapatite column chromatographies. It was found to be 2 fractions; F-A, not adsorbed on hydroxyapatite and F-B, adsorbed on hydroxyapatite. They were homogeneous in ordinary and SDS-PAGE and had similarities in some enzymatic and physico-chemical properties. The differences, however, were found in the N-terminal amino acid, which was only serine for F-A while it was serine and alanine for F-B, and also in their peptide mapping patterns of digests with trypsin, Achromobacter protease I, and cyanogen bromide. The results suggest that glucose isomerase from S. phaeochromogenes was composed of the two kinds of isozymes and that each of isozymes was a tetramer constituted of non-identical subunits.     

Purification and cloning of a novel serine protease, RNK-Met-1, from the granules of a rat natural killer cell leukemia.

We have purified a 30-kDa serine protease (designated RNK-Met-1) from the granules of the rat large granular lymphocyte leukemia cell line (RNK-16) that hydrolytically cleaves model peptide substrates after methionine, leucine, and norleucine (Met-ase activity). Utilizing molecular sieve chromatography, heparin-agarose, chromatography, and reverse-phase high pressure liquid chromatography, RNK-Met-1 was purified to homogeneity and 25 NH2-terminal amino acids were sequenced. By using the polymerase chain reaction, oligonucleotide primers derived from amino acids at position 14-25 and from a downstream active site conserved in other serine protease genes were used to generate a 534-base pair cDNA clone encoding a novel serine protease from RNK-16 mRNA. This cDNA clone was used to isolate a full-length 867-base pair RNK-Met-1 cDNA from an RNK-16 lambda-gt11 library. The open reading frame predicts a mature protein of 238 amino acids with two potential sites for N-linked glycosylation. The cDNA also encodes a leader peptide of at least 20 amino acids. The characteristic Ile-Ile-Gly-Gly amino acids of the NH2 terminus and the His, Asp, and Ser residues that form the catalytic triad of serine proteases were both conserved. The amino acid sequence has less than 45% identity with any other member of the serine protease family, indicating that RNK-Met-1 is distinct and may itself represent a new subfamily of serine proteases. Northern blot analysis of total cellular RNA detected a single 0.9-kilobase mRNA in the in vitro and in vivo variants of RNK-16 and in spleen-derived plastic-adherent rat lymphokine-activated killer cells. RNK-Met-1 mRNA was not detectable in freshly isolated rat splenocytes, thymocytes, brain, colon, and liver or activated nonadherent rat splenocytes and thymocytes. These data indicate that RNK-Met-1 is a serine protease with unique activity that is expressed in the granules of large granular lymphocytes.     

In vivo analysis of fibroin heavy chain signal peptide of silkworm Bombyx mori using recombinant baculovirus as vector

In order to investigate the functional signal peptide of silkworm fibroin heavy chain (FibH) and the effect of N- and C-terminal parts of FibH on the secretion of FibH in vivo, N- and C-terminal segments of fibh gene were fused with enhanced green fluorescent protein (EGFP) gene. The fused gene was then introduced into silkworm larvae and expressed in silk gland using recombinant AcMNPV (Autographa californica multiple nuclear polyhedrosis virus) as vector. The fluorescence of EGFP was observed with fluorescence microscope. FibH-EGFP fusion proteins extracted from silk gland were analyzed by Western blot. Results showed that the two alpha helices within N-terminal 163 amino acid residues and the C-terminal 61 amino acid residues were not necessary for cleavage of signal peptide and secretion of the fusion protein into silk gland. Then the C-terminal 61 amino acid residues were substituted with a His-tag in the fusion protein to facilitate the purification. N-terminal sequencing of the purified protein showed that the signal cleavage site is between position 21 and 22 amino acid residues.     

Cloning, overexpression, purification, and characterization of O-acetylserine sulfhydrylase-B from Escherichia coli

O-Acetylserine sulfhydrylase-B (OASS-B, EC 2.5.1.47) is one of the two isozymes produced by Escherichia coli that catalyze the synthesis of L-cysteine from O-acetyl-L-serine and sulfide. The cysM gene encoding OASS-B was cloned and the enzyme was overexpressed in E. coli using pUC19 with a lacUV5 promoter. The enzyme was purified to homogeneity, as evidenced by SDS-PAGE. Approximately 300 mg of purified OASS-B was obtained from 1600 mL of culture broth with a purification yield of 60% or higher. The purified OASS-B was characterized and its properties compared with OASS-A. OASS-B did not form a complex with E. coli serine acetyltransferase (SAT, EC 2.3.1.30) and showed a wide range of substrate specificity in nonproteinaceous amino acid synthesis.     

Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions

Each of the two isozymes, which are different in thermostability and quaternary structure, of isocitrate dehydrogenase (NADP+) [IDH: EC 1.1.1.42] was purified to an electrophoretically homogeneous state from an obligately psychrophilic marine bacterium, Vibrio sp. strain ABE-1. Hydrophobic chromatography was an efficient procedure to separate the two isozymes from each other. The isoelectric points of isozyme I (IDH-I; a dimer, Mr 88,100) and isozyme II (IDH-II; a monomer, Mr 80,500) were found to be pH 4.9 and 5.2, respectively. The two isozymes were similar in amino acid compositions, though there were slight differences in the contents of nonpolar and hydroxyl amino acids. However, their NH2-terminal amino acid sequences and immunochemical properties were clearly different from each other. The NH2-terminal amino acid sequence analysis also indicated that the subunits of IDH-I are chemically identical or highly homologous. Non-immuno-crossreactivity between the isozymes enabled us to measure the intracellular contents of the isozymes. IDH-I and -II were found to be differentially regulated in vivo by various growth conditions. IDH-I was induced by acetate, while IDH-II remained almost unchanged.     

D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Practical utility of an antibody specific to a synthetic peptide corresponding to the N-terminal amino acid sequence of human urine DNAse I for genetic analysis of human DNase I isozymes

An antibody specific to a synthetic peptide corresponding to the N-terminal 27 amino acid residues of human urine DNase I (anti-DNase I peptide) was obtained. The antibody did not inhibit the activity of the enzyme, but reacted well with the enzyme upon immunoblotting following electrophoresis. The urine DNase I isozyme patterns detected using this antibody were almost identical to those produced with an antibody specific to purified DNase I. Therefore, the anti-DNase I peptide antibody should prove to be valuable for genetic analysis of human DNase I isozymes.     

Comparative studies of two cathepsin B isozymes from porcine spleen. Isolation, polypeptide chain arrangements, and enzyme specificity

Our previous studies on carbohydrate structures of purified porcine spleen cathepsin B indicated that there are two cathepsin B isozymes, each containing a different carbohydrate (Takahashi, T., Schmidt, P.G., and Tang, J. (1984) J. Biol. Chem. 259, 6059-6062). We have now isolated these two enzymes and carried out a comparative study on their structures and enzymic properties. The major isozyme (CB-I) is a two-chain enzyme (Mr = 28,000) with a light chain (Mr = 5,000) and a heavy chain (Mr = 23,000), whereas the minor enzyme (CB-II) is a single chain enzyme (Mr = 27,000). The NH2-terminal amino acid residues of CB-I were leucine and valine for the light and heavy chain, respectively. However, the NH2-terminal residue of CB-II was not available for automated Edman degradation. In addition, peptide mapping experiments indicated a difference in the primary structure of these two proteins. Despite such structural differences, they are similar in many enzymic properties. CB-I was more catalytically efficient than CB-II toward synthetic substrates, except for the substrate benzoyl-L-arginine beta-naphthylamide for which the relative catalytic efficiency is reversed. Both isozymes degraded glucagon by a dipeptidyl carboxypeptidase activity. Under the same conditions, CB-I was 4-5 times more efficient than CB-II. The results indicate that the cathepsin B isozymes are two separate gene products, but they are similar in enzymic properties.     

Plant triose phosphate isomerase isozymes : purification, immunological and structural characterization, and partial amino Acid sequences

We report the first complete purifications of the cytosolic and plastid isozymes of triose phosphate isomerase (TPI; EC 5.3.1.1) from higher plants including spinach (Spinacia oleracea), lettuce (Lactuca sativa), and celery (Apium graveolens). Both isozymes are composed of two isosubunits with approximate molecular weight of 27,000; in spinach and lettuce the plastid isozyme is 200 to 400 larger than the cytosolic isozyme. The two isozymes, purified from lettuce, had closely similar amino acid compositions with the exception of methionine which was four times more prevalent in the cytosolic isozyme. Partial amino acid sequences from the N-terminus were also obtained for both lettuce TPIs. Nine of the 13 positions sequenced in the two proteins had identical amino acid residues. The partial sequences of the plant proteins showed high similarity to previously sequenced animal TPIs. Immunological studies, using antisera prepared independently against the purified plastid and cytosolic isozymes from spinach, revealed that the cytosolic isozymes from a variety of species formed an immunologically distinct group as did the plastid isozymes. However, both plastid and cytosolic TPIs shared some antigenic determinants. The overall similarity of the two isozymes and the high similarity of their partial amino acid sequences to those of several animals indicate that TPI is a very highly conserved protein.     

Primary structure of the silk fibroin light chain determined by cDNA sequencing and peptide analysis

A cDNA clone, pFL18, carrying a putative full-length fibroin light chain (L-chain) sequence was isolated and its nucleotide sequence was determined. This revealed the presence of an open reading frame corresponding to a polypeptide with 262 amino acid residues. The sequence was concluded to be that of the L-chain with its signal peptide because corresponding amino acid sequences for the seven tryptic and the four chymotryptic peptides from the purified L-chain were all included and an N-terminal region having typical properties of a signal peptide was present. The N terminus of the mature form of L-chain was identified as N-acetyl serine by analyzing the acyl-dansylhydrazide derived from the N-acyl-amino acid which had been released from the N-terminal blocked chymotryptic peptide by the acylamino acid-releasing enzyme. It was suggested that a signal peptide had cleaved between Pro18 and Ser19, yielding a mature L-chain polypeptide consisting of 244 amino acid residues. The molecular weight of the L-chain was calculated to be 25,800 including the N-acetyl group. The L-chain contained three Cys residues, two of which were suggested to form an intramolecular disulfide linkage, leaving the third one at the most C-terminal position and in a relatively hydrophilic region as the most probable site of disulfide linkage with the fibroin heavy chain.     

Characterization of the isozymes of glucuronoxylan xylanohydrolase in the presence of a native cell wall substrate

K. HAYASHI, M. INOUHE, C. AOYAGI AND D.J. NEVINS. 1996. A simple but accurate method for measuring glucuronoxylan xylanohydrolase activity was developed using coleoptile cell wall particles prepared from maize ( Zea mays L.). Two isozymes of glucuronoxylan xylanohydrolases designated as GX1 and GX2 (EC 3.2.1.136) were purified from a commercially available amylase preparation to electrophoretic homogeneity by three cation exchange chromatography steps. Upon characterization no significant differences between the two enzymes were detected: the molecular mass measured by MALDITOF mass spectrometry was 44 360 100 for GX1 and 44 370 50 for GX2 suggesting no difference in the total number of amino acid residues. Furthermore the N -terminal amino acid sequence for each of the isozymes was identical through the 37th amino acid residue. The values of pI were determined to be 9.0 for GX1 and 9.1 for GX2. The sensitivity to temperature, pH and to ionic strength was similar for both isozymes as were kinetic parameters including K _m and V _maxm No differences could be detected in substrate specificity.     

Liver isozyme of rabbit glycogen synthase. Amino acid sequences surrounding phosphorylation sites recognized by cyclic AMP-dependent protein kinase

Glycogen synthase, the rate-limiting enzyme in glycogen biosynthesis, has been postulated to exist as isozymes in rabbit liver and muscle (Camici, M., Ahmad, Z., DePaoli-Roach, A. A., and Roach, P. J. (1984) J. Biol. Chem. 259, 2466-2473). Both isozymes share a number of properties including multiple phosphorylation of the enzyme subunit. In the present study, we determined the amino acid sequences surrounding phosphorylation sites in the rabbit liver isozyme recognized by cyclic AMP-dependent protein kinase. Two dominant phosphopeptides (P-1 and P-2) were generated from tryptic digestion. Amino acid sequences of the purified peptides were determined by automated Edman degradation using a gas-phase sequenator. The locations of phosphorylated residues were identified by measuring 32Pi release during Edman degradation cycles. The NH2-terminal sequence of peptide P-1 is S-L-S(P)-V-T-S-L-G-G-L-P-Q-W-E-V-E-E-L-P-V-D-D-L-L-L-P-E-V. This sequence exhibits a strong homology to the site 2 region in the NH2 terminus of the muscle isozyme. The NH2-terminal sequence of peptide P-2 is M-Y-P-R-P-S(P)-S(P)-V-P-P-S-P-L-G-S-Q-A. This sequence shows strong homology to the site 3 region in the COOH terminus of the muscle isozyme. However, some interesting sequence differences were revealed in this region. For example, substitution of serine for alanine at position 6 of peptide P-2 created a new phosphorylation site for cyclic AMP-dependent protein kinase. Phosphorylation of the proline/serine-rich site 3 region correlated with inactivation of the liver isozyme and suggests an important role for this segment of the molecule in the regulation of glycogen synthase. No phosphorylation sites corresponding to sites 1a and 1b of the muscle isozyme were detected. In addition, the results provide definitive chemical proof that glycogen synthase from rabbit liver and muscle are isozymes encoded by distinct messages.     

Nucleotide sequence of cDNA for the eclosion hormone of the silkworm, Bombyx mori, and the expression in a brain.

The cDNAs encoding eclosion hormone (EH) of the silkworm, Bombyx mori, were isolated and sequenced. The results showed that the pre-EH molecule contains a 26-amino acid signal peptide and a 62-amino acid mature EH. The deduced amino acid sequence agreed with that previously determined by the peptide analysis. The presence of leucine residue at the carboxyl terminal of EH, which had not been detected directly by the peptide analysis, was proved. Primer extension and Northern hybridization analyses revealed that 0.9 kb mRNA is transcribed and it has a 66-nucleotide non-translated sequence at the 5'-end region. In situ hybridization showed that the EH gene is expressed in two pairs of nuerosecretory cells in the brain of 5th instar larva.     

Detection, purification and characterization of a human cancer-associated galactosyltransferase acceptor.

A low-molecular-weight acceptor of galactosyltransferase activity was detected in sera and effusions of patients with extensive maligant disease. This substance was purified to homogeneity from both human serum and effusion by using sequential charcoal/Celite and DEAE-cellulose column chromatography. The purified acceptor was shown to act as substrate for both purified normal and cancer-associated human galactosyltransferase (EC 2.4.1.22) isoenzymes, but had a higher affinity for the cancer-associated isoenzyme (Km = 20 microM) than for the normal isoenzyme (Km = 500 microM). The substrate was found to be a glycopeptide with mol.wt. approx. 3600 determined by polyacrylamide-gel chromatography. Carbohyydate analysis demonstrated only the presence of glucosamine and mannose. Amino acid analysis revealed that the peptide moiety consisted of eight different amino acids, including two residues of asparagine and one residue of serine, but no threonine. These structural data suggest that the acceptor is a fraction of an asparagine-glucosamine type of glycoprotein.     

Isolation, purification and characterization of silk protein sericin from cocoon peduncles of tropical tasar silkworm, Antheraea mylitta

A high molecular weight water-soluble glue protein, sericin was identified in the cocoon peduncle (a strong thread connecting the cocoons to the branches of the tree with a ring) of the tropical tasar silkworm, Antheraea mylitta. The sericin was isolated by 8 M urea containing 1% sodium dodecyl sulfate and β-mercaptoethenol (2%) or by 1% sodium chloride. The protein was purified by gel filtration chromatography. In SDS-PAGE, a single band of approximately 200 kDa was detected both in non-reducing and reducing conditions. Amino acid analysis showed that the protein is enriched in glycine and serine. There is a slight difference observed in amino acid composition between the sericin from cocoon peduncle and cocoon of A. mylitta. Secondary structure estimation by circular dichroism spectrometry showed 36.7% β-sheets, 52.7% random coils, 10.6% turns and no helices.     

A novel protease obtained from FBS-containing culture supernatant,that processes single chain form hepatocyte growth factor to two chain form in serum-free culture

The recombinant human hepatocyte growth factor (r-hHGF) produced by Chinese hamster ovary cells transfected with hHGF cDNA (CHO BD-24 cells) was the two chain form in fetal bovine serum (FBS) containing culture. However, in serum-free culture the non-processed r-hHGF, single chain form, was detected with two chain form r-hHGF. We purified the protease that proteolytically processed single chain r-hHGF to two chain form r-hHGF. A protease was purified to give a single peak from the culture supernatant by use of several column chromatographies. When this protease was added to serum-free culture of CHO BD-24 cells, the proteolytic processing of single chain r-hHGF to two chain form r-hHGF was completely achieved. This protease was found to be composed of two peptide chains with molecular mass of 38 kDa under non-reducing condition by SDS-PAGE. The results of N-terminal amino acid sequence analysis and inhibitor selectivity suggested that this protease was a novel serine protease originating from fetal bovine serum.     

“两广二号”家蚕抗病毒基因Bmlipase-1和BmSP-2克隆与原核表达

本研究以优良杂交品种"两广二号"家蚕为试材,克隆了该杂交品种家蚕两个抗家蚕核型多角体病毒(BmNPV)基因:脂肪酶基因Bmlipase-1和丝氨酸蛋白酶基因BmSP-2,测序并分别与不同品种蚕的同源基因序列进行比较。结果显示,"两广二号"家蚕Bmlipase-1基因ORF长度为885bp,编码294个氨基酸,BmSP-2扩增长度为855bp,编码284个氨基酸;它们的核苷酸和推导氨基酸序列同源性皆达92%以上,Bmlipase-1更保守,同源性大于99%";两广二号"家蚕的Bmlipase-1基因脂肪酶活化部位和BmSP-2基因酶催化三联体位点的氨基酸残基与不同品种蚕的完全相同。以上结果说明这两个抗病毒基因在蚕的遗传进化过程中高度保守,提示其可能在机体消化或者免疫防御方面起着重要生理作用。将这两个抗病毒基因在大肠杆菌BL21中进行融合表达,获得的融合Bmlipase-1和BmSP-2蛋白分子量分别为47kD和42kD左右。     

The small subunits of calcium dependent proteases with different calcium sensitivities are identical

The small subunits of two calcium dependent proteases from rabbit with different calcium sensitivities were isolated by high performance liquid chromatography (HPLC) and their properties were compared. The isolated subunits were indistinguishable on polyacrylamide gel electrophoresis and HPLC. Their amino acid compositions were identical, and the peptides obtained on their digestion with lysyl-endopeptidase showed identical peptide maps on HPLC. Furthermore, the amino acid compositions and partial amino acid sequences of the corresponding peptides purified by HPLC were the same. These results indicate that the two calcium protease isozymes possess the same small subunit.