Purification of an embryotrophic factor from commercial bovine serum albumin and its identification as citrate.

A factor of low M(r) with growth-promoting effects on rabbit embryos was extracted and purified from commercial bovine serum albumin (BSA). This embryotrophic factor was extracted from BSA dissolved in formic acid by membrane filtration (membrane cutoff of M(r) 10,000) and then freeze-drying of the filtrate. The extract was purified successively by chromatography on G-10 Sephadex, QAE-Sephadex A-25 anion exchange and high-performance liquid chromatography (HPLC) reverse-phase columns. Mass spectrometry of the active reverse-phase material indicated that the major component in this material had an M(r) of 192. The embryotrophic factor in the low M(r) extract of BSA was shown to be citrate, because: (i) the mass spectra of the active reverse-phase material and citrate were identical, (ii) the activity was eluted at the identical position to citrate on an analytical HPLC anion-exchange column, (iii) the original BSA sample was shown by enzyme assay to be heavily contaminated by citrate and (iv) citrate stimulated cell proliferation and expansion of blastocysts.     

A Novel Glial Growth Inhibitory Factor, Gliostatin, Derived from Neurofibroma

Neurofibroma tissue was investigated for the presence of glial growth modulators that would suppress the proliferation of glial cells. A novel endogenous polypeptide inhibitor of proliferation and DNA synthesis in glial cells, gliostatin, was purified from the extracts of neurofibroma by a procedure comprising dye and anion-exchange column chromatography, and HPLC. A monoclonal antibody raised against partially purified gliostatin showed no cross-reactivity with known cytokines, but adsorbed the growth inhibitory activity of gliostatin and immunochemically visualized the putative gliostatin bands on western blot analyses. Although the product showed an apparent M(r) of 100,000 accompanied by an inhibitory activity on gel filtration column chromatography, it migrated at a lower apparent M(r) of 50,000 under the reducing conditions on western blotting, indicating that a homodimeric structure of native gliostatin consisted of 50-kDa subcomponents. Gliostatin was a potent growth inhibitor acting at nanomolar concentrations against all glial tumor cells and glia maturation factor-stimulated astroblasts, but not neuronal cells.     

Structural analysis of a unique hybrid-type ganglioside with isoglobo-, neolacto-, and ganglio-core from the gills of the Pacific salmon (Oncorhynchus keta)

Monosialosyl gangliosides from the gills of the Pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. The unknown acidic glycolipids (M14 and M15) with slower mobility than GM1a on thin-layer chromatography were separated by Iatrobeads column chromatography and were characterized by compositional analysis, methylation analysis, chemical, and enzymatic degradation, negative-ion LSIMS, and (1)H nuclear magnetic resonance spectroscopy. Both M14 and M15 contained a same oligosaccharide core with isoglobo-, neolacto-, and ganglio-series as follows: [carbohydrate structure: see text]. The only difference between M14 and M15 was in fatty acid acylation. Analysis of the fatty acids indicated a predominance of C24:1 fatty acid in M14 and shorter chain saturated fatty acids, C14:0 and C16:0, in M15.     

Urokinase separation from cell culture broth of a human kidney cell line

A single step ion-exchange chromatography on a sulfo-propyl (SP)- Sepharose column was performed to separate both the high molecular weight (HMW)- and low molecular weight (LMW)- forms of enzymatically active urokinase type plasminogen activator from human kidney (HT1080) cell culture media. The level of urokinase secreted by the cell line reached to about 145 Plough units/ml culture broth within 48 h of cultivation. The conditioned cell culture media was applied directly to the column without any prior concentration steps. Polyacrylamide gel electrophoresis of the column eluates in the presence of sodium dodecyl sulphate showed that the cell line secretes three forms of two-chain high molecular weight (HMW) urokinase of molecular weights (M(r)) 64,000, 60,900 and 55,000. In addition, two low molecular weight (LMW) forms of M(r) 22,000 and 20,000; proteolytic cleavage products of HMW, were also found. The HMW and LMW forms had intrinsic plasminogen dependent proteolytic activity as judged by zymographic analysis. The specific activity of the pooled peak fractions increased (approximately 93-fold) to values as high as 1481 Plough units/ mg protein. Both HMW as well as LMW forms were obtained in significantly high yields.     

Involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing intestinal IEC-6 cell death

Monomeric 14-kDa bovine alpha-lactalbumin was purified with a preparation of lower molecular weight whey protein concentrate from Holstein cow normal milk followed by size exclusion chromatography. The protein showed a stimulatory rather than an inhibitory effect on the proliferation of a cultured IEC-6 cell line from the rat small intestine. But incubation in 30% trifluoroethanol/acetate buffer (pH 5.5) at 37 degrees C for 5 d in a slowly rotating test tube rendered it highly cytotoxic with concomitant appearance of SDS-stable 20- and 30-kDa forms of alpha-lactalbumin on electrophoresis. Furthermore, alpha-lactalbumin obtained by a one-step purification procedure by affinity chromatography on an anti-alpha-lactalbumin antibody column from the lower molecular weight whey protein concentrate, which had been found to contain several SDS-stable higher M(r) forms of alpha-lactalbumin, exhibited potent inhibitory activity on IEC-6 cell growth. These results indicate the involvement of SDS-stable higher M(r) forms of bovine normal milk alpha-lactalbumin in inducing cell death on the intestinal IEC-6 cell line.     

A serpin with M(r) 43,000 is a binding protein of M(r) 25,000 protein, a substrate for protein ser/thr kinase detected in Xenopus laevis oocytes

In an attempt to get some clue as to the function of M(r) 25,000 protein, a protein Ser/Thr kinase substrate detected in Xenopus laevis oocytes [Hashimoto, E. et al. (1995) J. Biochem. 118, 453-460], the binding protein was surveyed using the (32)P-labeled protein by casein kinase II as a screening probe. When the cytosolic proteins from oocytes were transferred to a polyvinylidene fluoride membrane and incubated with the labeled protein, only one protein with M(r) 43,000 was visualized on autoradiography. This protein was purified to a nearly homogeneous state through several column chromatography steps. The amino acid sequence of the amino-terminal region of this protein identified it as a kind of serine protease inhibitor (serpin) [Holland, L.J. et al. (1992) J. Biol. Chem. 267, 7053-7059]. However, the M(r) 25,000 protein did not have any effect on the inhibitory action of this serpin on alpha-chymotrypsin. In addition, several binding proteins were also detected in the particulate fraction of oocytes, although the exact identity of these proteins is not clear at this time. These results suggest that the M(r) 25,000 protein may play some role(s) by interacting with these binding proteins in Xenopus oocytes.     

Lipoprotein separation and low density lipoprotein molecular weight determination using high performance gel-filtration chromatography

Plasma lipoproteins were isolated at d less than 1.225 g/ml from nonhuman primates of three species, cynomolgus, rhesus, and African green (vervet) monkeys. Individual lipoprotein classes were separated by high performance gelfiltration chromatography and low density lipoprotein (LDL) molecular weight was determined. A comparison was made using column configurations including TSK 3000 SW, 4000 SW, and 5000 PW columns. Due to its relative simplicity, stability, and economy, a single 5000 PW column was selected for most of the work. The recovery of lipoprotein cholesterol from the column averaged 91 +/- 2.5%. A comparison of the immunologic, chemical, and electrophoretic properties of high density lipoproteins (HDL) and LDL isolated by this technique with those of HDL and LDL isolated by conventional agarose column chromatography indicated that lipoproteins isolated by high performance gel-filtration chromatography were intact and reasonably free of cross contamination. A standard preparation of 125I-labeled LDL was added to the d less than 1.225 g/ml lipoprotein fraction just prior to separation and a relative size index, r1, was determined. When r1 values for a large number of samples were compared with the log of the LDL molecular weight (determined by agarose column chromatography) a linear relationship was found with a correlation coefficient, r = 0.85. The regression equation for this relationship could be used to calculate LDL molecular weights from the r1 value. These values agreed with LDL molecular weight determined by flotation equilibrium analysis in the analytical ultracentrifuge. We conclude that high performance gel-filtration chromatography using the TSK 5000 PW column provides an analytical and preparative technique for simultaneous separation of individual lipoproteins and determination of LDL molecular weight.     

Purification and characterization of a novel enzyme, alpha-neoagarooligosaccharide hydrolase (alpha-NAOS hydrolase), from a marine bacterium, Vibrio sp. strain JT0107.

A novel enzyme, alpha-neoagarooligosaccharide hydrolase (EC 3.2.1.-), which hydrolyzes the alpha-1,3 linkage of neoagarooligosaccharides to yield agaropentaose (O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro-alpha-L-galactopyranosyl (1-->3)-D-galactose], agarotriose [O-beta-D-galactopyranosyl(1-->4)-O-3,6-anhydro- alpha-L-galactopyranosyl (1-->3)-D-galactose], agarobiose [O-beta-D-galactopyranosyl(1-->4)-3,6-anhydro-L-galactose], 3,6-anhydro-L-galactose, and D-galactose was isolated from the marine bacterium Vibrio sp. strain JT0107 and characterized. This enzyme was purified 383-fold from cultured cells by using a combination of ammonium sulfate precipitation, successive anion-exchange column chromatography, gel filtration, and hydroxyapatite chromatography, gel filtration, and hydroxyapatite chromatography. The purified protein gave a single band (M(r), 42,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Estimation of the M(r) by the gel filtration method gave a value of 84,000, indicating that the enzyme is dimeric. Amino acid sequence analysis revealed it to have a single N-terminal sequence that has no sequence homology to any other known agarases. The optimum temperature and pH were 30 degrees C and 7.7, respectively. The Km and maximum rate of metabolism for neoagarobiose were 5.37 mM and 92 U/mg of protein, respectively.     

Homologous expression of recombinant manganese peroxidase in Phanerochaete chrysosporium.

The promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene (gpd) was used to drive expression of mnp1, the gene encoding Mn peroxidase isozyme 1, in primary metabolic cultures of Phanerochaete chrysosporium. A 1,100-bp fragment of the P. chrysosporium gpd promoter region was fused upstream of the mnp1 gene to construct plasmid pAGM1, which contained the Schizophyllum commune ade5 gene as a selectable marker. pAGM1 was used to transform a P. chrysosporium ade1 auxotroph to prototrophy. Ade+ transformants were screened for peroxidase activity on a solid medium containing high carbon and high nitrogen (2% glucose and 24 mM NH4 tartrate) and o-anisidine as the peroxidase substrate. Several transformants that expressed high peroxidase activities were purified and analyzed further in liquid cultures. Recombinant Mn peroxidase (rMnP) was expressed and secreted by transformant cultures on day 2 under primary metabolic growth conditions (high carbon and high nitrogen), whereas endogenous wild-type mnp genes were not expressed under these conditions. Expression of rMnP was not influenced by the level of Mn in the culture medium, as previously observed for the wild-type Mn peroxidase (wtMnP). The amount of active rMnP expressed and secreted in this system was comparable to the amount of enzyme expressed by the wild-type strain under ligninolytic conditions. rMnP was purified to homogeneity by using DEAE-Sepharose chromatography, Blue Agarose chromatography, and Mono Q column chromatography. The M(r) and absorption spectrum of rMnP were essentially identical to the M(r) and absorption spectrum of wtMnP, indicating that heme insertion, folding, and secretion were normal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Restriction and modification in Bacillus subtilis: two DNA methyltransferases with BsuRI specificity. I. Purification and physical properties

Two S-adenosyl-L-methionine:DNA (cytosine 5)-methyltransferases, termed M.BsuRIa and M.BsuRIb, were purified 3,000- and 4,000-fold, respectively, from Bacillus subtilis strain OG3R (r+m+) by successive column chromatography. The molecular weights determined by gel filtration were 37,000 for M.BsuRIa and 40,000 for M.BsuRIb. The sedimentation coefficients s20,w were 3.55 for both enzymes as determined by glycerol gradient centrifugation, corresponding to molecular weights of 43,000. Analysis of the two methyltransferases by agarose gel electrophoresis under native conditions, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, showed correspondence of the M.BsuRIa activity with one protein band at a molecular weight of 41,000, whereas M.BsuRIb activity was associated with two protein bands with molecular weights of 42,000 and 39,000, respectively.     

Isolation and characterization of acidic glycosphingolipids from the gill of the Pacific Salmon (Oncorhynchus keta): A novel hybrid-type ganglioside with isoglobo- and neolacto-Series

Monosialosyl gangliosides and sulfoglycolipids in the gill of pacific salmon, Oncorhynchus keta, have been prepared by solvent extraction and DEAE-Sephadex column chromatography. Acidic glycolipid bands (M1-M13) detected by thin layer chromatography were separated by Iatrobeads column chromatography and 13 components were characterized by TLC, compositional analysis, methylation analysis, chemical and enzymatic degradation, liquid secondary ion mass spectrometry and (1)H nuclear magnetic resonance spectroscopy. In addition to the acidic glycolipids with known structures (SM4s, SM3, GM3, LM1, GM1b and V(3)alphaFuc,IV(3)betaGalNAc-GM1a), two fractions (M11 and M13) of unknown monosialosyl gangliosides with TLC mobility slower than GM1a were isolated and characterized as having the following structure with a hybrid of isoglobo- and neolacto-series. [formula: see text] Analysis of fatty acid indicated predominance of C24:1 fatty acid in the upper band (M11) and shorter chain saturated fatty acids in the lower band (M13). The tissue concentrations of M11 and M13 were 1.15 and 0.96 mumol/kg wet weight, respectively.     

Overexpression, purification, and characterization of the thermostable mevalonate kinase from Methanococcus jannaschii.

We report here the first overexpression and characterization of a thermostable mevalonate kinase from an archae, Methanococcus jannaschii, a strict anaerobe, which produces methane and grows at pressure of 200 atm and an optimum temperature near 85 degrees C. PCR-derived DNA fragments containing the structural gene for mevalonate kinase were cloned into an expression vector, pET28a, to form pETMVK. The mevalonate kinase was overexpressed from Escherichia coli pETMVK/BL21(DE3) (15-20% of total soluble protein) when induced with isopropyl beta-d-thiogalactopyranoside. The protein was purified by heat treatment (to denature E. coli proteins), followed by metal-affinity chromatography on Talon metal-affinity resin column. The purified protein had a dimeric structure composed of identical subunits, and the M(r) of the enzyme determined by gel chromatography was 68K. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the subunit M(r) was 36, 000. The pI for mevalonate kinase was 7.8. The Michaelis constant (K(m)) for (RS)-mevalonate was 68.5 microM and was 92 microM for ATP. The V(max) was 387 units mg(-1). The optimal temperature for mevalonate kinase activity was 70-75 degrees C.     

Two squid skin proteoglycans each containing chondroitin sulfates with different sulfation patterns.

Two chondroitin sulfate containing proteoglycans, amounting to approximately 6% of the tissue proteoglycans, were isolated from the skin of the squid. They were almost completely extracted by 4 M guanidine hydrochloride in the presence of proteinase inhibitors, and then they were separated by DEAE-Sephacel chromatography and isolated by further chromatography on Sepharose CL-4B. Each proteoglycan contained two types of chondroitin sulfates that differed in their sulfation patterns. One proteoglycan (molecular mass (M(r)) 5.6 x 10(5)) contained, on the average, four chondroitins (M(r) 8.4 x 10(4)) and five chondroitin sulfates (M(r) 3.4 x 10(4)), whereas the other proteoglycan (M(r) 5.2 x 10(5)) contained three chondroitin sulfates (M(r) 1.1 x 10(5)) and five oversulfated chondroitin sulfates (M(r) 4.3 x 10(4)). The glycosaminoglycans were released from the proteoglycans by treatment with alkaline borohydride, separated from the oligosaccharides by chromatography on Bio-Gel P-30, and isolated by chromatography on DEAE-Sephacel and Sepharose CL-6B. Chondroitin sulfates were degraded by chondroitinase AC to an extent of 70% and consisted of significant amounts of disaccharides sulfated at C-4 of the galactosamine, disulfated disaccharides, and small amounts of nonsulfated disaccharides, as well as disaccharides that bore sulfates at C-6. Oversulfated chondroitin sulfate was degraded by chondroitinase AC to only 40% and contained appreciable amounts of disulfated and trisulfated disaccharides. The glycosaminoglycans also contained neutral monosaccharides; glucose was the predominant neutral sugar. A part of the oligosaccharides of both proteoglycans was of identical structure to that of chondroitin sulfate.     

Histamine-releasing activity. V. Characterization and purification using high-performance liquid chromatography

The production of large quantities of the lymphokine(s) histamine-releasing activity (HRA) and its partial purification by Sephadex G-75 and ion-exchange chromatography on carboxymethyl (CM) Sepharose 6B have been detailed (M. A. Lett-Brown, D. O. Thueson, D. E. Plank, M. P. Langford, and J. A. Grant, Cell. Immunol. 87, 434-444, 1984). Two peaks of activity (HRA I and II) were recovered. Preparations of HRA have now been analyzed by high-performance liquid chromatography (HPLC). Thoracic duct lymphocytes stimulated with 200 U/ml streptokinase were used as a source of HRA. Gel-filtration HPLC on a TSK 3000 column separated HRA into two peaks of activity (10,000-20,000 and 1300 Da). Reverse-phase high-performance liquid chromatography using a Nucleosil C-8 column showed that HRA II (the activity eluted at a conductivity of 18-20 mmho on the CM-Sepharose column) eluted as a single sharp peak, the main protein contaminant being cytochrome c, the carrier protein added to enhance the yield of HRA. High-performance liquid chromatography was found to be a useful analytical tool and may be suitable for the large-scale purification of HRA.     

Purification and some properties of cycloinulo-oligosaccharide fructanotransferase from Bacillus circulans OKUMZ 31B

Cycloinulo-oligosaccharide fructanotransferase was purified from the cultured medium of Bacillus circulans OKUMZ 31B, to electrophoretic homogeneity, by anion-exchange column chromatography on DEAE-Toyopearl 650M, hydrophobic column chromatography on Butyl-Toyopearl 650M, gel-filtration column chromatography on Sephacryl S-2000HR and anion-exchenge column chromatography on SuperQ-Toyopearl 650M. The enzyme has a molecular weight of 132 000 and a pI of 4.1. The enzyme was most active at pH 7.5 and 40°C, and was stable at pH 6.0–9.0 and below 40°C. The enzyme catalyses the conversion of inulin into cycloinulohexaose and cycloinuloheptaose in the ratio of ca. 4:1, and a small amount of cycloinulo-octaose. The enzyme has an isoform which may be a proteolyticaly modified species of the CFTase because of its reduced molecular weight, 126 000.     

A protein factor from Bufo marinus erythrocytes cross-bridges microtubules in vitro

A microtubule cross-bridging factor was isolated from erythrocytes of the toad, Bufo marinus. Erythrocytes were lysed and their cytoskeletons disassembled by sonication and high salt extraction. The solubilized proteins were recovered and fractionated using Sephadex G-200 column chromatography. The protein fractions from the column were analysed by SDS-PAGE and pooled into three groups: high molecular weight (HMW) proteins that eluted from the column in the void volume and had a protein composition that included HMW polypeptides; intermediate MW proteins that were shown by SDS-PAGE to contain polypeptides smaller than 120,000 D; and low MW (LMW) proteins that contained polypeptides smaller than 70,000 D. Each group was further fractionated by phosphocellulose (PC) chromatography. The flow-through was recovered, and bound proteins were then eluted by a step gradient of salt (0.2, 0.4, 0.6 and 0.8 M KCl). To assay for microtubule cross-bridging activity, column fractions were incubated with taxol-stabilized microtubules, formed from PC-purified brain tubulin (PC microtubules). Negatively stained samples were examined in the electron microscope for the reconstitution of microtubule bundles with interconnecting cross-bridges. The HMW protein fraction from the G-200 column contained the cross-bridging factor. When these proteins were further fractionated by PC chromatography only the fraction eluted by 0.2 M KCl induced the formation of microtubule bundles with cross-bridges. No other protein fraction isolated by the described method revealed cross-bridges between microtubules in vitro.     

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.07M sodium phosphate buffer (pH 6.8). Chondroitinase C was then purified by affinity chromatography using dermatan sulfate-bound AH-Sepharose 4B coated with the same glycosaminoglycan. Purification of the enzyme was achieved 18-fold and in 73% yield. On the other hand, the activities of delta4,5glycosiduronase and sulfatase were decreased to 50 and 60%, respectively, as compared with those in the crude chondroitinase B fraction, after passing the fraction through a column of phosphono-cellulose pre-equilibrated with 0.1M sodium phosphate buffer (pH 6.8). The remaining activities of these two enzymes were then eliminated from chondroitinase B by affinity chromatography with heparin-bound AH-Sepharose 4B coated with dermatan sulfate. In the affinity chromatography used in the present study, non-covalent coating of the glycosaminoglycan-bound (covalently) AH-Sepharose 4B with the same or another glycosaminoglycan was found to be important.     

Characteristics of structure, composition, mass spectra, and iron release from the ferritin of shark liver (Sphyrna zygaena)

The ferritin consists of a protein shell constructed of 24 subunits and an iron core. The liver ferritin of Sphyrna zygaena (SZLF) purified by column chromatography is a protein composed of eight ferritins containing varying iron numbers ranging from 400+/-20 Fe3+/SZLF to 1890+/-20 Fe3+/SZLF within the protein shell. Nature SZLF (SZLFN) consisting of holoSZLF and SZLF with unsaturated iron (SZLFUI) to have been purified with polyacrylamide gel electrophoresis (PAGE) exhibited five ferritin bands with different pI values ranging from 4.0 to 7.0 in the gel slab of isoelectric focusing (IEF). HoloSZLF purified by PAGE (SZLFE) not only had 1890+/-20 Fe3+/SZLFE but also showed an identical size of iron core observed by transmission electron microscopy (TEM). Molecular weight of approximately 21 kDa for SZLFE subunit was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Four peaks of molecular ions at mass/charge (m/z) ratios of 10611.07, 21066.52, 41993.16, and 63555.64 that come from the SZLFE were determined by matrix-assisted laser desorption ionization/time-of-flight mass spectrometry (MALDI-TOF MS), which were identified as molecular ions of the ferritin subunit (M+) and its polymers, namely, [M]2+, [M]+, [2M]+, and [3M]+, respectively. Both SZLFE and a crude extract from shark liver of S. zygaena showed similar kinetic characteristics of complete iron release with biphasic behavior. In addition, a combined technique of visible spectrometry and column chromatography was used for studying ratio of phosphate to Fe3+ within the SZLFE core. Interestingly, this ratio maintained invariable even after the iron release, which differed from that of other mammal ferritins.     

Bmcystatin, a cysteine proteinase inhibitor characterized from the tick Boophilus microplus

The bovine tick Rhipicephalus (Boophilus) microplus is a blood-sucking animal, which is responsible for Babesia spp and Anaplasma marginale transmission for cattle. From a B. microplus fat body cDNA library, 465 selected clones were sequenced randomly and resulted in 60 Contigs. An open reading frame (ORF) contains 98 amino acids named Bmcystatin, due to 70% amino acid identity to a classical type 1 cystatin from Ixodes scapularis tick (GenBank Accession No. ). The Bmcystatin amino acid sequence analysis showed two cysteine residues, theoretical pI of 5.92 and M(r) of 11 kDa. Bmcystatin gene was cloned in pET 26b vector and the protein expressed using bacteria Escherichia coli BL21 SI. Recombinant Bmcystatin (rBmcystatin) purified by affinity chromatography on Ni-NTA-agarose column and ionic exchange chromatography on HiTrap Q column presented molecular mass of 11 kDa, by SDS-PAGE and the N-terminal amino acid sequenced revealed unprocessed N-terminal containing part of pelB signal sequence. Purified rBmcystatin showed to be a C1 cysteine peptidase inhibitor with K(i) value of 0.1 and 0.6 nM for human cathepsin L and VTDCE (vitellin degrading cysteine endopeptidase), respectively. The rBmcystatin expression analyzed by semi-quantitative RT-PCR confirmed the amplification of a specific DNA sequence (294 bp) in the fat body and ovary cDNA preparation. On the other hand, a protein band was detected in the fat body, ovary, and the salivary gland extracts using anti-Bmcystatin antibody by Western blot. The present results suggest a possible role of Bmcystatin in the ovary, even though the gene was cloned from the fat body, which could be another site of this protein synthesis.