A protein with a low molecular mass of 6027 was purified from cocoon shell of silkworm, Bombyx mori. Twodimensional polyacrylamide gel electrophoresis (2D/PAGE) resolved this protein into a single spot with pI 4.3 and M r 6000. Amino acid sequence analysis revealed that this protein consists of 55 amino acids, six of these being cysteine residues and is highly homologous to bovine pancreatic trypsin inhibitor-type inhibitors. The 6-kDa protein is heat stable and acid stable and inhibits bovine trypsin by forming a low-dissociation complex with trypsin in a 1 : 1 molar ratio (K i = 2.8 £ 10 ±10 ), but does not a-chymotrypsin. This cocoon shell-associated trypsin inhibitor (CSTI) was thus concluded to belong to the bovine pancreatic trypsin inhibitor class. CSTI was developmentally regulated in the silk gland at the final stage of larval growth, and its specific distribution in the middle silk gland, an organ in which silk proteins are stored during the final larval instar, occurred before the onset of spinning. This inhibitor protects the tryptic degradation of fibroin light (L) chain in vitro. These results suggest that this trypsin inhibitor may play an important part on regulating proteolytic activity in the silk gland or protecting silk proteins from degradation during histolysis.Keywords: amino acid sequence; Bombyx mori; cocoon; trypsin inhibitor.The silkworm Bombyx mori produces massive amounts of silk proteins during the final stage of larval development (fifth instar), and these proteins are stored in the middle silk gland until they are discharged through the anterior duct and the spinneret at the end of the fifth instar. As major components of cocoons, two kinds of silk proteins have been distinguished: fibroin, a fibrous protein composed of one heavy (H) chain of about 350 kDa and a L-chain of about 25 kDa linked by disulfide bond(s); and sericin, serving as an adhesive to unite fibroin for making cocoons. The crystalline structures, chemical compositions and physical properties of the silk proteins have been extensively studied, but it is believed that these proteins in the cocoon shell are biologically inactive molecules. We previously have reported that four low-molecular-mass proteins are present in a water-soluble extract from the cocoon shell as minor components. These proteins with relative molecular masses of 10 kDa, 6 kDa, 4 kDa and 3 kDa were found to be distinct from both types of silk proteins on the basis of amino acid composition analysis [1,2]. N-terminal amino acid sequence analysis revealed that the 4-kDa protein was highly homologous to Kazal-type trypsin inhibitors [3]. Biologically active proteins would thus appear to quite likely coexist with silk proteins in the cocoon shell. Although trypsin and a-chymotrypsin inhibitors in the hemolymph of insects such as B. mori [4,5], Antheraea pernyi [6], Drosophila melanogaster [7] and Manduca sexta [8] have been demonstrated, such protease inhibitor has yet to be shown present in the cocoon shell.We here report the purification, protein se...

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Acid-degraded sericin powder (AC-SP) was prepared from aqueous solution containing citric acid-degraded sericin polypeptides of Bombyx mori. The morphological and biochemical properties of AC-SP were compared with those of alkali-degraded sericin powder (AL-SP) and hot-water degraded sericin powder (HW-SP). Based on an SEM analysis, AC-SP showed a thin film structure of 10-100 microm with good dispersity while AL-SP and HW-SP had a much larger thin film structure (<500 microm). The extract of AC-SP showed stronger trypsin inhibitor activity due to cocoon shell trypsin inhibitor (CSTI-IV) than that of HW-SP. The extract of AL-SP showed no CSTI-IV activity. It was found that AC-SP was a trypsin inhibitor complex powder and that the release of CSTI-IV from AC-SP depended on pH and ion strength. Similar powder materials were obtained when such organic acids as tartaric acid and succinic acid were used. These results suggest that the acid-degraded sericin polypeptides work as a protein matrix to which CSTI-IV may bind ionically.

Prevention of arterial thrombotic disease has high priority in developed countries. As inappropriate diet predispose to acute thrombotic events, regular intake of an antithrombotic diet may be a convenient and effective way of prevention. The present study was performed to examine antithrombotic effect of mulberry varieties. A shear-induced in vitro platelet reactivity/thrombolysis test (Gorog Thrombosis Test) was used to screen for antiplatelet and thrombolytic activities. In case of effectiveness, it was followed by an in vivo test of laser-induced thrombosis in mice. Antioxidant capacity was assessed by superoxide anion and radical scavenging activities. Total polyphenolics, anthocyanin and citrate contents were also measured. The tested varieties showed different effect in vitro on platelet reactivity and endogenous thrombolytic activity. Varieties inhibiting platelet reactivity were antithrombotic in vivo regardless inhibition or enhancement of thrombolysis. Those mulberry varieties, which enhanced platelet reactivity in vitro, were prothrombotic only if inhibitory activity on endogenous thrombolysis coexisted with the platelet effect. Antioxidant activities and polyphenolics content did not affect platelets and the overall thrombotic status. However, antioxidant activities and polyphenolics content significantly correlated with the endogenous thrombolytic activity. These data showed that mulberry varieties can be grouped into subclasses with either anti- or prothrombotic activities. Antioxidant activities and polyphenolic contents do not affect platelets but may enhance endogenous thrombolysis, thus causing an overall antithrombotic effect.

: An emulsion (SR-EM) was prepared from vitamin E and aqueous sericin solution obtained from cocoons of Bombyx mori. Properties such as emulsifying degree, heat stability and radical scavenging activity of SR-EM were compared with those of CS-EM and AL-EM, vitamin E emulsions prepared from casein and bovine serum albumin, respectively. The degree of emulsifying for SR-EM was three times higher than that for CS-EM. CS-EM and AL-EM were a heat-stable emulsion, whereas SR-EM was a heat-labile emulsion because absorbance spectra changed exclusively in SR-EM after treated from 115 to 127!. These results indicated that the oil-water interface of SR-EM was unstable under the high pressure-high temperature treatments compared to those of CS-EM and AL-EM. Each emulsion showed lower radical scavenging activity than a vitamin E solution,suggesting that the interaction of vitamin E and a radical molecule may be interfered by a protective protein colloid. SR-EM and CS-EM were more stable in 60% ethanol solution at ambient temperature than AL-EM because the radical scavenging activity of SR-EM and CS-EM was some 50% lower than that of AL-EM.