Kuo-Chang Zen - Academia.edu (original) (raw)

Papers by Kuo-Chang Zen

Research paper thumbnail of Nucleotide sequence of a cDNA clone that encodes the maize inhibitor of trypsin and activated Hageman factor

Plant Molecular Biology, 1992

Research paper thumbnail of Site-Directed Sulfhydryl Labeling of the Lactose Permease of Escherichia coli:  Helix X

Biochemistry, 2000

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic f... more Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.

Research paper thumbnail of Induction of chitinases and beta-l,3-glucanases in Rhizoctonia solani-infected rice plants: Isolation of an infection-related chitinase cDNA clone

Physiologia Plantarum, 1996

Extracts from several rice cultivars (Oryza saliva L. cvs IR58, 74586 and SC33) infected with the... more Extracts from several rice cultivars (Oryza saliva L. cvs IR58, 74586 and SC33) infected with the sheath blight pathogen Rhizoctonia solani, were analyzed to determine the isozyme distribution of chitinases (EC 3.2.1.14) and β-1,3-glucanases (EC 3.2.1.39). Upon infection by the fungal pathogen, two chitinases of 28 and 35 kDa and two β-1,3-glucanases of 30 and 32 kDa were shown to be induced in all cultivars. They resolved into multiple isozymes under nondenaturing electrophoretic conditions. Wounding, but not bacterial infection, resulted in the induction of these hydrolytic enzymes. Even though fungal infection resulted in induction of chitinases and β-glucanases in all cultivars, some cultivars that were moderately resistant to R. solani appeared to have higher levels of specific isozymes. A chitinase cDNA clone was identified by screening a library, prepared from RNA isolated from R. solani-infected rice plants, with an antibody to a bean chitinase. This cDNA encoded a 35-kDa chitinase which was significantly different in amino acid sequence from other rice chitinases described so far. Northern blot analysis of RNA from infected rice plants indicated that transcripts corresponding to this chitinase gene were induced upon fungal infection.

Research paper thumbnail of A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosp... more A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Research paper thumbnail of Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta

Insect Biochemistry and Molecular Biology, 1992

The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx ... more The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta, during the larval-pupal transformation was determined by immunoblot analysis using rabbit polyclonal antibodies for B. mori chitinase (EC 3.2.1.14) and fl-N-acetylglucosaminidase (EC 3.2.1.30) as probes. Similar temporal patterns of appearance and immunoreactivities of chitinase-like and fl-N-acetylglucosaminidase-like proteins were observed in the two species. Several chitinase-like proteins (MWapp 50-200 kDa) were resolved by denaturing electrophoresis. During the latter part of the fifth larval stadium, the major immunoreactive protein in B. mori integument and molting fluid had an apparent molecular mass of 88 kDa, which was previously observed (Koga et al., 1989). In M. sexta integument on days 6-8 of the fifth larval stadium and in molting fluid, the major immunoreactive protein had an apparent molecular mass of 97 kDa, which was 22 kDa larger than the 75 kDa chitinase detected in molting fluid (Koga et al., 1983). In integument on days 3-7 of the fifth larval stadium, another immunoreactive protein with an apparent molecular mass of 119 kDa was present. In contrast to multiple immunoreactive chitinase-like proteins, only a single major fl-N-acetylglucosaminidase-immunoreactive protein was detected in integument and molting fluid from either species. The immunoreactive fl-N-acetylglucosaminidase-like proteins had an apparent molecular mass of 67.5 kDa and a pI of 5.0, which are identical values to those of fl-N-acetylglucosaminidases determined previously. The fl-N-acetylglucosaminidases cleaved N-acetylchitooligosaccharides from the non-reducing end in an exo-fashion. The results of this study suggest that chitinases are synthesized in B. mori and M. sexta integuments as zymogens, which are activated by limited proteolysis, whereas fl-N-acetylglucosaminidases are not.

Research paper thumbnail of Nucleotide sequence of a cDNA clone that encodes the maize inhibitor of trypsin and activated Hageman factor

Plant Molecular Biology, 1992

Research paper thumbnail of Site-Directed Sulfhydryl Labeling of the Lactose Permease of Escherichia coli:  Helix X

Biochemistry, 2000

Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic f... more Site-directed sulfhydryl modification in situ is employed to investigate structural and dynamic features of transmembrane helix VII and the beginning of the periplasmic loop between helices VII and VIII (loop VII/VIII). Essentially all of the Cys-replacement mutants in the periplasmic half of the helix and the portion of loop VII/VIII tested are labeled by N-[(14)C]ethylmaleimide (NEM). In contrast, with the exception of two mutants at the cytoplasmic end of helix VII, none of the mutants in the cytoplasmic half react with the alkylating agent. Labeling of most of the mutants is unaltered by ligand at 25 degrees C. However, at 4 degrees C, conformational changes induced by substrate binding become apparent. In the presence of ligand, permease mutants with a Cys residue at position 241, 242, 244, 245, 246, or 248 undergo a marked increase in labeling, while the reactivity of a Cys at position 238 is slightly decreased. Labeling of the remaining Cys-replacement mutants is unaffected by ligand. Studies with methanethiosulfonate ethylsulfonate (MTSES), a hydrophilic impermeant thiol reagent, show that most of the positions that react with NEM are accessible to MTSES; however, the two NEM-reactive mutants at the cytoplasmic end of helix VII and position 236 in the middle of the membrane-spanning domain are not. The findings demonstrate that positions in helix VII that reflect ligand-induced conformational changes are located in the periplasmic half and accessible to the aqueous phase from the periplasmic face of the membrane. In the following papers in this issue (Venkatesan, P., Lui, Z., Hu, Y., and Kaback H. R.; Venkatesan, P., Hu, Y., and Kaback H. R.), the approach is applied to helices II and X.

Research paper thumbnail of Induction of chitinases and beta-l,3-glucanases in Rhizoctonia solani-infected rice plants: Isolation of an infection-related chitinase cDNA clone

Physiologia Plantarum, 1996

Extracts from several rice cultivars (Oryza saliva L. cvs IR58, 74586 and SC33) infected with the... more Extracts from several rice cultivars (Oryza saliva L. cvs IR58, 74586 and SC33) infected with the sheath blight pathogen Rhizoctonia solani, were analyzed to determine the isozyme distribution of chitinases (EC 3.2.1.14) and β-1,3-glucanases (EC 3.2.1.39). Upon infection by the fungal pathogen, two chitinases of 28 and 35 kDa and two β-1,3-glucanases of 30 and 32 kDa were shown to be induced in all cultivars. They resolved into multiple isozymes under nondenaturing electrophoretic conditions. Wounding, but not bacterial infection, resulted in the induction of these hydrolytic enzymes. Even though fungal infection resulted in induction of chitinases and β-glucanases in all cultivars, some cultivars that were moderately resistant to R. solani appeared to have higher levels of specific isozymes. A chitinase cDNA clone was identified by screening a library, prepared from RNA isolated from R. solani-infected rice plants, with an antibody to a bean chitinase. This cDNA encoded a 35-kDa chitinase which was significantly different in amino acid sequence from other rice chitinases described so far. Northern blot analysis of RNA from infected rice plants indicated that transcripts corresponding to this chitinase gene were induced upon fungal infection.

Research paper thumbnail of A novel cereal storage protein: molecular genetics of the 19 kDa globulin of rice

A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosp... more A lambda gt11 cDNA library, constructed from poly(A)+ RNA isolated from immature rice seed endosperm, was screened with affinity-purified antibodies against the rice storage protein called alpha-globulin (previously), or the 19 kDa globulin (our term). A positive clone was isolated and sequenced and shown to encode a 21 kDa precursor for the 19 kDa globulin, based on the identity of portions of the inferred amino acid sequence and the sequence of three cyanogen bromide peptides of the 19 kDa globulin. Analysis of genomic DNA by Southern blotting using the cDNA clone probe revealed one hybridizing band in Eco RI, Hind III, and Bam HI digests. This strongly suggests that the 19 kDa globulin is encoded by a single-copy gene. Because of its single-copy nature and its abundance of Arg and lack of Lys, the 19 kDa rice globulin appears to be a particularly attractive target for genetically engineering increased Lys content in rice seeds.

Research paper thumbnail of Immunoblot analysis of chitinolytic enzymes in integument and molting fluid of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta

Insect Biochemistry and Molecular Biology, 1992

The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx ... more The occurrence of proteins related to chitinolytic enzymes in integument of the silkworm, Bombyx mori, and the tobacco hornworm, Manduca sexta, during the larval-pupal transformation was determined by immunoblot analysis using rabbit polyclonal antibodies for B. mori chitinase (EC 3.2.1.14) and fl-N-acetylglucosaminidase (EC 3.2.1.30) as probes. Similar temporal patterns of appearance and immunoreactivities of chitinase-like and fl-N-acetylglucosaminidase-like proteins were observed in the two species. Several chitinase-like proteins (MWapp 50-200 kDa) were resolved by denaturing electrophoresis. During the latter part of the fifth larval stadium, the major immunoreactive protein in B. mori integument and molting fluid had an apparent molecular mass of 88 kDa, which was previously observed (Koga et al., 1989). In M. sexta integument on days 6-8 of the fifth larval stadium and in molting fluid, the major immunoreactive protein had an apparent molecular mass of 97 kDa, which was 22 kDa larger than the 75 kDa chitinase detected in molting fluid (Koga et al., 1983). In integument on days 3-7 of the fifth larval stadium, another immunoreactive protein with an apparent molecular mass of 119 kDa was present. In contrast to multiple immunoreactive chitinase-like proteins, only a single major fl-N-acetylglucosaminidase-immunoreactive protein was detected in integument and molting fluid from either species. The immunoreactive fl-N-acetylglucosaminidase-like proteins had an apparent molecular mass of 67.5 kDa and a pI of 5.0, which are identical values to those of fl-N-acetylglucosaminidases determined previously. The fl-N-acetylglucosaminidases cleaved N-acetylchitooligosaccharides from the non-reducing end in an exo-fashion. The results of this study suggest that chitinases are synthesized in B. mori and M. sexta integuments as zymogens, which are activated by limited proteolysis, whereas fl-N-acetylglucosaminidases are not.