RESUMO
Human epidermal growth factor receptor 2 (HER-2) is overexpressed in many malignant tumors. The anti-HER2 antibody trastuzumab has been approved for treating HER2-positive early and metastatic breast cancers. Pseudomonas exotoxin A (PE), a bacterial toxin of Pseudomonas aeruginosa, consists of an A-domain with enzymatic activity and a B-domain with cell binding activity. Recombinant immunotoxins comprising the HER2(scFv) single-chain Fv from trastuzumab and the PE24B catalytic fragment of PE display promising cytotoxic effects, but immunotoxins are typically insoluble when expressed in the cytoplasm of Escherichia coli, and thus they require solubilization and refolding. Herein, a recombinant immunotoxin gene was fused with maltose binding protein (MBP) and overexpressed in a soluble form in E. coli. Removal of the MBP yielded stable HER2(scFv)-PE24B at 91% purity; 0.25 mg of pure HER2(scFv)-PE24B was obtained from a 500 mL flask culture. Purified HER2(scFv)-PE24B was tested against four breast cancer cell lines differing in their surface HER2 level. The immunotoxin showed stronger cytotoxicity than HER2(scFv) or PE24B alone. The IC50 values for HER2(scFv)-PE24B were 28.1 ± 2.5 pM (n = 9) and 19 ± 1.4 pM (n = 9) for high HER2-positive cell lines SKBR3 and BT-474, respectively, but its cytotoxicity was lower against MDA-MB-231 and MCF7. Thus, fusion with MBP can facilitate the soluble expression and purification of scFv immunotoxins.
Assuntos
ADP Ribose Transferases , Antineoplásicos Imunológicos/farmacologia , Toxinas Bacterianas , Exotoxinas , Imunotoxinas/farmacologia , Proteínas Ligantes de Maltose , Receptor ErbB-2/antagonistas & inibidores , Proteínas Recombinantes de Fusão/farmacologia , Anticorpos de Cadeia Única , Fatores de Virulência , ADP Ribose Transferases/genética , Toxinas Bacterianas/genética , Linhagem Celular Tumoral , Sobrevivência Celular , Relação Dose-Resposta a Droga , Escherichia coli/genética , Escherichia coli/metabolismo , Exotoxinas/genética , Expressão Gênica , Engenharia Genética , Vetores Genéticos/genética , Humanos , Imunotoxinas/genética , Imunotoxinas/isolamento & purificação , Proteínas Ligantes de Maltose/genética , Espectrometria de Massas , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/isolamento & purificação , Anticorpos de Cadeia Única/genética , Fatores de Virulência/genética , Exotoxina A de Pseudomonas aeruginosaRESUMO
BACKGROUND: The 3D8 single chain variable fragment (scFv) is a mini-antibody sequence that exhibits independent nuclease activity against all types of nucleic acids. In this research, crossing a 3D8 scFv G1 transgenic rooster with wild-type hens produced 3D8 scFv G2 transgenic chickens to evaluate suppression of viral transmission. RESULT: The transgenic chickens were identified using genomic PCR and immunohistochemistry. To evaluate Newcastle disease virus (NDV) protection conferred by 3D8 scFv expression, transgenic, non-transgenic, and specific pathogen-free (SPF) chickens were challenged with virulent NDV by direct injection or aerosol exposure. The three groups of chickens showed no significant differences (p < 0.05) in mean death time after being directly challenged with NDV; however, in contrast to chickens in the non-transgenic and SPF groups, chickens in the transgenic group survived after aerosol exposure. Although the transgenic chickens did not survive after direct challenge, we found that the chickens expressing the 3D8 scFv survived aerosol exposure to NDV. CONCLUSIONS: Our finding suggest that the 3D8 scFv could be a useful tool to prevent chickens from spreading NDV and control virus transmission.
Assuntos
Galinhas/genética , Doença de Newcastle/transmissão , Vírus da Doença de Newcastle/fisiologia , Doenças das Aves Domésticas/virologia , Animais , Animais Geneticamente Modificados , Galinhas/imunologia , Feminino , Masculino , Doença de Newcastle/virologia , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/transmissão , Anticorpos de Cadeia Única , Organismos Livres de Patógenos EspecíficosRESUMO
Viral protein neutralizing antibodies have been developed but they are limited only to the targeted virus and are often susceptible to antigenic drift. Here, we present an alternative strategy for creating virus-resistant cells and animals by ectopic expression of a nucleic acid hydrolyzing catalytic 3D8 single chain variable fragment (scFv), which has both DNase and RNase activities. HeLa cells (SCH07072) [corrected] expressing 3D8 scFv acquired significant resistance to DNA viruses. Virus challenging with Herpes simplex virus (HSV) in 3D8 scFv transgenic cells and fluorescence resonance energy transfer (FRET) assay based on direct DNA cleavage analysis revealed that the induced resistance in HeLa cells was acquired by the nucleic acid hydrolyzing catalytic activity of 3D8 scFv. In addition, pseudorabies virus (PRV) infection in WT C57BL/6 mice was lethal, whereas transgenic mice (STG90) that expressed high levels of 3D8 scFv mRNA in liver, muscle, and brain showed a 56% survival rate 5 days after PRV intramuscular infection. The antiviral effects against DNA viruses conferred by 3D8 scFv expression in HeLa cells as well as an in vivo mouse system can be attributed to the nuclease activity that inhibits viral genome DNA replication in the nucleus and/or viral mRNA translation in the cytoplasm. Our results demonstrate that the nucleic-acid hydrolyzing activity of 3D8 scFv confers viral resistance to DNA viruses in vitro in HeLa cells and in an in vivo mouse system.
Assuntos
DNA Viral/metabolismo , Desoxirribonucleases/imunologia , RNA Viral/metabolismo , Ribonucleases/imunologia , Anticorpos de Cadeia Única/genética , Animais , Anticorpos Neutralizantes/imunologia , Anticorpos Antivirais/imunologia , Antivirais/metabolismo , Linhagem Celular Tumoral , Infecções por Vírus de DNA/imunologia , Vírus de DNA/genética , Vírus de DNA/imunologia , Desoxirribonucleases/genética , Transferência Ressonante de Energia de Fluorescência , Células HeLa , Herpesvirus Suídeo 1/genética , Herpesvirus Suídeo 1/imunologia , Humanos , Hidrólise , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pseudorraiva/genética , Pseudorraiva/imunologia , Ribonucleases/genética , Simplexvirus/genética , Simplexvirus/imunologia , Anticorpos de Cadeia Única/imunologiaRESUMO
Crotamine, one of the major toxins present in the venom of the South American rattlesnake Crotalus durissus terrificus, exhibits potent cytotoxic properties and has been suggested for cancer therapy applications. However, its selectivity for cancer cells needs to be improved. This study designed and produced a novel recombinant immunotoxin, HER2(scFv)-CRT, composed of crotamine and single-chain Fv (scFv) derived from trastuzumab targeting human epidermal growth factor receptor 2 (HER2). The recombinant immunotoxin was expressed in Escherichia coli and purified using various chromatographic techniques. The cytotoxicity of HER2(scFv)-CRT was assessed in three breast cancer cell lines, demonstrating enhanced specificity and toxicity in HER2-expressing cells. These findings suggest that the crotamine-based recombinant immunotoxin has the potential to expand the repertoire of recombinant immunotoxin applications in cancer therapy.
Assuntos
Venenos de Crotalídeos , Imunotoxinas , Neoplasias , Animais , Humanos , Venenos de Crotalídeos/química , Crotalus , Imunotoxinas/metabolismo , Neoplasias/tratamento farmacológico , Linhagem Celular TumoralRESUMO
Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression and protein modification. Proteome changes of tumor tissue were investigated after intraperitoneal administration of a high concentration of ascorbic acid in BALB/C mice implanted with CT-26 cancer cells using two-dimensional gel electrophoresis and mass spectrometry. Eighteen protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, eukaryotic translation initiation factor 3 subunit 1, nucleophosmin, latexin, actin-related protein 2/3 complex subunit 5, M2-type pyruvate kinase, vimentin, tumor protein translationally-controlled 1, RAS oncogene family Ran, plastin 3 precursor, ATPase, Rho GDT dissociation inhibitor ß, and proteasome activator subunit 2 expression were quantitatively up-regulated. The increase in the level of these proteins was accompanied by an increase in mRNA level. The cytoskeleton protein actin, vimentin, and tumor protein translationally-controlled 1 showed quantitative expression profile differences. A change in actin cytoskeleton distribution, functionally relevant to the proteome result, was observed after treatment with ascorbic acid. These results suggest a previously undefined role of ascorbic acid in the regulation of cytoskeleton remodeling in tumor tissues.
Assuntos
Ácido Ascórbico/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Neoplasias/metabolismo , Proteoma/análise , Citoesqueleto de Actina/efeitos dos fármacos , Animais , Linhagem Celular Tumoral , Eletroforese em Gel Bidimensional , Injeções Intraperitoneais , Camundongos , Camundongos Endogâmicos BALB C , Neoplasias/patologia , RNA Mensageiro/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Bioassays and biomarkers have been previously developed to assess the effects of heavy metal contaminants on the early life stages of the sea urchin. In this study, malformation in the early developmental processes was observed in sea urchin (Strongylocentrotus intermedius) larvae exposed to 10 ppm Ni for over 30 h. The most critical stage at which the triggering of nickel effects takes place is thought to be the blastula stage, which occurs after fertilization in larval development. To investigate the molecular-level responses of sea urchin exposed to heavy metal stress and to explore the differentially expressed genes that are induced or repressed by nickel, differential display polymerase chain reaction (DD-PCR) was used with sea urchin mRNAs. The malformation-related genes expressed in the early life stages of the sea urchin were cloned from larvae exposed to 10 ppm of nickel for 15 h, and accessed via DD-PCR. Sequence analysis results revealed that each of the genes evidenced high homology with EGF2, PCSK9, serine/threonine protein kinase, apolipophorin precursor protein, and MGC80921 protein/transcript variant 2. This result may prove useful in the development of novel biomarkers for the assessment of heavy metal stresses on sea urchin embryos.
Assuntos
Reação em Cadeia da Polimerase , Ouriços-do-Mar/efeitos dos fármacos , Ouriços-do-Mar/genética , Animais , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Larva , Metais Pesados/farmacologia , Níquel/farmacologia , Ouriços-do-Mar/embriologia , Homologia de Sequência de AminoácidosRESUMO
A new honeysuckle yellow vein geminivirus (HYVV) isolate (2,763 nucleotides) and the HYVV-ß satellite (1,227 nucleotides) from Korea were cloned and characterized from symptomatic leaves of Lonicera japonica with a yellow net mosaic appearance. Phylogenetic analysis of HYVV and 13 other begomoviruses revealed that HYVV has the highest nucleotide sequence homology to HYVV-UK2. Grafting challenge of a virus-free L. japonica (scion) on an HYVV-infected L. japonica (stock) resulted in the appearance of typical HYVV disease symptoms on the newly developed leaves of the scion. Two pMon521 plasmids containing 1.3 copies and 2.0 copies of the cloned HYVV isolate, respectively, were infectious and produced disease symptoms in Nicotiana benthamiana after agroinoculation. We conclude that the causal agent of yellow net mosaic in L. japonica is a new Korean isolate of HYVV, based on sequence comparisons, agroinoculation-induced disease symptoms, electron microscopy, and phylogenetic analysis.
Assuntos
Begomovirus/patogenicidade , Lonicera/virologia , Doenças das Plantas/virologia , Begomovirus/genética , Begomovirus/isolamento & purificação , Clonagem Molecular , Análise por Conglomerados , DNA Viral/química , DNA Viral/genética , Coreia (Geográfico) , Dados de Sequência Molecular , Filogenia , Folhas de Planta/virologia , Análise de Sequência de DNA , Nicotiana/virologiaRESUMO
The virus behind the current pandemic, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is responsible for the etiology of novel coronavirus disease (COVID-19) and poses a critical public health threat worldwide. Effective therapeutics and vaccines against multiple coronaviruses remain unavailable. Single-chain variable fragment (scFv), a recombinant antibody, exhibits broad-spectrum antiviral activity against DNA and RNA viruses owing to its nucleic acid-hydrolyzing property. The antiviral activity of 3D8 scFv against SARS-CoV-2 and other coronaviruses was evaluated in Vero E6 cell cultures. Viral growth was quantified with quantitative RT-qPCR and plaque assay. The nucleic acid-hydrolyzing activity of 3D8 was assessed through abzyme assays of in vitro viral transcripts and cell viability was determined by MTT assay. We found that 3D8 inhibited the replication of SARS-CoV-2, human coronavirus OC43 (HCoV-OC43), and porcine epidemic diarrhea virus (PEDV). Our results revealed the prophylactic and therapeutic effects of 3D8 scFv against SARS-CoV-2 in Vero E6 cells. Immunoblot and plaque assays showed the reduction of coronavirus nucleoproteins and infectious particles, respectively, in 3D8 scFv-treated cells. These data demonstrate the broad-spectrum antiviral activity of 3D8 against SARS-CoV-2 and other coronaviruses. Thus, it could be considered a potential antiviral countermeasure against SARS-CoV-2 and zoonotic coronaviruses.
Assuntos
Antivirais/farmacologia , SARS-CoV-2/efeitos dos fármacos , Anticorpos de Cadeia Única/farmacologia , Animais , COVID-19/prevenção & controle , Sobrevivência Celular/genética , Chlorocebus aethiops , Coronavirus/efeitos dos fármacos , Coronavirus/fisiologia , Relação Dose-Resposta a Droga , Hidrólise , RNA Viral/metabolismo , SARS-CoV-2/fisiologia , Células Vero , Carga Viral/efeitos dos fármacos , Replicação Viral/efeitos dos fármacos , Tratamento Farmacológico da COVID-19RESUMO
To test the carcinostatic effects of ascorbic acid, we challenged the mice of seven experimental groups with 1.7 x 10(-4) mol high dose concentration ascorbic acid after intraperitoneal administrating them with sarcoma S-180 cells. The survival rate was increased by 20% in the group that received high dose concentration ascorbic acid, compared to the control. The highest survival rate was observed in the group in which 1.7 x 10(-4) mol ascorbic acid had been continuously injected before and after the induction of cancer cells, rather than just after the induction of cancer cells. The expression of three angiogenesis-related genes was inhibited by 0.3 times in bFGF, 7 times in VEGF and 4 times in MMP2 of the groups with higher survival rates. Biopsy Results, gene expression studies, and wound healing analysis in vivo and in vitro suggested that the carcinostatic effect induced by high dose concentration ascorbic acid occurred through inhibition of angiogenesis.
Assuntos
Antineoplásicos/uso terapêutico , Ácido Ascórbico/uso terapêutico , Neoplasias/metabolismo , Neovascularização Patológica/tratamento farmacológico , Sarcoma/tratamento farmacológico , Animais , Antineoplásicos/farmacologia , Antioxidantes/farmacologia , Antioxidantes/uso terapêutico , Ascite/tratamento farmacológico , Ascite/patologia , Ácido Ascórbico/farmacologia , Linhagem Celular Tumoral , Movimento Celular , Relação Dose-Resposta a Droga , Fator 2 de Crescimento de Fibroblastos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Metaloproteinase 2 da Matriz/genética , Camundongos , Camundongos Endogâmicos BALB C , Células NIH 3T3 , Transplante de Neoplasias , Neoplasias/genética , Neoplasias/patologia , Taxa de Sobrevida , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
Infectious bronchitis (IB) generated by the infectious bronchitis virus (IBV) causes economic difficulties for livestock farmers. The 3D8 single chain variable fragment (scFv) protein is a recombinant antibody with nuclease activity that shows antiviral effects against various DNA and RNA viruses in mice and chickens. In this experiment, 3D8 scFv G2 transgenic chickens produced by crossing 3D8 scFv G1 transgenic rooster and wild type hens were screened by genomic PCR and immunohistochemistry analysis. 3D8 scFv transgenic chickens, wild type sibling chickens, and SPF chickens were directly infected with IBV (5 chickens per group) and indirectly infected by airborne propagation (15 chickens per group). The relative IBV shedding titers were measured by quantitative real-time PCR using oropharyngeal and cloacal swabs on days 3 and 5 after intraocular infection. The viral load was significantly decreased in the 3D8 scFv transgenic chickens from the contact transmission group. Additionally, blood was collected from each group on day 17 post-infection. The ELISA results showed a marked reduction of the antibody titer against IBV in the 3D8 scFv transgenic chickens from the contact transmission group. These results suggest that the 3D8 scFv protein potentially inhibits infectious bronchitis virus transmission in chickens.
Assuntos
Galinhas/genética , Infecções por Coronavirus/veterinária , Vírus da Bronquite Infecciosa/fisiologia , Doenças das Aves Domésticas/virologia , Eliminação de Partículas Virais/genética , Animais , Animais Geneticamente Modificados , Antivirais/farmacologia , Galinhas/imunologia , Infecções por Coronavirus/transmissão , Infecções por Coronavirus/virologia , Ensaio de Imunoadsorção Enzimática , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/transmissão , Proteínas Recombinantes , Anticorpos de Cadeia Única , Carga Viral/efeitos dos fármacos , Eliminação de Partículas Virais/imunologiaRESUMO
The antiviral effect of a catalytic RNA-hydrolyzing antibody, 3D8 scFv, for intranasal administration against avian influenza virus (H1N1) was described. The recombinant 3D8 scFv protein prevented BALB/c mice against H1N1 influenza virus infection by degradation of the viral RNA genome through its intrinsic RNA-hydrolyzing activity. Intranasal administration of 3D8 scFv (50 µg/day) for five days prior to infection demonstrated an antiviral activity (70% survival) against H1N1 infection. The antiviral ability of 3D8 scFv to penetrate into epithelial cells from bronchial cavity via the respiratory mucosal layer was confirmed by immunohistochemistry, qRT-PCR, and histopathological examination. The antiviral activity of 3D8 scFv against H1N1 virus infection was not due to host immune cytokines or chemokines, but rather to direct antiviral RNA-hydrolyzing activity of 3D8 scFv against the viral RNA genome. Taken together, our results suggest that the RNase activity of 3D8 scFv, coupled with its ability to penetrate epithelial cells through the respiratory mucosal layer, directly prevents H1N1 virus infection in a mouse model system.
Assuntos
Anticorpos Catalíticos/administração & dosagem , Antivirais/administração & dosagem , Células Epiteliais/imunologia , Vírus da Influenza A Subtipo H1N1/imunologia , Infecções por Orthomyxoviridae/prevenção & controle , Ribonucleases/administração & dosagem , Anticorpos de Cadeia Única/administração & dosagem , Administração Intranasal , Animais , Antivirais/farmacocinética , Hidrólise , Camundongos Endogâmicos BALB C , RNA Viral/metabolismo , Anticorpos de Cadeia Única/farmacocinética , Resultado do TratamentoRESUMO
3D8 single chain variable fragment (scFv) is a recombinant monoclonal antibody with nuclease activity that was originally isolated from autoimmune-prone MRL mice. In a previous study, we analyzed the nuclease activity of 3D8 scFv and determined that a HeLa cell line expressing 3D8 scFv conferred resistance to herpes simplex virus type 1 (HSV-1) and pseudorabies virus (PRV). In this study, we demonstrate that 3D8 scFv could be delivered to target tissues and cells where it exerted a therapeutic effect against PRV. PRV was inoculated via intramuscular injection, and 3D8 scFv was injected intraperitoneally. The observed therapeutic effect of 3D8 scFv against PRV was also supported by results from quantitative reverse transcription polymerase chain reaction, southern hybridization, and immunohistochemical assays. Intraperitoneal injection of 5 and 10 µg 3D8 scFv resulted in no detectable toxicity. The survival rate in C57BL/6 mice was 9% after intramuscular injection of 10 LD50 PRV. In contrast, the 3D8 scFv-injected C57BL/6 mice showed survival rates of 57% (5 µg) and 47% (10 µg). The results indicate that 3D8 scFv could be utilized as an effective antiviral agent in several animal models.
Assuntos
Anticorpos Monoclonais/farmacologia , Antivirais/farmacologia , Pseudorraiva/prevenção & controle , Anticorpos de Cadeia Única/farmacologia , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/farmacocinética , Antivirais/química , Antivirais/farmacocinética , Linhagem Celular Tumoral , Desoxirribonucleases/química , Avaliação Pré-Clínica de Medicamentos , Feminino , Herpesvirus Suídeo 1/efeitos dos fármacos , Herpesvirus Suídeo 1/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ribonucleases/química , Anticorpos de Cadeia Única/química , Anticorpos de Cadeia Única/farmacocinética , Distribuição Tecidual , Replicação Viral/efeitos dos fármacosRESUMO
Paclitaxel is used extensively as a chemotherapeutic agent against a broad range of tumors but often leads to the early termination of treatment due to severe toxic side effects. In this study, we hypothesized that ascorbic acid could reduce the toxic side effects without interfering with the anticancer effect of paclitaxel. To demonstrate this, we examined the effect of the combinational treatment of ascorbic acid and paclitaxel using H1299 (a non-small cell lung cancer cell line) and BALB/c mice implanted with or without sarcoma 180 cancer cells. In H1299 cells, the anticancer effects of the combinational treatment with paclitaxel and ascorbic acid were up to 1.7-foldhigher than those of single-agent paclitaxel treatment. In addition, it was shown that the viability of the HEL299 normal cells was up to 1.6-fold higher with the combinational treatment than with paclitaxel treatment alone. In vivo mouse experiments also showed that mice co-treated with paclitaxel and ascorbic acid did not exhibit the typical side effects induced by paclitaxel, such as a reduction in the numbers of white blood cells and red blood cells and the level of hemoglobin (P < .05). The analysis of cancer-related gene expression by quantitative real-time polymerase chain reaction and immunohistochemistry revealed that the combinational treatment suppressed cancer cell multiplication. Taken together, these results suggest that combinational chemotherapy with ascorbic acid and paclitaxel not only does not block the anticancer effects of paclitaxel but also alleviates the cytotoxicity of paclitaxel in vivo and in vitro.
Assuntos
Antineoplásicos/uso terapêutico , Ácido Ascórbico/uso terapêutico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Paclitaxel/toxicidade , Sarcoma 180/tratamento farmacológico , Vitaminas/uso terapêutico , Animais , Antineoplásicos/farmacologia , Antineoplásicos/toxicidade , Protocolos de Quimioterapia Combinada Antineoplásica , Ácido Ascórbico/farmacologia , Contagem de Células Sanguíneas , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sinergismo Farmacológico , Feminino , Expressão Gênica/efeitos dos fármacos , Hemoglobinas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Paclitaxel/uso terapêutico , Vitaminas/farmacologiaRESUMO
Invertase (EC 3.2.1.26) catalyzes the hydrolysis of sucrose into D-glucose and D-fructose. Insoluble acid invertase (INAC-INV) was purified from pea (Pisum sativum L.) by sequential procedures entailing ammonium sulfate precipitation, ion exchange chromatography, absorption chromatography, reactive green-19 affinity chromatography, and gel filtration. The purified INAC-INV had a pH optimum of 4.0 and a temperature optimum of 45 °C. The effects of various concentrations of Tris-HCl, HgCl(2), and CuSO(4) on the activities of the purified invertase were examined. INAC-INV was not affected by Tris-HCl and HgCl(2). INAC-INV activity was inhibited by 6.2 mM CuSO(4) up to 50%. The enzymes display typical hyperbolic saturation kinetics for sucrose hydrolysis. The K(m) and V(max) values of INAC-INV were determined to be 4.41 mM and 8.41 U (mg protein)(-1) min(-1), respectively. INAC-INV is a true member of the ß-fructofuranosidases, which can react with sucrose and raffinose as substrates. SDS-PAGE and immunoblotting were used to determine the molecular mass of INAC-INV to be 69 kDa. The isoelectric point of INAC-INV was estimated to be about pH 8.0. Taken together, INAC-INV is a pea seedling invertase with a stable and optimum activity at lower acid pH and at higher temperature than other invertases.