RESUMO
Actinobacillus seminis, a commensal of ovine and caprine reproductive organs, is able to induce epididymitis in the small ruminants that it infects. In this work, we characterised two protein bands of approximately 150 kDa and 65 kDa. These proteins cross-reacted with a polyclonal serum against Gallibacterium anatis hemagglutinin and with a polyclonal serum from sheep with epididymitis, indicating that the proteins are expressed in vivo; the two proteins also interacted with biotin-labeled sheep fibrinogen and fibronectin, suggesting that they may function as adhesins. The participation of these proteins as adhesins was confirmed by a cultured human bladder cell-A. seminis adhesion assay and adherence inhibition by preincubation of A. seminis with polyclonal antiserum to the 150 kDa protein. Both proteins presented sequence identity with an A. seminis GroEL protein by mass spectrometry analysis and agglutinated glutaraldehyde-fixed sheep red blood cells. Immunogold labeling was observed by transmission electron microscopy on bacterial cells that were negatively stained, and a peroxidase reaction was detected in A. seminis biofilms, when an anti-A. seminis 150 kDa protein serum was used, indicating the presence of this protein on the surface of A. seminis and in biofilms. The A. seminis GroEL-homologue is a multifunctional protein that likely acts as a hemagglutinin.
Assuntos
Actinobacillus seminis/fisiologia , Eritrócitos/imunologia , Proteínas de Choque Térmico/imunologia , Proteínas de Choque Térmico/metabolismo , Aglutinação , Testes de Aglutinação , Animais , Anticorpos Antibacterianos , Proteínas da Membrana Bacteriana Externa/imunologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Biofilmes , Adesão Celular , Eritrócitos/metabolismo , Proteínas de Choque Térmico/isolamento & purificação , Hemaglutinação , Hemaglutininas/metabolismo , OvinosRESUMO
Chaperones belonging to the small heat shock protein (sHSP) family are ubiquitous and exhibit elevated expression under stresses conditions to protect proteins against aggregation, thereby contributing to the stress tolerance of the organism. Tropical plants are constantly exposed to high temperatures, and the mechanisms by which these plants tolerate heat stress are of foremost importance to basic science as well as applied agrobiotechnology. Therefore, this study aims to characterize sHSPs from different organelles from sugarcane, an important crop that is associated with sugar and bioenergy production. An expression sequence tag database of sugarcane was searched, and sHsp genes of mitochondrial and chloroplast organelles were selected and cloned. The proteins were expressed in Escherichia coli and isolated and purified by two chromatographic steps with high purity as single species. Circular dichroism and fluorescence spectroscopy showed that both proteins were purified in their folded states with a predominant ß-sheet secondary structure. Determination of the molecular weight, diffusion coefficient and Stokes radius parameters showed that both chaperones form large spherical-like oligomers in solution. The two sHSPs had different oligomeric states and substrate specificities. The mitochondrial sHSP was a 20-mer with ability to protect model substrates that differ from that of the 16-meric sHSP from chloroplasts. These results indicate that both sHSPs are key agents to protect against stress confirming the importance of the great diversity of sHSP chaperones in plants for homeostasis maintenance. Moreover, to our knowledge, this is the first report about small HSPs from sugarcane organelles.
Assuntos
Cloroplastos/metabolismo , Proteínas de Choque Térmico/metabolismo , Mitocôndrias/metabolismo , Proteínas de Plantas/metabolismo , Saccharum/metabolismo , Cromatografia em Gel , Clonagem Molecular , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/isolamento & purificação , Saccharum/genética , Alinhamento de Sequência , Espectrometria de Fluorescência , Especificidade por SubstratoRESUMO
INTRODUCTION: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. OBJECTIVE: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. MATERIALS AND METHODS: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. RESULTS: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. CONCLUSION: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.
Assuntos
Antiprotozoários/farmacologia , Mefloquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/biossíntese , Quinina/farmacologia , Compostos de Espiro/farmacologia , Triterpenos/farmacologia , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Eritrócitos/parasitologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Técnicas In Vitro , Concentração Inibidora 50 , Dados de Sequência Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteoma , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Introduction: Despite efforts to control malaria, around 10% of the world population is at risk of acquiring this disease. Plasmodium falciparum accounts for the majority of severe cases and deaths. Malaria control programs have failed due to the therapeutic failure of first-line antimalarials and to parasite resistance. Thus, new and better therapeutic alternatives are required. Proteomic analysis allows determination of protein expression levels under drug pressure, leading to the identification of new therapeutic drug targets and their mechanisms of action. Objective: The aim of this study was to analyze qualitatively the expression of P.falciparum trophozoite proteins (strain ITG2), after exposure to antimalarial drugs, through a proteomic approach. Materials and methods: In vitro cultured synchronized parasites were treated with quinine, mefloquine and the natural antiplasmodial diosgenone. Protein extracts were prepared and analyzed by two-dimensional electrophoresis. The differentially expressed proteins were selected and identified by MALDI-TOF mass spectrometry. Results: The following proteins were identified among those differentially expressed in the parasite in the presence of the drugs tested: enolase (PF10_0155), calcium-binding protein (PF11_0098), chaperonin (PFL0740c), the host cell invasion protein (PF10_0268) and proteins related to redox processes (MAL8P1.17). These findings are consistent with results of previous studies where the parasite was submitted to pressure with other antimalarial drugs. Conclusion: The observed changes in the P. falciparum trophozoite protein profile induced by antimalarial drugs involved proteins mainly related to the general stress response.
Introducción. A pesar de los esfuerzos para controlar la malaria, esta sigue siendo un problema de salud pública. Plasmodium falciparum es responsable de la mayoría de los casos graves y de las muertes. Los programas de control de la malaria han sido cuestionados debido al fracaso del tratamiento y a la resistencia del parásito a los antipalúdicos de primera línea, por lo que se requieren nuevas y mejores alternativas. El análisis proteómico permite identificar y determinar los niveles de expresión de las proteínas bajo la presión de los medicamentos, lo que posibilita la identificación de nuevos blancos terapéuticos y mecanismos de acción. Objetivo. Analizar cualitativamente la expresión diferencial de proteínas del citosol del trofozoíto de P. falciparum bajo tratamiento con quinina, mefloquina y el compuesto natural diosgenona mediante una aproximación proteómica. Materiales y métodos. Se trataron trofozoítos sincronizados y cultivados in vitro de P. falciparum (cepa ITG2) con quinina, mefloquina y el compuesto natural diosgenona. Los extractos proteicos se prepararon y analizaron por electroforesis bidimensional. Las proteínas con aparente expresión diferencial se seleccionaron e identificaron mediante espectrometría de masas MALDI-TOF. Resultados. Se encontraron las siguientes proteínas diferencialmente expresadas en el trofozoíto: la enolasa (PF10_0155), la proteína de unión a calcio (PF11_0098), la chaperonina (PFL0740c), la proteína de invasión a la célula del huésped (PF10_0268) y la proteína relacionada con procesos de reducción y oxidación (redox) (MAL8P1.17). Estos hallazgos son congruentes con resultados previos de estudios en los que el parásito fue presionado con otros medicamentos antipalúdicos. Conclusión. Los cambios observados en el perfil de proteínas del trofozoíto de P. falciparum tratado con antipalúdicos involucraron preferencialmente proteínas relacionadas con la respuesta al estrés general.
Assuntos
Humanos , Antiprotozoários/farmacologia , Mefloquina/farmacologia , Plasmodium falciparum/efeitos dos fármacos , Proteínas de Protozoários/biossíntese , Quinina/farmacologia , Compostos de Espiro/farmacologia , Triterpenos/farmacologia , Sequência de Aminoácidos , Eletroforese em Gel Bidimensional , Eritrócitos/parasitologia , Regulação da Expressão Gênica/efeitos dos fármacos , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Técnicas In Vitro , Dados de Sequência Molecular , Proteoma , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
Heat shock proteins (Hsp) are molecular chaperones with the capability to interact with a wide range of other proteins and are thus often found coupled with other heat shock and non-heat shock proteins. This can be an advantage to study specific interactions between a chaperone and other proteins and to generate an antitumoral immune response. In this chapter, we present two protocols to isolate Hsp. One involves column chromatography with hydroxyapatite and the other employs immunoprecipitation with antibodies coupled to magnetic beads. In both cases, we specifically want to isolate Hsp coupled with other proteins and use the Hsp complexes as intermediaries to present the coupled peptides/proteins to the immune system, or to explore the associations of a particular Hsp with other proteins.
Assuntos
Cromatografia de Afinidade/métodos , Proteínas de Choque Térmico/isolamento & purificação , Imunoprecipitação/métodos , Materiais Biocompatíveis , Linhagem Celular Tumoral , Durapatita , Proteínas de Choque Térmico/química , Humanos , Domínios e Motivos de Interação entre ProteínasRESUMO
The Salmon Rickettsia syndrome (SRS) remains a major infectious disease in the Chilean aquaculture. A limited number of Piscirickettsia salmonis proteins have been characterized so far for their use as potential candidates for vaccines studies. In this study, we identified and expressed a highly immunogenic protein of P. salmonis extracted by selective hydrophobicity from crude-cell macerates of naturally infected salmonid fish. One and two-D PAGE gels followed by Western blot analysis with a battery of polyclonal anti-P. salmonis antibodies have allowed the isolation of the target protein. Basic local alignment search (BLAST) done after partial sequencing of the pure protein identified it as a member of the heat-shock protein (HSP) family of prokaryotes. The protein, named ChaPs, was cloned as a single open reading frame encoding 545 amino acid residues with a predicted molecular mass of 57.3 kDa. The amplicon representing the entire novel gene was expressed in vitro in different heterologous systems: the PurePro Caulobacter crescentus expression system from where most of the characterization was attained, and also in the Escherichia coli BL-21 CodonPlus model for commercially potential purposes. The immunologic potential of ChaPs was determined with serum from naturally infected fish.
Assuntos
Proteínas de Bactérias/imunologia , Proteínas de Choque Térmico/imunologia , Oncorhynchus kisutch , Piscirickettsia/imunologia , Infecções por Piscirickettsiaceae/veterinária , Sequência de Aminoácidos , Animais , Anticorpos Antibacterianos/sangue , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Proteínas de Bactérias/isolamento & purificação , Western Blotting , Caulobacter/genética , Clonagem Molecular , DNA Bacteriano/química , DNA Bacteriano/genética , Eletroforese em Gel Bidimensional , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Doenças dos Peixes/imunologia , Doenças dos Peixes/microbiologia , Expressão Gênica , Proteínas de Choque Térmico/isolamento & purificação , Dados de Sequência Molecular , Peso Molecular , Infecções por Piscirickettsiaceae/imunologia , Infecções por Piscirickettsiaceae/microbiologia , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
The hspA gene (XAC1151) from Xanthomonas axonopodis pv. citri encodes a protein of 158 amino acids that belongs to the small heat-shock protein (sHSP) family of proteins. These proteins function as molecular chaperones by preventing protein aggregation. The protein was crystallized using the sitting-drop vapour-diffusion method in the presence of ammonium phosphate. X-ray diffraction data were collected to 1.65 angstroms resolution using a synchrotron-radiation source. The crystal belongs to the rhombohedral space group R3, with unit-cell parameters a = b = 128.7, c = 55.3 angstroms. The crystal structure was solved by molecular-replacement methods. Structure refinement is in progress.
Assuntos
Proteínas de Bactérias/química , Proteínas de Choque Térmico/química , Xanthomonas/química , alfa-Cristalinas/química , Proteínas de Bactérias/isolamento & purificação , Cristalização , Cristalografia por Raios X , Proteínas de Choque Térmico/isolamento & purificaçãoRESUMO
sHsps are ubiquitous ATP-independent molecular chaperones, which efficiently prevent the unspecific aggregation of non-native proteins. Here, we described the purification of the small heat shock protein Hsp26 from a Saccharomyces cerevisiae strain harboring a multicopy plasmid carrying HSP26 gene under the control of its native promoter. A 26 kDa protein was purified to apparent homogeneity with a recovery of 74% by a very reproducible three steps procedure consisting of ethanol precipitation, sucrose gradient ultracentrifugation, and heat inactivation of residual contaminants. The purified polypeptide was unequivocally identified as Hsp26 using a specific Hsp26 polyclonal antibody as a probe. The analysis of the purified protein by electron microscopy revealed near spherical particles with a diameter of 12.0 nm (n=57, standard deviation +/-1.6 nm), displaying a dispersion in size ranging from 9.2 to 16.1 nm, identical to Methanococcus jannaschii Hsp16.5 and in the range of the size estimated for yeast Hsp26, in a previous report. Purified yeast Hsp26 was able to suppress 72% of the heat-induced aggregation of citrate synthase at a ratio of 1:1 (Hsp26 24-mer complex to citrate synthase dimer), and 86% of the heat-induced aggregation of lysozyme at a molar ratio of 1:16 (Hsp26 24-mer complex to lysozyme monomer). In conclusion, the Hsp26 protein purified as described here has structure and activity similar to the previously described preparations. As advantages, this new protocol is very reproducible and requires simple apparatuses which are found in all standard biochemistry laboratories.
Assuntos
Proteínas de Choque Térmico/isolamento & purificação , Complexos Multiproteicos/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas Arqueais/química , Proteínas Arqueais/ultraestrutura , Citrato (si)-Sintase/química , Proteínas de Choque Térmico/biossíntese , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/ultraestrutura , Complexos Multiproteicos/biossíntese , Complexos Multiproteicos/ultraestrutura , Muramidase/química , Dobramento de Proteína , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Proteins that belong to the heat shock protein (Hsp) 40 family assist Hsp70 in many cellular functions and are important for maintaining cell viability. A knowledge of the structural and functional characteristics of the Hsp40 family is therefore essential for understanding the role of the Hsp70 chaperone system in cells. In this work, we used small angle x-ray scattering and analytical ultracentrifugation to study two representatives of human Hsp40, namely, DjA1 (Hdj2/dj2/HSDJ/Rdj1) from subfamily A and DjB4 (Hlj1/DnaJW) from subfamily B, and to determine their quaternary structure. We also constructed low resolution models for the structure of DjA1-(1-332), a C-terminal-deleted mutant of DjA1 in which dimer formation is prevented. Our results, together with the current structural information of the Hsp40 C-terminal and J-domains, were used to generate models of the internal structural organization of DjA1 and DjB4. The characteristics of these models indicated that DjA1 and DjB4 were both dimers, but with substantial differences in their quaternary structures: whereas DjA1 consisted of a compact dimer in which the N and C termini of the two monomers faced each other, DjB4 formed a dimer in which only the C termini of the two monomers were in contact. The two proteins also differed in their ability to bind unfolded luciferase. Overall, our results indicate that these representatives of subfamilies A and B of human Hsp40 have different quaternary structures and chaperone functions.
Assuntos
Proteínas de Choque Térmico/química , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Sequência de Aminoácidos , Cristalografia por Raios X , Dimerização , Proteínas de Choque Térmico HSP40 , Proteínas de Choque Térmico HSP70/química , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/isolamento & purificação , Humanos , Modelos Moleculares , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/isolamento & purificação , Dados de Sequência Molecular , Dobramento de Proteína , Alinhamento de Sequência , SoluçõesRESUMO
The small heat shock proteins (smHSPs) belong to a family of proteins that function as molecular chaperones by preventing protein aggregation and are also known to contain a conserved region termed alpha-crystallin domain. Here, we report the expression, purification, and partial characterization of a novel smHSP (HSP17.9) from the phytopathogen Xylella fastidiosa, causal agent of the citrus variegated chlorosis (CVC). The gene was cloned into a pET32-Xa/LIC vector to over-express the protein coupled with fusion tags in Escherichia coli BL21(DE3). The expressed HSP17.9 was purified by immobilized metal affinity chromatography (IMAC) and had its identity determined by mass spectrometry (MALDI-TOF). The correct folding of the purified recombinant protein was verified by circular dichroism spectroscopy. Finally, the HSP17.9 protein also proved to efficiently prevent induced aggregation of insulin, strongly indicating a chaperone-like activity.
Assuntos
Proteínas de Choque Térmico/isolamento & purificação , Proteínas de Choque Térmico/metabolismo , Xylella/genética , Sequência de Aminoácidos , Dicroísmo Circular , Escherichia coli/citologia , Escherichia coli/genética , Expressão Gênica , Vetores Genéticos , Proteínas de Choque Térmico/química , Insulina/metabolismo , Dados de Sequência Molecular , Oligonucleotídeos/química , Plasmídeos , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Relação Estrutura-Atividade , Fatores de Tempo , Transformação BacterianaRESUMO
Prions are composed of an isoform of a normal sialoglycoprotein called PrP(c), whose physiological role has been under investigation, with focus on the screening for ligands. Our group described a membrane 66 kDa PrP(c)-binding protein with the aid of antibodies against a peptide deduced by complementary hydropathy. Using these antibodies in western blots from two-dimensional protein gels followed by sequencing the specific spot, we have now identified the molecule as stress-inducible protein 1 (STI1). We show that this protein is also found at the cell membrane besides the cytoplasm. Both proteins interact in a specific and high affinity manner with a K(d) of 10(-7) M. The interaction sites were mapped to amino acids 113-128 from PrP(c) and 230-245 from STI1. Cell surface binding and pull-down experiments showed that recombinant PrP(c) binds to cellular STI1, and co-immunoprecipitation assays strongly suggest that both proteins are associated in vivo. Moreover, PrP(c) interaction with either STI1 or with the peptide we found that represents the binding domain in STI1 induce neuroprotective signals that rescue cells from apoptosis.
Assuntos
Apoptose , Proteínas de Choque Térmico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Animais , Anisomicina/antagonistas & inibidores , Anisomicina/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Cobre/metabolismo , AMP Cíclico/fisiologia , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas do Olho/química , Proteínas do Olho/metabolismo , Proteínas de Choque Térmico/química , Proteínas de Choque Térmico/isolamento & purificação , Interações Hidrofóbicas e Hidrofílicas , Laminina/metabolismo , Substâncias Macromoleculares , Proteínas de Membrana/metabolismo , Camundongos , Chaperonas Moleculares/química , Chaperonas Moleculares/isolamento & purificação , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/isolamento & purificação , Neurônios/citologia , Técnicas de Cultura de Órgãos , Fragmentos de Peptídeos/metabolismo , Ligação Proteica , Mapeamento de Interação de Proteínas , Proteínas Recombinantes de Fusão/metabolismo , Retina/citologia , Retina/efeitos dos fármacos , Transdução de SinaisRESUMO
Estrogen, through estrogen receptors (ERs), may regulate the synthesis of progesterone receptors (PRs) and of a heat shock estrogen receptor-associated protein (hsp27). In female breast carcinoma (FBC) both proteins serve as surrogate indicators for the presence of functional ERs. In addition, the expression of these proteins was related to other prognostic indicators of value in female breast tumours. Endocrine disorders, hormone therapy and altered estrogen metabolism have been associated with the development of male breast cancer (MBC), suggesting that evaluation of the expression of ER, PR and hsp27 might improve our understanding of the biology of this tumour. ER and PR status and hsp27 expression were evaluated by immunohistochemistry in 16 primary MBC patients. The interrelationships between these parameters were established and compared with the clinicopathological data on the tumours. Ten (56%) MBC patients were ER-positive, 69% were PR-positive and all samples were hsp27-positive. Our series of MBC patients showed a positive correlation between ERs and PRs, however there was a lack of correlation between hsp27 and ERs or PRs. MBCs did not exhibit any correlation between the biomarkers studied and known prognostic indicators for females (e.g. Scarff-Bloom-Richardson (SBR) or modified SBR (MSBR) grade, T stage, lymph node status). This is the first published series reporting the incidence of hsp27 in MBC. The lack of association between the expression of ERs and hsp27 found in MBC differs from the results reported for FBC, moreover the expression of ERs, PRs or hsp27 did not correlate with the clinicopathological parameters that have prognostic value in females. Although the data were obtained from a relatively small sample population, our findings suggest that MBC and FBC are biologically different tumours with respect to the expression of the studied proteins.
Assuntos
Neoplasias da Mama Masculina/química , Carcinoma/química , Proteínas de Choque Térmico/isolamento & purificação , Receptores de Estrogênio/isolamento & purificação , Receptores de Progesterona/isolamento & purificação , Adulto , Idoso , Neoplasias da Mama Masculina/patologia , Neoplasias da Mama Masculina/terapia , Carcinoma/patologia , Carcinoma/terapia , Expressão Gênica , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , PrognósticoRESUMO
A three step purification procedure for trehalase from Saccharomyces cerevisiae with a recovery of 76% of the original activity is presented. The enzyme was activated by a heat shock treatment prior to homogenization of the cells. A mutant strain deleted in SUC genes was used to avoid contamination by invertase. The lyophylized enzyme was stable for, at least, 5 months and could be used to determine trehalose in the range 25 to 500 nmol. The preparation was free of inspecific phosphatases allowing for trehalose determinations in yeast cell free extracts and in insect hemolymph.