Bazedoxifene - a promising brain active SERM that crosses the blood brain barrier and enhances spatial memory.

Over 20 years of accumulated evidence has shown that the major female sex hormone 17ß-estradiol can enhance cognitive functioning. However, the utility of estradiol as a therapeutic cognitive enhancer is hindered by its unwanted peripheral effects (carcinogenic). Selective estrogen receptor modulators (SERMs) avoid the unwanted effects of estradiol by acting as estrogen receptor antagonists in some tissues such as breast and uterus, but as agonists in others such as bone, and are currently used for the treatment of osteoporosis. However, understanding of their actions in the brain are limited. The third generation SERM bazedoxifene has recently been FDA approved for clinical use with an improved biosafety profile. However, whether bazedoxifene can enter the brain and enhance cognition is unknown. Using mice, the current study aimed to explore if bazedoxifene can 1) cross the blood-brain barrier, 2) rescue ovariectomy-induced hippocampal-dependent spatial memory deficit, and 3) activate neural estrogen response element (ERE)-dependent gene transcription. Using liquid chromatography-mass spectrometry (LC-MS), we firstly demonstrate that a peripheral injection of bazedoxifene can enter the brain. Secondly, we show that an acute intraperitoneal injection of bazedoxifene can rescue ovariectomy-induced spatial memory deficits. And finally, using the ERE-luciferase reporter mouse, we show in vivo that bazedoxifene can activate the ERE in the brain. The evidence shown here suggest bazedoxifene could be a viable cognitive enhancer with promising clinical applicability.

Comparative Metabolomics of Mycoplasma bovis and Mycoplasma gallisepticum Reveals Fundamental Differences in Active Metabolic Pathways and Suggests Novel Gene Annotations.

Mycoplasmas are simple, but successful parasites that have the smallest genome of any free-living cell and are thought to have a highly streamlined cellular metabolism. Here, we have undertaken a detailed metabolomic analysis of two species, Mycoplasma bovis and Mycoplasma gallisepticum, which cause economically important diseases in cattle and poultry, respectively. Untargeted gas chromatography-mass spectrometry and liquid chromatography-mass spectrometry analyses of mycoplasma metabolite extracts revealed significant differences in the steady-state levels of many metabolites in central carbon metabolism, while 13C stable isotope labeling studies revealed marked differences in carbon source utilization. These data were mapped onto in silico metabolic networks predicted from genome wide annotations. The analyses elucidated distinct differences, including a clear difference in glucose utilization, with a marked decrease in glucose uptake and glycolysis in M. bovis compared to M. gallisepticum, which may reflect differing host nutrient availabilities. The 13C-labeling patterns also revealed several functional metabolic pathways that were previously unannotated in these species, allowing us to assign putative enzyme functions to the products of a number of genes of unknown function, especially in M. bovis. This study demonstrates the considerable potential of metabolomic analyses to assist in characterizing significant differences in the metabolism of different bacterial species and in improving genome annotation. IMPORTANCE Mycoplasmas are pathogenic bacteria that cause serious chronic infections in production animals, resulting in considerable losses worldwide, as well as causing disease in humans. These bacteria have extremely reduced genomes and are thought to have limited metabolic flexibility, even though they are highly successful persistent parasites in a diverse number of species. The extent to which different Mycoplasma species are capable of catabolizing host carbon sources and nutrients, or synthesizing essential metabolites, remains poorly defined. We have used advanced metabolomic techniques to identify metabolic pathways that are active in two species of Mycoplasma that infect distinct hosts (poultry and cattle). We show that these species exhibit marked differences in metabolite steady-state levels and carbon source utilization. This information has been used to functionally characterize previously unknown genes in the genomes of these pathogens. These species-specific differences are likely to reflect important differences in host nutrient levels and pathogenic mechanisms.

Investigation of the cytotoxic capacity of some adherent opportunistic enterobacterial strains by the MTT assay and transmission electron microscopy.

The purpose of this study was to determine the cytotoxic effect on CaCo-2 intestinal cells of dialysates obtained from bacterial cultures of some enterobacterial opportunistic strains with different sources of isolation (food, stool culture, acute diarrhoea, urine culture), previously tested and selected for their intensive adherence and invasion capacity to the cellular substratum and also for their cytotoxic effect on cell monolayers. In this study the level of cytotoxicity was measured quantitatively by means of the MTT assay and qualitatively by transmission electron microscopy (TEM). The MTT method uses a tetrazolium salt for the quantitative spectrophotometric assay of CaCo-2 cells survival and proliferation rates in the presence of bacterial dialysates. This test detects the viable cells, which are able to reduce the tetrazolium salt and offers the advantages of a very simple, rapid and precise method. For TEM examination the ultrathin sections were prepared following the standard protocols. The most cytotoxic strains proved to be Citrobacter freundii 93 strain isolated from stool culture, and Enterobacter cloacae 43, isolated from food followed by E. coli 115 strain isolated from acute diarrhoea. These results correlate well with TEM results pointing out the cytotoxic effect of Enterobacter cloacae 43 strain and also its ability to induce attachment and to destroy the cell surface (A/E) of HEp-2 cells. Besides their great adherence and invasion capacity, the production and release of cytotoxic factors into the extracellular medium represent virulence factors in these strains. This could be responsible for the increase of the pathogenic potential of opportunistic bacteria and explain their implication in the etiology of severe infections and food-borne diseases. This study proved that the virulence of opportunistic pathogens is not correlated with the strain's origin, the most evident virulence features being exhibited by an Enterobacter cloacae strain isolated from food.

Virulence spectra of typed strains of Campylobacter jejuni from different sources: a blinded in vivo study.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. The aims of the present blinded study were to measure and compare the in vivo properties of 40 serotyped, biotyped and genotyped C. jejuni isolates from different sources and genetic makeup. An 11-day-old chick embryo lethality assay, which measured embryo deaths and total viable bacteria over 72 h following inoculation of bacteria into the chorioallantoic membrane, revealed a spectrum of activity within the C. jejuni strains. Human and chicken isolates showed similar high virulence values for embryo deaths while the virulence of the bovine isolates was less pronounced. A one-way ANOVA comparison between the capacity of the strains to kill the chick embryos after 24 h with cytotoxicity towards cultured CaCo-2 cells was significant (P=0.025). After inoculation with a Campylobacter strain, mouse ligated ileal loops were examined histologically and revealed degrees of villous atrophy, abnormal mucosa, dilation of the lumen, congestion and blood in lumen, depending on the isolate examined. A 'total pathology score', derived for each C. jejuni strain after grading the pathology features for degree of severity, showed no apparent relationship with the source of isolation. Some relationship was found between amplified fragment length polymorphism groups and total ileal loop pathology scores, and a one-way ANOVA comparison of the mouse pathology scores against total chick embryo deaths after 72 h was significant (P=0.049).

Comparison of virulence-associated in vitro properties of typed strains of Campylobacter jejuni from different sources.

Campylobacter jejuni is a major cause of human diarrhoeal disease, but specific virulence mechanisms have not been well defined. This blinded study was undertaken with 40 C. jejuni isolates from different sources to determine their haemolytic, cytotoxic and adhesion and invasion activities towards mammalian cells. The results were correlated with source of isolation and genetic makeup by amplified fragment length polymorphism (AFLP) typing. The isolates had variable degrees of haemolytic activity against rabbit erythrocytes and cytotoxicity towards CaCo-2, HeLa and Vero cells. The data indicated that the haemolytic and cytotoxic activities were due to separate factors. A range of cytotoxicity was exhibited, whereby some strains had no activity against the target cells and others had activity against all three cell lines. Certain strains had activity against CaCo-2 cells but little or no activity against the other cells, while others exhibited the opposite phenotype. The data suggested that the cytotoxicity assay with the different cell lines may have detected more than one cytotoxin. A wide variation between isolates was observed for both adherence and invasion with all three cell lines, yet, overall, the strains showed a significantly greater invasion capacity for CaCo-2. There was no clear relationship between source of isolation or disease manifestation and possession of statistically significantly higher levels of particular virulence-associated factors although, in some cases, a correlation between cytotoxicity and cell invasion was evident. Five AFLP clusters, each representing two to eleven isolates with similar profiles, were observed at the 90 % similarity level. Some AFLP groups contained isolates with a common serotype, but each group had C. jejuni isolates from more than one source with the exception of group IV, which contained only human isolates. Isolates with high cytotoxic activity against CaCo-2 cells were confined to groups I, III and IV and a group of unrelated strains (U). Group II isolates had uniformly low cytotoxicity. Isolates in groups I, V and U were more invasive for CaCo-2 cells than isolates in groups II, III and IV. The strain differences in cytotoxicity or invasion did not correlate with source of isolation.

Scanning CO2 laser bacterial inactivation systems.

AIMS: The performance of three scanning CO(2) laser inactivation systems was assessed and included: a gantry system, a rapidly rotating mirror and a low-power hybrid system combining an oscillating mirror and rotary motion of the sample. METHODS AND RESULTS: Escherichia coli and Staphylococcus aureus were the target organisms on stainless steel, nutrient agar or moist collagen film and the laser power was varied from 2 to 1060 W (two laser sources). In general, a threshold energy density was identified, above which no inactivation was observed because the scanning velocity was too high (10 cm s(-1) for stainless steel, 660 W). Reducing the velocity increased the inactivation process until complete inactivation was observed at 1.3 cm s(-1) (E. coli, approximately 10(6) CFU per sample) and 0.82 cm s(-1) (S. aureus, approximately 10(8) CFU per sample); consequently, S. aureus organisms showed a greater resistance to laser irradiation. For the nutrient agar and collagen samples, the averages of the width of clearing were measured as a function of the translation velocity and the rates of inactivation (I(R), cm(2) s(-1)) were found; an optimum velocity was observed that produced the maximum rate of inactivation. At a laser power of 1060 W, the maximum value of I(R) was 140 cm(2) s(-1) ( approximately 10(7) CFU cm(-2)) for S. aureus on collagen and slightly less on nutrient agar (114 cm(2) s(-1), estimated from a best-fit polynomial, r(2) = 0.98). CONCLUSIONS: A comparison of the low- and high-power lasers produced values of 0.09 cm(2) s(-1) W(-1) (i.e. I(R) per Watt delivered) for S. aureus on nutrient agar with the low-power laser at 13 W and on collagen 0.13 cm(2) s(-1) W(-1) for 1060 W. The rate of inactivation was found to be a function of the laser power, translation velocity and properties of the substrate media. The three laser inactivation systems successfully demonstrated the potential speed, efficiency and application of such systems. SIGNIFICANCE AND IMPACT OF THE STUDY: Laser scanning systems offer the potential for rapid and efficient inactivation of surfaces, eliminating the need for chemical treatment.

Effect of laser and environmental parameters on reducing microbial contamination of stainless steel surfaces with Nd:YAG laser irradiation.

AIMS: The effect of laser (pulse repetition frequency, pulse energy and exposure time) and environmental parameters (pH, NaCl concentration and wet or dry samples) of Nd:YAG laser decontamination of stainless steel inoculated with Escherichia coli, Staphylococcus aureus and Listeria monocytogenes was investigated. METHODS AND RESULTS: Stainless steel discs were inoculated with the bacterial samples and exposed to laser energy densities to about 900 J cm(-2). These inactivation curves allowed selection of laser parameters for two-level multifactorial designed experiments, the results of which allowed comparisons to be made between effects of individual and combined parameters on the laser inactivation efficiency. Escherichia coli was inactivated most effectively as a wet film with L. monocytogenes and S. aureus showing similar response. For the multifactorial experiments all laser parameters were significant and were smallest for S. aureus as a wet film. CONCLUSIONS: pH and NaCl concentration had little effect on the efficacy of laser inactivation but dry or wet states and all laser parameters were significant. SIGNIFICANCE AND IMPACT OF THE STUDY: Such systems may prove to be applicable in industrial processes where stainless steel may be contaminated with acidic solutions or salt, e.g. in the food industry with laser inactivation seeming to be independent of these parameters. Parameters have been identified that allow optimization of the treatment process.

Characteristics of Vibrio cholerae proteinases: potential, candidate vaccine antigens.

Vibrio cholerae extracellular proteinases (proteases) have been studied as potential candidate antigens for acellular cholera vaccines. Proteinases from V. cholerae NCTC 10732 were prepared from batch culture either by ammonium sulphate precipitation and G100 Sephadex gel filtration or by isoelectric focusing (IEF). Proteinase activity was at a maximum level after 24 h, coincident with the late exponential phase and early stationary phase. Three major IEF peaks of activity were resolved with specific activities in the range 17.2-195 EU ml(-1 )mg(-1). Sodium dodecyl sulphate-polyacrylamide gel electrophoreses (SDS-PAGE) of these fractions revealed 42, 45, 57 and 75 kDa bands in which proteinase activity was demonstrable. Peptide digest analysis suggested different catalytic specificities for each proteinase fraction. Metalloproteinase and serine proteinase inhibitors, alpha(2)-macroglobulin (alpha(2)-M), the thiol proteinase inhibitor and N-ethylmaleimide inhibited the proteinases. The proteinases nicked Escherichia coli heat-labile toxin to yield catalytically active sub-units, confirmed by the measurement of intrinsic ADP-ribosylation activity. The possible value of these putative V. cholerae antigens in an acellular vaccine is discussed.

Specificity modulation of barley alpha-amylase through biased random mutagenesis involving a conserved tripeptide in beta --> alpha loop 7 of the catalytic (beta/alpha)(8)-barrel domain.

The relative specificity and bond cleavage pattern of barley alpha-amylase 1 (AMY1) were dramatically changed by mutation in F(286)VD that connected beta-strand 7 of the catalytic (beta/alpha)(8)-barrel to a succeeding 3(10)-helix. This conserved tripeptide of the otherwise variable beta --> alpha segment 7 lacked direct ligand contact, but the nearby residues His290 and Asp291 participated in transition-state stabilization and catalysis. On the basis of sequences of glycoside hydrolase family 13, a biased random mutagenesis protocol was designed which encoded 174 putative F(286)VD variants of C95A-AMY1, chosen as the parent enzyme to avoid inactivating glutathionylation by the yeast host. The FVG, FGG, YVD, LLD, and FLE mutants showed 12-380 and 1.8-33% catalytic efficiency (k(cat)/K(m)) toward 2-chloro-4-nitrophenyl beta-D-maltoheptaoside and amylose DP17, respectively, and 0.5-50% activity for insoluble starch compared to that of C95A-AMY1. K(m) and k(cat) were decreased 2-9- and 1.3-83-fold, respectively, for the soluble substrates. The starch:oligosaccharide and amylose:oligosaccharide specificity ratios were 13-172 and 2.4-14 for mutants and 520 and 27 for C95A-AMY1, respectively. The FVG mutant released 4-nitrophenyl alpha-D-maltotrioside (PNPG(3)) from PNPG(5), whereas C95A-AMY1 produced PNPG and PNPG(2). The mutation thus favored interaction with the substrate aglycon part, while products from PNPG(6) reflected the fact that the mutation restored binding at subsite -6 which was lost in C95A-AMY1. The outcome of this combined irrational and rational protein engineering approach was evaluated considering structural accommodation of mutant side chains. FVG and FGG, present in the most active variants, represented novel sequences. This emphasized the worth of random mutagenesis and launched flexibility as a goal for beta --> alpha loop 7 engineering in family 13.

Extensive N-glycosylation reduces the thermal stability of a recombinant alkalophilic bacillus alpha-amylase produced in Pichia pastoris.

Alkalophilic Bacillus alpha-amylase (ABA) was produced in the yeast Pichia pastoris with a yield of 50 mg L(-1) of culture supernatant. The recombinant protein, rABA, was glycosylated at seven of the nine sites for potential N-glycosylation as identified by automated peptide sequencing and MALDI-TOF MS of tryptic fragments. The number of hexose units within each glycan chain was found to vary from 8 to 18 as calculated from the masses of glycosylated peptide fragments. Temperature stability measurements in the absence of substrate showed that the T(50) of glycosylated rABA and its endoglycosidase H-deglycosylated form was 76 degrees C while that of ABA purified from Bacillus was 89 degrees C thus demonstrating that the original temperature stability of ABA was not retained by rABA. The relative thermoperformance, i.e., the activity at 80 degrees C relative to that at 37 degrees C was 0.9 +/- 0.3 for rABA. Removal of all seven N-linked glycans by endoglycosidase H increased the relative thermoperformance to 2.4 +/- 0.6, compared to the value of 3.5 +/- 1.1 for ABA. Thus, removal of the N-linked glycans did not improve the thermostability of rABA but modified its thermoperformance to approach that of the original Bacillus enzyme. rABA had the highest activity around pH 6. Treatment of rABA with endoglycosidase H shifted the pH activity profile in a more alkaline direction approaching the pH activity profile of ABA.

Bactericidal action of high-power Nd:YAG laser light on Escherichia coli in saline suspension.

Infra-red light (1064 nm) from a high-power Nd:YAG laser caused more than 90% loss of viability of Escherichia coli during exposures that raised the temperature of PBS suspensions of the bacteria to 50 C in a thermocouple-equipped cuvette. In contrast, there was minimal loss of viability after heating the same suspensions to 50 degrees C in a water-bath, or in a PCR thermal cycler. The mechanism of laser killing at 50 degrees C was explored by differential scanning calorimetry, by laser treatment of transparent and turbid bacterial suspensions, and by optical absorbency studies of E. coli suspensions at 1064 nm. Taken together, the data suggested that the bactericidal action of Nd:YAG laser light at 50 degrees C was due partly to thermal heating and partly to an additional, as yet undefined, mechanism. Scanning electron microscopy revealed localized areas of surface damage on laser-exposed E. coli cells.

Heat transfer analysis of staphylococcus aureus on stainless steel with microwave radiation.

Staphylococcus aureus (NCTC 6571; Oxford strain) on stainless steel discs was exposed to microwave radiation at 2450 MHz and up to 800 W. Cell viability was reduced as the exposure time increased, with complete bacterial inactivation at 110 s, attaining a temperature of 61.4 degrees C. The low rate of temperature rise, RT, of the bacterial suspension as compared with sterile distilled water or nutrient broth suggests a significant influence of the microwave sterilization efficacy on the thermal properties of the micro-organisms. The heat transfer kinetics of thermal microwave irradiation suggest that the micro-organism has a power density at least 51-fold more than its surrounding liquid suspension. When the inoculum on the stainless steel disc was subjected to microwave radiation, heat conduction from the stainless steel to the inoculum was the cause of bacteriostasis with power absorbed at 23.8 W for stainless steel and 0.16 W for the bacteria-liquid medium. This report shows that the microwave killing pattern of Staph. aureus on stainless steel was mainly due to heat transfer from the stainless steel substrate and very little direct energy was absorbed from the microwaves.

The Production of Polyclonal Antibodies in Laboratory Animals. The Report and Recommendations of ECVAM Workshop 35.

This is the report of the thirty-fifth of a series of workshops organised by the European Centre for the Validation of Alternative Methods (ECVAM). ECVAM's main goal, as defined in 1993 by its Scientific Advisory Committee, is to promote the scientific and regulatory acceptance of alternative methods which are of importance to the biosciences and which reduce, refine or replace the use of laboratory animals. One of the first priorities set by ECVAM was the implementation of procedures which would enable it to become well informed about the state-of-the-art of non-animal test development and validation, and the potential for the possible incorporation of alternative tests into regulatory procedures. It was decided that this would be best achieved by the organisation of ECVAM workshops on specific topics, at which small groups of invited experts would review the current status of various types of in vitro tests and their potential uses, and make recommendations about the best ways forward (1). This joint ECVAM/FELASA (Federation of European Laboratory Animal Science Associations) workshop on The Immunisation of Laboratory Animals for the Production of Polyclonal Antibodies was held in Utrecht (The Netherlands), on 20-22 March 1998, under the co-chairmanship of Coenraad Hendriksen (RIVM, Bilthoven, The Netherlands) and Wim de Leeuw (Inspectorate for Health Protection, The Netherlands). The participants, all experts in the fields of immunology, laboratory animal science, or regulation, came from universities, industry and regulatory bodies. The aims of the workshop were: a) to discuss and evaluate current immunisation procedures for the production of polyclonal antibodies (including route of injection, animal species and adjuvant ); and b) to draft recommendations and guidelines to improve the immunisation procedures, with regard both to animal welfare and to the optimisation of immunisation protocols. This report summarises the outcome of the discussions and includes a number of recommendations and a set of draft guidelines (included in Appendix 1).

Enzymatic properties of the cysteinesulfinic acid derivative of the catalytic-base mutant Glu400-->Cys of glucoamylase from Aspergillus awamori.

The pKa of the catalytic base was lowered and its distance to the general acid catalyst, Glu179, was increased in the glucoamylase from Aspergillus awamori by replacing the catalytic base Glu400 with cysteine followed by oxidation to cysteinesulfinic acid [Fierobe, H.-P., Mirgorodskaya, E., McGuire, K. A., Roepstorff, P., Svensson, B. and Clarke, A. J. (1998) Biochemistry 37, 3743-3752. 1H NMR spectroscopy demonstrated that the oxidized mutant Glu400-->Cys-SO2H glucoamylase, like the wild-type, catalyzed hydrolysis with inversion of the anomeric configuration of the product. Relative to the catalytic base mutant Glu400-->Cys, the Cys400-SO2H glucoamylase had 700 times higher kcat toward maltose, while K(m) was unchanged. Compared to wild-type glucoamylase, the Cys400-SO2H derivative had kcat values of 150-190% and 85-320% on malto- and isomaltooligosaccharides, respectively, while K(m) values were similar to those of wild-type with the two disaccharides and 3.5-5.5- and 1.8-2.5-fold higher for the longer malto- and isomaltooligosaccharides substrates, respectively. The pH-activity dependence at saturating concentration of maltose indicated that the pKa of the catalytic base Cys400-SO2H was about 0.5 pH unit lower than that of wild-type Glu400. The Ki of Cys400-SO2H glucoamylase for the pseudotetrasaccharide and potent inhibitor acarbose increased more than 10(4)-fold, but Ki values of the mono- and disaccharide analogues 1-deoxynojirimycin and beta-O-methylacarviosinide were unchanged, suggesting perturbation at binding subsites beyond the catalytic center. A distinct property of Cys400-SO2H glucoamylase was the catalysis of the condensation of beta-D-glucopyranosyl fluoride and subsequent hydrolysis of the product to beta-glucose, under conditions where this was not detected for the wild-type enzyme.

Mechanistic consequences of mutation of active site carboxylates in a retaining beta-1,4-glycanase from Cellulomonas fimi.

The exoglucanase/xylanase Cex from Cellulomonas fimi is a retaining glycosidase which functions via a two-step mechanism involving the formation and hydrolysis of a covalent glycosyl-enzyme intermediate. The roles of three conserved active site carboxylic acids in this enzyme have been probed by detailed kinetic analysis of mutants modified at these three positions. Elimination of the catalytic nucleophile (E233A) results in an essentially inactive enzyme, consistent with the important role of this residue. However addition of small anions such as azide or formate restores activity, but as an inverting enzyme since the product formed under these conditions is the alpha-glycosyl azide. Shortening of the catalytic nucleophile (E233D) reduces the rates of both formation and hydrolysis of the glycosyl-enzyme intermediate some 3000-4000-fold. Elimination of the acid/base catalyst (E127A) yields a mutant for which the deglycosylation step is slowed some 200-300-fold as a consequence of removal of general base catalysis, but with little effect on the transition state structure at the anomeric center. Effects on the glycosylation step due to removal of the acid catalyst depend on the aglycon leaving group ability, with minimal effects on substrates requiring no general acid catalysis but large (> 10(5)-fold) effects on substrates with poor leaving groups. The Brønsted beta 1g value for hydrolysis of aryl cellobiosides was much larger (beta 1g approximately -1) for the mutant than for the wild-type enzyme (beta 1g = -0.3), consistent with removal of protonic assistance. The pH-dependence was also significantly perturbed. Mutation of a third conserved active site carboxylic acid (E123A) resulted in rate reductions of up to 1500-fold on poorer substrates, which could be largely restored by addition of azide, but without the formation of glycosyl azide products. These results suggest a simple strategy for the identification of the key active site nucleophile and acid/base catalyst residues in glycosidases without resort to active site labeling.

Overexpression, purification, and characterization of recombinant barley alpha-amylases 1 and 2 secreted by the methylotrophic yeast Pichia pastoris.

Recombinant barley alpha-amylase isozymes 1 and 2 were secreted by Pichia pastoris at up to 50 and 1 mg/liter, respectively, representing approximately a 50-fold increase compared to the levels of the heterologous expression by Saccharomyces cerevisiae. The cDNA clones E or pM/C encoding isozymes 1 and 2, respectively, were placed under the control of regulatory sequences from the Pichia AOX1 gene in the vector pHIL-D2. Both isozymes were effectively secreted to the medium as directed by their own signal sequences and easily purified to homogeneity in quantitative yield by affinity chromatography on beta-cyclodextrin-Sepharose. The N-terminal sequence, pI, and Mr indicated that native-like processing took place. Electrospray ionization mass spectrometry, however, revealed microheterogeneity for recombinant isozyme 1. While Mr of one recombinant isozyme 1 form of 45,452 was in excellent agreement with a value of 45,447 calculated from the sequence, liquid chromatography/mass spectrometry of endo Lys C-generated peptides followed by tandem mass spectrometry on a nanoelectrospray ionization/mass spectrometry/mass spectrometry system identified additional recombinant isozyme 1 forms to be glycosylated on Thr410, N-acetylated on His1, S-glutathionylated on Cys95, or C-terminally truncated of -412RS, -411QRS, and -410LQRS. The recombinant enzymes and the alpha-amylases from barley malt closely resembled each other in enzymatic activity on insoluble Blue Starch, amylose of degree of polymerization 17, and 2-chloro-4-nitrophenyl beta-D-maltoheptaoside as well as in Ca2+ dependency of activity. Pichia pastoris thus produced in high yields recombinant alpha-amylase that is similar with respect to structure and function to the enzyme purified from malt extracts. This greatly facilitates future mutational analysis of barley alpha-amylase in order to probe structure/function relationships.