Increased encapsulated cell biodelivery of nerve growth factor in the brain by transposon-mediated gene transfer.

Nerve growth factor (NGF) is a potential therapeutic agent for Alzheimer's disease (AD) as it has positive effects on the basal forebrain cholinergic neurons whose degeneration correlates with the cognitive decline in AD. We have previously described an encapsulated cell biodelivery device, NsG0202, capable of local delivery of NGF by a genetically modified human cell line, NGC-0295. The NsG0202 devices have shown promising safety and therapeutic results in a small phase 1b clinical study. However, results also show that the NGF dose could advantageously be increased. We have used the sleeping beauty transposon expression technology to establish a new clinical grade cell line, NGC0211, with at least 10 times higher NGF production than that of NGC-0295. To test whether encapsulation of this cell line provides a relevant dose escalation step in delivering NGF for treatment of the cognitive decline in AD patients, we have validated the bioactivity of devices with NGC0211 and NGC-0295 cells in normal rat striatum as well as in the quinolinic acid striatal lesion model. These preclinical animal studies show that implantation of devices with NGC0211 cells lead to significantly higher NGF output, which in both cases correlate with highly improved potency.

Effect of acute and delayed hyperbaric oxygen therapy on cyanide whole blood levels during acute cyanide intoxication.

Cyanide and carbon monoxide, which are often found in fire victims, are toxic gases emitted from fires. Cyanide and carbon monoxide have similar molecular structure. Cyanide binds to the enzyme cytochrome oxidase a, a3 similar to carbon monoxide, thus blocking the mitochondrial respiration chain causing depletion of adenosine triphosphate. Hyperbaric oxygen (HBO2) is recommended for treating carbon monoxide poisoning. The therapeutic effect is due to a high oxygen pressure removing carbon monoxide from the cells. We hypothesise that HBO2 induces changes in whole-blood-cyanide by a competitive mechanism forcing cyanide out of cellular tissues. A rat model was developed to study this effect. Female Sprague Dawley rats were anesthetized with a fentanyl + fluanizone combination and midazolam given subcutaneously (s.c.). Rats were poisoned with 5.4 mg/kg KCN injected intra-peritoneally in Group 1 and intra-arterially in Group 2. Blood samples were taken immediately after poisoning, and at one and a half, three and five hours. Blood was drawn from a jugular vein in Group 1 and from a femoral artery in Group 2. Group 1 rats were divided into a control group of 12 rats without HBO2, 10 rats had acute HBO2 immediately after poisoning and a group of 10 rats had HBO2 one and a half hours after poisoning. Group 2 rats were divided into a control group and an acute HBO2 group, with 10 rats in both groups. Whole-blood-cyanide concentrations were measured using the Conway method based on diffusion and the subsequent formation of cyanocobalamin measured by a spectrophotometer. Results showed that whole-blood-cyanide concentration in Group 1 controls and acute HBO2 initially rose and then fell towards zero. In rats treated with delayed HBO2, the reduction in whole-blood-cyanide concentration was significantly less as compared to controls and acute HBO2-treated rats. Group 2 controls whole-blood-cyanide concentration decreased towards zero throughout the observation period. However, in Group 2 acute HBO2-treated rats a secondary rise in whole-blood-cyanide was observed. The study indicates that HBO2 can move cyanide from tissue to blood. These findings may be of clinical importance, as combined HBO2 and antidote treatment, may accelerate detoxification.

Mutation in APOA1 predicts increased risk of ischaemic heart disease and total mortality without low HDL cholesterol levels.

OBJECTIVES: To determine whether mutations in APOA1 affect levels of high-density lipoprotein (HDL) cholesterol and to predict risk of ischaemic heart disease (IHD) and total mortality in the general population. BACKGROUND: Epidemiologically, risk of IHD is inversely related to HDL cholesterol levels. Mutations in apolipoprotein (apo) A-I, the major protein constituent of HDL, might be associated with low HDL cholesterol and predispose to IHD and early death. DESIGN: We resequenced APOA1 in 190 individuals and examined the effect of mutations on HDL cholesterol, risk of IHD, myocardial infarction (MI) and mortality in 10 440 individuals in the prospective Copenhagen City Heart Study followed for 31 years. Results were validated in an independent case-control study (n = 16 035). Additionally, we determined plasma ratios of mutant to wildtype (WT) apoA-I in human heterozygotes and functional effects of mutations in adenovirus-transfected mice. RESULTS: We identified a new mutation, A164S (1 : 500 in the general population), which predicted hazard ratios for IHD, MI and total mortality of 3.2 [95% confidence interval (CI): 1.6-6.5], 5.5 (95% CI: 2.6-11.7) and 2.5 (95% CI: 1.3-4.8), respectively, in heterozygotes compared with noncarriers. Mean reduction in survival time in heterozygotes was 10 years (P < 0.0001). Results for IHD and MI were confirmed in the case-control study. Furthermore, the ratio of mutant S164 to WT A164 apoA-I in plasma of heterozygotes was reduced. In addition, A164S heterozygotes had normal plasma lipid and lipoprotein levels, including HDL cholesterol and apoA-I, and this finding was confirmed in adenovirus-transfected mice. CONCLUSIONS: A164S is the first mutation in APOA1 to be described that predicts an increased risk of IHD, MI and total mortality without low HDL cholesterol levels.

Identification of a major protein on the cytosolic face of caveolae.

Cav-p60, a specific and ubiquitous caveolar protein, was immunoprecipitated from solubilized rat adipocyte plasma membranes and identified as similar to a GeneBank entry annotated mouse polymerase transcript release factor (PTRF) by MALDI-TOF and MS-MS of major fragments. Cloning and virtual translation of the corresponding rat adipocyte cDNA sequence revealed 98.7% identity with mouse PTRF. In vitro translation of this sequence produced a protein, which was recognized by antibodies to both cav-p60 and PTRF. EM gold labeling studies showed that a rabbit antiserum against murine PTRF immunolabeled caveolae specifically in adipocytes from both mouse and rat. In view of the reported function of the protein, which is exerted in the cell nucleus, its subcellular localization was investigated. We found that the protein could be purified by differential solubilization of a plasma membrane fraction followed by SDS-PAGE, and that the protein was as abundant as caveolin in this fraction. We were unable to detect the protein in cell nuclei by subcellular fractionation or fluorescence microscopy. The results show that in a large number of cell types, PTRF is essentially located to caveolae, and that each caveola harbors many copies of the protein. Consequently, we suggest the name Cavin for this protein.

The (ProB27, ThrB28) human insulin analogue is more potent and more rapidly absorbed from subcutis than human insulin.

OBJECTIVE: To test the physiological properties of human insulin in which the amino acids Thr (B27) and Pro (B28) are interchanged (PT insulin). This was hypothesised to prevent dimerisation and accelerate the absorption from s.c. tissue without altering the affinity for the insulin receptor. DESIGN: PT insulin was expressed in Pichia pastoris and processed in vitro. The purified compound was used for physiological investigations. METHODS: Receptor binding activity to insulin and IGF receptors was evaluated in a competition assay using iodinated PT insulin and recombinant receptors while growth induction properties were evaluated by thymidine incorporation. Absorption kinetics from pig subcutis was investigated by measuring the disappearance of iodinated PT insulin. The potency was evaluated by measuring the blood glucose lowering activity in mice. RESULTS: The absorption of PT insulin was accelerated compared with human insulin, although still slower than Asp (B28) insulin. Human and PT insulin had similar affinities for the human insulin receptor (K(d)=3.6 x 10(-12) vs 5.2 x 10(-12) mol/l) while the affinity for the IGF receptor was four times higher for PT insulin than for human insulin (K(d)=3.4 x 10(-8) vs 1.3 x 10(-7) mol/l). This resulted in a slightly higher DNA synthesis when assayed in intermediary insulin concentrations. The blood glucose lowering effect in mice exceeded the effect of human insulin (integral 0-60 min: 61.4+/-7 vs 30+/-4, n=6, P=0.046). CONCLUSIONS: PT insulin is absorbed faster and is more potent than human insulin. Although PT insulin stimulates growth more than human insulin, this will not prevent its use in the clinic, but the main interest will probably focus on investigations to clarify the paradox of full biological activity in connection with the recently described lack of structure in the B-chain.

Post-translational modifications of heterologously expressed cholecystokinin in Saccharomyces cerevisiae.

The vertebrate neuroendocrine peptide cholecystokinin (CCK) is subjected to numerous post-translational modifications upon maturation to bioactive CCK. However, the current knowledge of the proteolytic processing of proCCK, as reviewed here, is not complete. We have chosen Saccharomyces cerevisiae as a model to study these endoproteolytic processing events. Expression of proCCK as a fusion protein to the prepro leader peptide of alpha-mating factor directed the protein through the secretory pathway and resulted in nanomolar concentrations of secreted glycine-extended CCK. The CCK peptides showed many correlations to the known endoproteolytic processing products of proCCK in endocrine cells. Especially the processing to the abundant form, CCK-22, was investigated and it is suggested that a novel enzyme is responsible for its production. Thus, yeast may be used to study the proteolysis of proCCK with the aim to identify mammalian homologues involved in maturation of CCK.

Advantages of external accumulation for electron capture dissociation in Fourier transform mass spectrometry.

A combination of external accumulation (XA) with electron capture dissociation (ECD) improves the electron capture efficiency, shortens the analysis time, and allows for rapid integration of multiple scans in Fourier transform mass spectrometry. This improves the signal-to-noise ratio and increases the number of detected products, including structurally important MS3 fragments. With XA-ECD, the range of the labile species amenable to ECD is significantly extended. Examples include the first-time determination of the positions of six GalNAc groups in a 60-residue peptide, five sialic acid and six O-linked GalNAc groups in a 25-residue peptide, and the sulfate group position in a 11-residue peptide. Even weakly bound supramolecular aggregates, including nonspecific peptide complexes, can be analyzed with XA-ECD. Preliminary results are reported on high-rate XA-ECD that uses an indirectly heated dispenser cathode as an electron source. This shortens the irradiation time to > or = 1 ms and increases the acquisition rate to 3 scans/s, an improvement by a factor of 10-100.

Human cathelicidin, hCAP-18, is processed to the antimicrobial peptide LL-37 by extracellular cleavage with proteinase 3.

Cathelicidins are a family of antimicrobial proteins found in the peroxidase-negative granules of neutrophils. The known biologic functions reside in the C-terminus, which must be cleaved from the holoprotein to become active. Bovine and porcine cathelicidins are cleaved by elastase from the azurophil granules to yield the active antimicrobial peptides. The aim of this study was to identify the physiological setting for cleavage of the only human cathelicidin, hCAP-18, to liberate the antibacterial and cytotoxic peptide LL-37 and to identify the protease responsible for this cleavage. Immunoelectron microscopy demonstrated that both hCAP-18 and azurophil granule proteins were present in the phagolysosome. Immunoblotting revealed no detectable cleavage of hCAP-18 in cells after phagocytosis. In contrast, hCAP-18 was cleaved to generate LL-37 in exocytosed material. Of the 3 known serine proteases from azurophil granules, proteinase 3 was solely responsible for cleavage of hCAP-18 after exocytosis. This is the first detailed study describing the generation of a human antimicrobial peptide from a promicrobicidal protein, and it demonstrates that the generation of active antimicrobial peptides from common proproteins occurs differently in related species. (Blood. 2001;97:3951-3959)

Identification and distribution of CCK-related peptides and mRNAs in the rainbow trout, Oncorhynchus mykiss.

Peptides homologous to mammalian cholecystokinin (CCK), and their corresponding cDNAs, have been isolated and sequenced from the rainbow trout, Oncorhynchus mykiss. Three cDNAs encoding CCK-like preprohormones were identified from the brain. The cDNAs encode three different putative CCK-8 peptides containing Asn, Leu or Thr, in position 6 (counting from the C-terminus). Hence, the trout CCKs are named CCK-N, CCK-L and CCK-T respectively. RT-PCR showed differential expression of the three mRNAs although all were detected in the brain and intestine, similar to the expression pattern of CCK in tetrapods. In situ hybridization on trout brain sections using (35)S-labeled gene-specific antisense oligonucleotides showed that the three mRNAs were present in different parts, suggesting that the three CCK peptides may have different functions in the brain. Purification of CCK-immunoreactive material from the trout brain resulted in two CCK octapeptides: DYNGWMDF(.)NH2 (CCK-N) and DYLGWMDF(.)NH2 (CCK-L) present in equal amounts. In the pyloric caeca, three forms of CCK-L were identified, consisting of 7, 8 and 21 residues, respectively. The last was dominating and had the sequence ASGPGPSHKIKDRDYLGWMDF(.)NH2. All isolated peptides were fully sulfated. The trout is the first species in which three different CCK-like cDNAs have been identified.

A processing enzyme cleaving avian progastrin at post-Phe bonds.

Neuroendocrine peptides mature partly through endoproteolytic processing of long precursor forms. Best characterised is cleavage at mono- and dibasic residues, but additional sites also exist. Among these is post-Phe cleavage, first suggested to participate in the processing of chicken progastrin. In order to characterise this new mechanism, antibodies recognising the processing products of post-Phe cleavage of chicken progastrin were produced for radioimmunoassay measurements and immunocytochemistry. High concentrations of the carboxyamidated C-terminus and the N-terminus of gastrin-53 were measured in extracts of the antrum. In addition, significant amounts were detected using an assay specific for the N-terminus of gastrin-30 and with another assay for the C-terminus of the corresponding peptide, gastrin-53(1-23), obtained after cleavage at the Phe(23)-Ala(24) bond of gastrin-53. Colocalisation in antral G-cells of the N-termini of gastrin-53 and gastrin-30 and of the C-terminus of gastrin-53(1-23) was confirmed by immunohistochemistry. Finally, we identified the intact N-terminal 1-23 fragment of gastrin-53 complementary to gastrin-30, verifying endoproteolytic cleavage at the Phe(23)-Ala(24) bond. Taken together, the results support the existence of vertebrate endoprotease cleaving hormone precursors at post-Phe sites.

Biochemical characterization of the penta-EF-hand protein grancalcin and identification of L-plastin as a binding partner.

Grancalcin is a recently described Ca(2+)-binding protein especially abundant in human neutrophils. Grancalcin belongs to the penta-EF-hand subfamily of EF-hand proteins, which also comprises calpain, sorcin, peflin, and ALG-2. Penta-EF-hand members are typified by two novel types of EF-hands: one that binds Ca(2+) although it has an unusual Ca(2+) coordination loop and one that does not bind Ca(2+) but is directly involved in homodimerization. We have developed a novel method for purification of native grancalcin and found that the N terminus of wild-type grancalcin is acetylated. This posttranslational modification does not affect the secondary structure or conformation of the protein. We found that both native and recombinant grancalcin always exists as a homodimer, regardless of the Ca(2+) load. Flow dialysis showed that recombinant grancalcin binds two Ca(2+) per subunit with positive cooperativity and moderate affinity ([Ca(2+)](0.5) of 25 and 83 microm in the presence and absence of octyl glycoside, respectively) and that the sites are of the Ca(2+)-specific type. Furthermore, we showed, by several independent methods, that grancalcin undergoes important conformational changes upon binding of Ca(2+) and subsequently exposes hydrophobic amino acid residues, which direct the protein to hydrophobic surfaces. By affinity chromatography of solubilized human neutrophils on immobilized grancalcin, L-plastin, a leukocyte-specific actin-bundling protein, was found to interact with grancalcin in a negative Ca(2+)-dependent manner. This was substantiated by co-immunoprecipitation of grancalcin by anti-L-plastin antibodies and vice versa.

Calreticulin is an interleukin-3-sensitive calcium-binding protein in human basophil leukocytes.

BACKGROUND: IL-3 enhances basophil histamine release upon stimulation with any known secretagogue. The molecular mechanism behind this regulation is not known, although some observations suggest that IL-3 modulates the calcium part of the signal transduction mechanism. The inhibitory action of glucocorticoids on basophils can be reversed by stimulation with IL-3. METHODS: Calcium-binding proteins in the basophil cell line KU812 were identified by two-dimensional gel electrophoresis, Calcium-overlay assay, N-terminal sequence analysis, and mass spectometry. The presence of the same proteins in purified human basophil leukocytes was established by comigration of KU812 and human basophil proteins on the two-dimensional gels. The expression of the calcium-binding proteins in the absence and presence of IL-3 and/or anti-IgE was determined by densitometric measurement of the spots on the two-dimensional gels. RESULTS: Calreticulin was identified on the two-dimensional gel of KU812 proteins. A protein with exactly the same migration pattern was found on the gels of proteins from purified human basophils. Immunoblotting with a specific antihuman calreticulin antibody confirmed that this protein was calreticulin. Subsequent analysis showed that the expression of calreticulin in the basophils is upregulated twofold upon stimulation with rhIL-3, even in doses below those needed for enhancement of histamine release. CONCLUSIONS: The expression of calreticulin in human basophil leukocytes is regulated by IL-3. Calreticulin is known to modulate IP3-dependent Ca2+ influx in different cell systems, and calreticulin overexpression inhibits steroid-induced transcriptional activation. Therefore, modulation of calreticulin expression may be one mechanism by which IL-3 exerts its effects on human basophils.

Identification of ostrich and chicken cholecystokinin cDNA and intestinal peptides.

The cDNAs encoding the preprohormones of the regulatory peptides cholecystokinin (CCK) and the related gastrin have been identified in a number of vertebrate species. However, from birds only chicken preprogastrin is known. In the present study preproCCK cDNA was identified in two species of birds, ostrich and chicken. In addition, the molecular forms of the bioactive peptides expressed in the small intestine were characterized. Both preproCCKs contain mono basic processing sites for the production of CCK-70 and -8 as seen in turtle and bullfrog. However, compared to these species an unusually large proportion was processed to the small forms CCK-7 and -8 and only minute amounts to larger forms. The encoded preprohormones are very similar to each other and to turtle CCK. Furthermore, they also show a high degree of similarity to the CCKs identified in more distant vertebrates. This confirms that CCK is highly conserved among vertebrates while the structure of gastrin, the other member of the CCK/gastrin family, is considerably more variable.

Structure, measurement, and secretion of human glucagon-like peptide-2.

By using radioimmunoassays toward the cDNA-predicted amino acid sequence of human glucagon-like peptide-2, a peptide was isolated from extracts of human ileum. By mass spectrometry and Edman sequencing, this peptide was identified as human proglucagon 126-158. High-performance liquid chromatography analyses indicated that a similar immunoreactive peptide (iGLP-2) was present in human plasma. Human plasma concentrations of iGLP-2 were elevated 3- to 4-fold at 1 to 2 h after ingestion of 800 to 1200 kcal meals.

Sulfakinin neuropeptides in a crustacean. Isolation, identification andtissue localization in the tiger prawn Penaeus monodon.

The sulfakinin (SK) family of neuropeptides are characterized by a C-terminal octapeptide sequence that begins with two acidic residues (most commonly DD), and ends with YGHMRF-NH2, usually with the tyrosyl residue sulfated. So far, sulfakinins have only been identified in insects and the present study was initiated to investigate if the family is more widely distributed within the arthropods. Purification of an extract of the central nervous system of the giant tiger prawn Penaeus monodon has revealed three novel members of the sulfakinin peptide family. One of the peptides, Pem SKI, has the sequence

Isolation, structural characterization, and bioactivity of a novel neuromedin U analog from the defensive skin secretion of the Australasian tree frog, Litoria caerulea.

We report the isolation of a novel bioactive peptide, neuromedin U-23 (NmU-23), from the defensive skin secretion of the Australasian tree frog, Litoria caerulea. The primary structure of the peptide was established by a combination of microsequencing, mass spectroscopy and site-directed antiserum immunoreactivity as SDEEVQVPGGVISNGYFLFRPRN-amide (M(r) 2580.6). A synthetic replicate of frog NmU-23 displaced monoradioiodinated rat NmU-23 from uterine membranes in a dose-dependent fashion indistinguishable from nonisotopically labeled rat NmU-23. In a rat uterine smooth muscle strip preparation, synthetic frog NmU-23 produced dose-dependent contractions identical to porcine NmU-25. However, in a preparation of human urinary bladder muscle strip, the synthetic frog peptide was more potent than porcine NmU-25 in eliciting contraction and produced desensitization of the preparation to the latter peptide. This report demonstrates that the defensive skin secretion of a frog contains a novel peptide exhibiting a high degree of primary structural similarity to the endogenous vertebrate peptide, NmU, and that this frog skin analog displays biological activity in mammalian tissues.

A highly selective CC chemokine receptor (CCR)8 antagonist encoded by the poxvirus molluscum contagiosum.

The MC148 CC chemokine from the human poxvirus molluscum contagiosum (MCV) was probed in parallel with viral macrophage inflammatory protein (vMIP)-II encoded by human herpesvirus 8 (HHV8) in 16 classified human chemokine receptors. In competition binding using radiolabeled endogenous chemokines as well as radiolabeled MC148, MC148 bound with high affinity only to CCR8. In calcium mobilization assays, MC148 had no effect on its own on any of the chemokine receptors, but in a dose-dependent manner blocked the stimulatory effect of the endogenous I-309 chemokine on CCR8 without affecting chemokine-induced signaling of any other receptor. In contrast, vMIP-II acted as an antagonist on 10 of the 16 chemokine receptors, covering all four classes: XCR, CCR, CXCR, and CX(3)CR. In chemotaxis assays, MC148 specifically blocked the I-309-induced response but, for example, not stromal cell-derived factor 1alpha, monocyte chemoattractant protein 1, or interleukin 8-induced chemotaxis. We thus concluded that the two viruses choose two different ways to block the chemokine system: HHV8 encodes the broad-spectrum chemokine antagonist vMIP-II, whereas MCV encodes a highly selective CCR8 antagonist, MC148, conceivably to interfere with monocyte invasion and dendritic cell function. Because of its pharmacological selectivity, the MC148 protein could be a useful tool in the delineation of the role played by CCR8 and its endogenous ligand, I-309.

Identification and expression of gastrin and cholecystokinin mRNAs from the turtle, Pseudemys scripta: evidence of tissue-specific tyrosyl sulfation(1).

Gastrin and cholecystokinin (CCK) are related peptide hormones expressed in the brain and gut of vertebrates. In this study, complementary DNAs have been characterised from the red-eared slider turtle, Pseudemys scripta. The encoded preproCCK contains mono and dibasic endoproteolytic processing sites for formation of the previously identified CCK-70, CCK-40 and CCK-8 products, whereas preprogastrin contains two dibasic processing sites for the generation of gastrin-52. Alignment of the predicted preprohormone structures with those of other species, showed that preproCCK has been well conserved among all vertebrates, whereas progastrin is less conserved. Both gastrin and CCK mRNA display expression patterns similar to their mammalian counterparts, with CCK being expressed in the brain, duodenum and small intestine, and gastrin in the antrum. Heterologous expression of turtle preprogastrin in a mammalian endocrine cell line led to production of carboxyamidated gastrin-52 as observed in turtle antrum. However, in contrast to the non-sulfated endogenous peptide, the heterologously expressed gastrin was completely Tyr sulfated. Consequently, it appears that either gastrin producing cells in the turtle gut do not express tyrosylprotein sulfotransferases or the enzyme(s) present in turtle antrum is unable to sulfate turtle gastrin.

Identification of pituitary adenylate cyclase-activating polypeptide1-38-binding factor in human plasma, as ceruloplasmin.

125I-Pituitary adenylate cyclase-activating polypeptide (PACAP) 1-38 is able to bind a factor in human plasma, which can be displaced by unlabelled PACAP 1-38 and PACAP 28-38 but not by the other biologically active form, PACAP 1-27. Likewise, 125I-PACAP 28-38 binds this plasma factor, whereas 125I-PACAP 1-27 does not. Apparent Kd values were measured to be 12.0+/-1.3 and 3.4+/-1.5 nM for PACAP 1-38 and PACAP 28-38, respectively, using a competition assay with 125I-PACAP 28-38. Purification of the PACAP 1-38-binding factor from human blood was made by ethanol precipitation of serum followed by Ni2+-chelating and anion-exchange chromatography. A 120-kDa band on SDS/PAGE, as well as some proteolytic products, was blotted on to PVDF membrane and their N-terminal amino acid sequences determined. In combination with a mass-spectrometric fingerprinting of a tryptic digest of the 120-kDa band, this PACAP 1-38-binding factor was identified as ceruloplasmin. Purified commercial ceruloplasmin shows identical mobility on SDS/PAGE to the PACAP 1-38-binding factor and the same binding characteristics to PACAP 1-38, 1-27 and 28-38, using the same amount of ceruloplasmin as was expected to be found in the human plasma. Furthermore, the ability of plasma to bind 125I-PACAP 1-38 or 28-38 disappeared when ceruloplasmin was immunoprecipitated from plasma with rabbit anti-human ceruloplasmin Ig.

Terminal truncations in amp C beta-lactamase from a clinical isolate of Pseudomonas aeruginosa.

AmpC beta-lactamases from strains of Pseudomonas aeruginosa have previously been shown to be heterogeneous with respect to their isoelectric point (pI). In order to elucidate the origin of this heterogeneity enzymes were isolated from a clinical isolate of a multiresistant P. aeruginosa strain and biochemically characterized. The purification was accomplished in four chromatographic steps comprising dye-affinity, size-exclusion, hydrophobic interaction chromatography, and chromatofocusing; this resulted in five forms with pI values of 9.1, 8.7, 8.3, 8.2, and 7.6. When analysed by SDS/PAGE and agarose IEF each separated beta-lactamase appeared to be both size- and charge-homogeneous. The specific activities of the variants were very similar. MS of each isolated beta-lactamase form showed minor differences in molecular mass (range 40.0-40.8 kDa). MS of the beta-lactamase with a pI of 8.2 demonstrated the presence of two subforms. The N-terminal sequences of three of the beta-lactamases were identical to the published sequence [Lodge, J.M. , Minchin, S.D., Piddock, L.J.V. & Busby, J.W. (1990) Biochem. J. 272, 627-631], while two variants were truncated by two amino-acid residues, one of which was acidic. The previously published sequence contains an alanine as the ultimate residue, but two of the beta-lactamases showed a substitution of Ala371 for arginine, whereas in the remaining forms C-terminal truncations by one and three residues were found. Our results indicate that the P. aeruginosa strain does not harbour multiple copies of the ampC gene, but rather that the five beta-lactamase isoforms are products of a single structural gene. The combinations of the identified N- and/or C-terminal truncations explained the multiple pI values of the beta-lactamase isoforms.