The metalloprotease ADAMTS4 generates N-truncated Aß4-x species and marks oligodendrocytes as a source of amyloidogenic peptides in Alzheimer's disease.

Brain accumulation and aggregation of amyloid-ß (Aß) peptides is a critical step in the pathogenesis of Alzheimer's disease (AD). Full-length Aß peptides (mainly Aß1-40 and Aß1-42) are produced through sequential proteolytic cleavage of the amyloid precursor protein (APP) by ß- and γ-secretases. However, studies of autopsy brain samples from AD patients have demonstrated that a large fraction of insoluble Aß peptides are truncated at the N-terminus, with Aß4-x peptides being particularly abundant. Aß4-x peptides are highly aggregation prone, but their origin and any proteases involved in their generation are unknown. We have identified a recognition site for the secreted metalloprotease ADAMTS4 (a disintegrin and metalloproteinase with thrombospondin motifs 4) in the Aß peptide sequence, which facilitates Aß4-x peptide generation. Inducible overexpression of ADAMTS4 in HEK293 cells resulted in the secretion of Aß4-40 but unchanged levels of Aß1-x peptides. In the 5xFAD mouse model of amyloidosis, Aß4-x peptides were present not only in amyloid plaque cores and vessel walls, but also in white matter structures co-localized with axonal APP. In the ADAMTS4-/- knockout background, Aß4-40 levels were reduced confirming a pivotal role of ADAMTS4 in vivo. Surprisingly, in the adult murine brain, ADAMTS4 was exclusively expressed in oligodendrocytes. Cultured oligodendrocytes secreted a variety of Aß species, but Aß4-40 peptides were absent in cultures derived from ADAMTS4-/- mice indicating that the enzyme was essential for Aß4-x production in this cell type. These findings establish an enzymatic mechanism for the generation of Aß4-x peptides. They further identify oligodendrocytes as a source of these highly amyloidogenic Aß peptides.

Zinc and Copper Differentially Modulate Amyloid Precursor Protein Processing by γ-Secretase and Amyloid-ß Peptide Production.

Recent evidence suggests involvement of biometal homeostasis in the pathological mechanisms in Alzheimer's disease (AD). For example, increased intracellular copper or zinc has been linked to a reduction in secreted levels of the AD-causing amyloid-ß peptide (Aß). However, little is known about whether these biometals modulate the generation of Aß. In the present study we demonstrate in both cell-free and cell-based assays that zinc and copper regulate Aß production by distinct molecular mechanisms affecting the processing by γ-secretase of its Aß precursor protein substrate APP-C99. We found that Zn2+ induces APP-C99 dimerization, which prevents its cleavage by γ-secretase and Aß production, with an IC50 value of 15 µm Importantly, at this concentration, Zn2+ also drastically raised the production of the aggregation-prone Aß43 found in the senile plaques of AD brains and elevated the Aß43:Aß40 ratio, a promising biomarker for neurotoxicity and AD. We further demonstrate that the APP-C99 histidine residues His-6, His-13, and His-14 control the Zn2+-dependent APP-C99 dimerization and inhibition of Aß production, whereas the increased Aß43:Aß40 ratio is substrate dimerization-independent and involves the known Zn2+ binding lysine Lys-28 residue that orientates the APP-C99 transmembrane domain within the lipid bilayer. Unlike zinc, copper inhibited Aß production by directly targeting the subunits presenilin and nicastrin in the γ-secretase complex. Altogether, our data demonstrate that zinc and copper differentially modulate Aß production. They further suggest that dimerization of APP-C99 or the specific targeting of individual residues regulating the production of the long, toxic Aß species, may offer two therapeutic strategies for preventing AD.

Shedding of neurexin 3ß ectodomain by ADAM10 releases a soluble fragment that affects the development of newborn neurons.

Neurexins are transmembrane synaptic cell adhesion molecules involved in the development and maturation of neuronal synapses. In the present study, we report that Nrxn3ß is processed by the metalloproteases ADAM10, ADAM17, and by the intramembrane-cleaving protease γ-secretase, producing secreted neurexin3ß (sNrxn3ß) and a single intracellular domain (Nrxn3ß-ICD). We further completed the full characterization of the sites at which Nrxn3ß is processed by these proteases. Supporting the physiological relevance of the Nrxn3ß processing, we demonstrate in vivo a significant effect of the secreted shedding product sNrxn3ß on the morphological development of adult newborn neurons in the mouse hippocampus. We show that sNrxn3ß produced by the cells of the dentate gyrus increases the spine density of newborn neurons whereas sNrxn3ß produced by the newborn neuron itself affects the number of its mossy fiber terminal extensions. These results support a pivotal role of sNrxn3ß in plasticity and network remodeling during neuronal development.

Inhibition of Notch pathway arrests PTEN-deficient advanced prostate cancer by triggering p27-driven cellular senescence.

Activation of NOTCH signalling is associated with advanced prostate cancer and treatment resistance in prostate cancer patients. However, the mechanism that drives NOTCH activation in prostate cancer remains still elusive. Moreover, preclinical evidence of the therapeutic efficacy of NOTCH inhibitors in prostate cancer is lacking. Here, we provide evidence that PTEN loss in prostate tumours upregulates the expression of ADAM17, thereby activating NOTCH signalling. Using prostate conditional inactivation of both Pten and Notch1 along with preclinical trials carried out in Pten-null prostate conditional mouse models, we demonstrate that Pten-deficient prostate tumours are addicted to the NOTCH signalling. Importantly, we find that pharmacological inhibition of γ-secretase promotes growth arrest in both Pten-null and Pten/Trp53-null prostate tumours by triggering cellular senescence. Altogether, our findings describe a novel pro-tumorigenic network that links PTEN loss to ADAM17 and NOTCH signalling, thus providing the rational for the use of γ-secretase inhibitors in advanced prostate cancer patients.

Production of active glycosylation-deficient γ-secretase complex for crystallization studies.

Alzheimer's disease (AD)-associated γ-secretase is a ubiquitously expressed multi-subunit protease complex embedded in the lipid bilayer of cellular compartments including endosomes and the plasma membrane. Although γ-secretase is of crucial interest for AD drug discovery, its atomic structure remains unresolved. γ-Secretase assembly and maturation is a multistep process, which includes extensive glycosylation on nicastrin (NCT), the only γ-secretase subunit having a large extracellular domain. These posttranslational modifications lead to protein heterogeneity that likely prevents the three-dimensional (3D) crystallization of the protease complex. To overcome this issue, we have engineered a Chinese hamster ovary (CHO) cell line deficient in complex sugar modifications (CHO lec1) to overexpress the four subunits of γ-secretase as a functional complex. We purified glycosylation-deficient γ-secretase from this recombinant cell line (CL1-9) and fully glycosylated γ-secretase from a recombinant CHO DG44-derived cell line (SS20). We characterized both complexes biochemically and pharmacologically in vitro. Interestingly, we found that the complex oligosaccharides, which largely decorate the extracellular domain of fully glycosylated NCT, are not involved in the proper assembly and maturation of the complex, and are dispensable for the specific generation, in physiological ratios, of the amyloid precursor protein (APP) cleavage products. In conclusion, we propose a novel bioengineering approach for the production of functional glycosylation-deficient γ-secretase, which may be suitable for crystallization studies. We expect that these findings will contribute both to solving the high-resolution 3D structure of γ-secretase and to structure-based drug discovery for AD.

The adipocyte differentiation protein APMAP is an endogenous suppressor of Aß production in the brain.

The deposition of amyloid-beta (Aß) aggregates in the brain is a major pathological hallmark of Alzheimer's disease (AD). Aß is generated from the cleavage of C-terminal fragments of the amyloid precursor protein (APP-CTFs) by γ-secretase, an intramembrane-cleaving protease with multiple substrates, including the Notch receptors. Endogenous modulation of γ-secretase is pointed to be implicated in the sporadic, age-dependent form of AD. Moreover, specifically modulating Aß production has become a priority for the safe treatment of AD because the inhibition of γ-secretase results in adverse effects that are related to impaired Notch cleavage. Here, we report the identification of the adipocyte differentiation protein APMAP as a novel endogenous suppressor of Aß generation. We found that APMAP interacts physically with γ-secretase and its substrate APP. In cells, the partial depletion of APMAP drastically increased the levels of APP-CTFs, as well as uniquely affecting their stability, with the consequence being increased secretion of Aß. In wild-type and APP/ presenilin 1 transgenic mice, partial adeno-associated virus-mediated APMAP knockdown in the hippocampus increased Aß production by ∼20 and ∼55%, respectively. Together, our data demonstrate that APMAP is a negative regulator of Aß production through its interaction with APP and γ-secretase. All observed APMAP phenotypes can be explained by an impaired degradation of APP-CTFs, likely caused by an altered substrate transport capacity to the lysosomal/autophagic system.

Identification of new Presenilin-1 phosphosites: implication for γ-secretase activity and Aß production.

An important pathological hallmark of Alzheimer's disease (AD) is the deposition of amyloid-beta (Aß) peptides in the brain parenchyma, leading to neuronal death and impaired learning and memory. The protease γ-secretase is responsible for the intramembrane proteolysis of the amyloid-ß precursor protein (APP), which leads to the production of the toxic Aß peptides. Thus, an attractive therapeutic strategy to treat AD is the modulation of the γ-secretase activity, to reduce Aß42 production. Because phosphorylation of proteins is a post-translational modification known to modulate the activity of many different enzymes, we used electrospray (LC-MS/MS) mass spectrometry to identify new phosphosites on highly purified human γ-secretase. We identified 11 new single or double phosphosites in two well-defined domains of Presenilin-1 (PS1), the catalytic subunit of the γ-secretase complex. Next, mutagenesis and biochemical approaches were used to investigate the role of each phosphosite in the maturation and activity of γ-secretase. Together, our results suggest that the newly identified phosphorylation sites in PS1 do not modulate γ-secretase activity and the production of the Alzheimer's Aß peptides. Individual PS1 phosphosites shall probably not be considered therapeutic targets for reducing cerebral Aß plaque formation in AD. In this study, we identified 11 new phosphosites in Presenilin-1 (PS1), the catalytic subunit of the Alzheimer's γ-secretase complex. By combining a mutagenesis approach with cell-based and cell-free γ-secretase assays, we demonstrate that the new phosphosites do not modulate the maturation and activity of γ-secretase. Individual PS1 phosphosites shall thus not be considered therapeutic targets for reducing cerebral Aß plaque formation in Alzheimer's Disease. Aß, amyloid beta.

Perturbations of the straight transmembrane α-helical structure of the amyloid precursor protein affect its processing by γ-secretase.

The amyloid precursor protein (APP) is a widely expressed type I transmembrane (TM) glycoprotein present at the neuronal synapse. The proteolytic cleavage by γ-secretase of its C-terminal fragment produces amyloid-ß (Aß) peptides of different lengths, the deposition of which is an early indicator of Alzheimer disease. At present, there is no consensus on the conformation of the APP-TM domain at the biological membrane. Although structures have been determined by NMR in detergent micelles, their conformation is markedly different. Here we show by using molecular simulations that the APP-TM region systematically prefers a straight α-helical conformation once embedded in a membrane bilayer. However, APP-TM is highly flexible, and its secondary structure is strongly influenced by the surrounding lipid environment, as when enclosed in detergent micelles. This behavior is confirmed when analyzing in silico the atomistic APP-TM population observed by residual dipolar couplings and double electron-electron resonance spectroscopy. These structural and dynamic features are critical in the proteolytic processing of APP by the γ-secretase enzyme, as suggested by a series of Gly(700) mutants. Affecting the hydration and flexibility of APP-TM, these mutants invariantly show an increase in the production of Aß38 compared with Aß40 peptides, which is reminiscent of the effect of γ-secretase modulators inhibitors.

Alzheimer's disease mutations in APP but not γ-secretase modulators affect epsilon-cleavage-dependent AICD production.

Pathological amino-acid substitutions in the amyloid precursor protein (APP) and chemical γ-secretase modulators affect the processing of APP by the γ-secretase complex and the production of the amyloid-beta peptide Aß42, the accumulation of which is considered causative of Alzheimer's disease. Here we demonstrate that mutations in the transmembrane domain of APP causing aggressive early-onset familial Alzheimer's disease affect both γ- and ε-cleavage sites, by raising the Aß42/40 ratio and inhibiting the production of AICD50-99, one of the two physiological APP intracellular domains (ICDs). This is in sharp contrast to γ-secretase modulators, which shift Aß42 production towards the shorter Aß38, but unequivocally spare the ε-site and APP- and Notch-ICDs production. Molecular simulations suggest that familial Alzheimer's disease mutations modulate the flexibility of the APP transmembrane domain and the presentation of its γ-site, modifying at the same time, the solvation of the ε-site.

Highly efficient production of the Alzheimer's γ-secretase integral membrane protease complex by a multi-gene stable integration approach.

Inefficient production of membrane-embedded multi-protein complexes by conventional methods has largely prevented the generation of high-resolution structural information and the performance of high-throughput drug discovery screens for this class of proteins. Not exempt from this rule is γ-secretase, an intramembrane-cleaving protease complex regulating a multitude of signaling pathways and biological processes by influencing gene transcription. γ-Secretase is also implicated in the pathogenesis of Alzheimer's disease and several types of cancer. As an additional challenge, the reconstitution of the protease complex in its active form requires an intricate assembly and maturation process, including a highly regulated endoproteolytic processing of its catalytic component. In this article we report the application of a transposon-mediated multigene stable integration technology to produce active γ-secretase in mammalian cells in amounts adequate for crystallization studies and drug screening. Our strategy is expected to help elucidate the molecular mechanisms of intramembrane proteolysis. It is further expected to be widely used for the production of other multi-protein complexes for applications in structural biology and drug development.

The role of γ-secretase activating protein (GSAP) and imatinib in the regulation of γ-secretase activity and amyloid-ß generation.

γ-Secretase is a large enzyme complex comprising presenilin, nicastrin, presenilin enhancer 2, and anterior pharynx-defective 1 that mediates the intramembrane proteolysis of a large number of proteins including amyloid precursor protein and Notch. Recently, a novel γ-secretase activating protein (GSAP) was identified that interacts with γ-secretase and the C-terminal fragment of amyloid precursor protein to selectively increase amyloid-ß production. In this study we have further characterized the role of endogenous and exogenous GSAP in the regulation of γ-secretase activity and amyloid-ß production in vitro. Knockdown of GSAP expression in N2a cells decreased amyloid-ß levels. In contrast, overexpression of GSAP in HEK cells expressing amyloid precursor protein or in N2a cells had no overt effect on amyloid-ß generation. Likewise, purified recombinant GSAP had no effect on amyloid-ß generation in two distinct in vitro γ-secretase assays. In subsequent cellular studies with imatinib, a kinase inhibitor that reportedly prevents the interaction of GSAP with the C-terminal fragment of amyloid precursor protein, a concentration-dependent decrease in amyloid-ß levels was observed. However, no interaction between GSAP and the C-terminal fragment of amyloid precursor protein was evident in co-immunoprecipitation studies. In addition, subchronic administration of imatinib to rats had no effect on brain amyloid-ß levels. In summary, these findings suggest the roles of GSAP and imatinib in the regulation of γ-secretase activity and amyloid-ß generation are uncertain.

Generation of monoclonal antibody fragments binding the native γ-secretase complex for use in structural studies.

A detailed understanding of γ-secretase structure is crucially needed to elucidate its unique properties of intramembrane protein cleavage and to design therapeutic compounds for the safe treatment of Alzheimer's disease. γ-Secretase is an enzyme complex composed of four membrane proteins, and the scarcity of its supply associated with the challenges of crystallizing membrane proteins is a major hurdle for the determination of its high-resolution structure. This study addresses some of these issues, first by adapting CHO cells overexpressing γ-secretase to growth in suspension, thus yielding multiliter cultures and milligram quantities of highly purified, active γ-secretase. Next, the amounts of γ-secretase were sufficient for immunization of mice and allowed generation of Nicastrin- and Aph-1-specific monoclonal antibodies, from which Fab fragments were proteolytically prepared and subsequently purified. The amounts of γ-secretase produced are compatible with robot-assisted crystallogenesis using nanoliter technologies. In addition, our Fab fragments bind exposed regions of native γ-secretase in a dose-dependent manner without interfering with its catalytic properties and can therefore be used as specific tools to facilitate crystal formation.

Label-free imaging of cerebral ß-amyloidosis with extended-focus optical coherence microscopy.

We demonstrate label-free imaging of cerebral ß-amyloidosis ex vivo and in a living mouse model of Alzheimer's disease using extended-focus Fourier domain optical coherence microscopy (xfOCM). xfOCM provides 3D, high-resolution images of individual ß-amyloid plaques in the brain parenchyma and vasculature and requires no staining of the alzheimeric sample under investigation. xfOCM also opens the possibility to perform minimally invasive studies of ß-amyloid pathology in vivo, without the use of labeling methods, which potentially confound experimental findings.

Structural comparison of the poplar plastocyanin isoforms PCa and PCb sheds new light on the role of the copper site geometry in interactions with redox partners in oxygenic photosynthesis.

Plastocyanin (PC) from poplar leaves is present in two isoforms, PCa and PCb, which differ in sequence by amino acid replacements at locations remote from the copper center and simultaneously act in the photosynthetic electron-transport chain. We describe ultra-high resolution structures of PCa and high-resolution structures of PCb, both under oxidizing and reducing conditions at pH 4, 6 and 8. The docking on cytochrome f and photosystem I, respectively, has been modeled for both isoforms. PCa and PCb exhibit closely similar overall and active-site structures, except for a difference in the relative orientation of the acidic patches. The isoforms exhibit substantial differences in the dependence of the reduced (Cu(I)) geometry on pH. In PCa, the decrease in pH causes a gradual dissociation of His87 from Cu(I) at low pH, probably adopting a neutral tautomeric state. In PCb, the histidine remains covalently bound to Cu(I) and may adopt a doubly protonated state at low pH. The fact that both isoforms have similar although not identical functions in photosynthetic electron flows suggests that the His87 imidazole does not play a crucial role for the pathway of electron transport from cytochrome f to oxidized PC.

Mercury is a direct and potent γ-secretase inhibitor affecting Notch processing and development in Drosophila.

Prenatal exposure to mercury causes neurodevelopmental disorders and neurological pathologies in infants, such as microcephaly and mental retardation. Despite critical importance, the molecular interactions leading to mercury toxicity are yet to be elucidated. We first used a cell-free assay to investigate mercury effects on purified γ-secretase activity. Next, we treated adult Drosophila melanogaster with mercury and collected control and mercury-treated embryos, which were subjected to mild hypotonic protein extraction, or immunostained to reveal nervous system morphology. Embryos left to develop into adults were examined for wing phenotypes. Relative to control metals, we found that mercury strongly inhibits in vitro γ-secretase processing of both amyloid-ß precursor protein (APP) and Notch. Mercury inhibited APP and Notch cleavage in a dose-dependent manner, with IC(50) values of 50-125 nM, and is therefore comparable in potency to benchmark γ-secretase inhibitors. Immunoblot analysis of embryonic protein extracts showed that mercury inhibits Notch cleavage by γ-secretase in vivo. This is accompanied by severe neurodevelopmental abnormalities in embryos and adult wing-notching phenotypes. Our findings provide first evidence that mercury is a direct and potent γ-secretase inhibitor and suggest that inhibition of γ-secretase and disruption of the Notch developmental pathway potentially contribute to mercury-induced toxicity in the nervous system.

Isolation and identification of poplar isoplastocyanins.

An improved four-stage isolation and purification procedure for preparing poplar isoplastocyanins is described in detail. Absorbance (UV-VIS) spectroscopy and isoelectric focusing (IEF) are used to determine the protein purity and identity. The present procedure increases twice the total plastocyanin (PC) yield. Four PC isoform fractions are consecutively isolated at the third chromatographic step: oxidized PCa(II) and PCb(II) and reduced PCb(I) and PCa(I). PCa(II) and PCb(II) obtained at the fourth chromatographic step are highly purified PC isoforms which show the purity index (p.i.) A278/A597 < or = 0.85. Isoelectric points (pl values) of the PC isoforms are found to be at pH 3.92 +/- 0.04 for PCa and at pH 3.85 +/- 0.02 for PCb. The results of appropriate biological experiments that include the highly purified poplar PC isoforms could give answers to the questions about the physiological significance of PC dimorphism for photosynthesis.

Oxidation of oxymyoglobin by poplar plastocyanins a and b.

Oxidation of oxymyoglobin [MbO2(Fe2+)] by isoplastocyanins a (PCa) and b (PCb) was experimentally investigated and the corresponding redox reaction was modeled using the physicochemical parameters of the isoforms to study the effect of the dimorphism. The kinetic curve of oxidation of MbO2 (Fe2+) by oxidized PCa [PCa(Cu2+)] and PCb [PCb(Cu2+)] and the pH-dependence of the rate constant kI were determined. In the range of pH 4.8-9.0, PCb reacts with higher k1, compared with PCa. For example, at pH 7.0, k1(PCb) = 4 x 10(2) M(-1)s(-1), whereas k1(PCa)= 2 x 10(2) M(-1)S(-1). The observed values of deltaE(0) for the reaction pairs Mb-PCa and Mb-PCb were -304 mV and -319 mV, respectively. The effect of the ionic strength (mu) on the rate of the electron transfer was also studied. It was found that: (i) the net charge Z1 of PCa and PCb fully corresponds to that calculated by their primary structures and Z2 of Mb corresponds to that calculated by its titration curve; (ii) the In k as function of mean square of mu was similar for both PCa and PCb; (iii) the curve of the reaction PCb <----(e(-1)) Mb (pH 7.0) wasshifted towards higher values of k, in agreement with the larger net negative charge of PCb; and (iv) the character of the electrostatic interactions remained unchanged by a replacement of PCa by PCb and by the change of pH from 7.0 to 4.8.

Evidence for inhibition of cholinesterases in insect and mammalian nervous systems by the insect repellent deet.

BACKGROUND: N,N-Diethyl-3-methylbenzamide (deet) remains the gold standard for insect repellents. About 200 million people use it every year and over 8 billion doses have been applied over the past 50 years. Despite the widespread and increased interest in the use of deet in public health programmes, controversies remain concerning both the identification of its target sites at the olfactory system and its mechanism of toxicity in insects, mammals and humans. Here, we investigated the molecular target site for deet and the consequences of its interactions with carbamate insecticides on the cholinergic system. RESULTS: By using toxicological, biochemical and electrophysiological techniques, we show that deet is not simply a behaviour-modifying chemical but that it also inhibits cholinesterase activity, in both insect and mammalian neuronal preparations. Deet is commonly used in combination with insecticides and we show that deet has the capacity to strengthen the toxicity of carbamates, a class of insecticides known to block acetylcholinesterase. CONCLUSION: These findings question the safety of deet, particularly in combination with other chemicals, and they highlight the importance of a multidisciplinary approach to the development of safer insect repellents for use in public health.

Some physicochemical peculiarities of poplar plastocyanins a and b.

The redox potentials of poplar plastocyanins a and b (PCa, PCb) were determined by spectrophotometric titrations of their reduced forms with [Fe(CN)6]3-. It was found that the two isoforms have the following millimolar extinction coefficients epsilon597 equilibrium constants Keq of one-electron exchange with [Fe(CN)6]4-/[Fe(CN)6]3-, and standard electron potentials E0: PCa: epsilon597 = (4.72 +/- 0.08) mM(-1) cm(-1), Keq = 0.133 +/- 0.009, E0' = (354 +/- 11) mV; PCb: epsilon597 = (5.23 +/- 0.16) mM(-1) cm(-1), Keq = 0.175 +/- 0.010, E0' = (363 +/- 12) mV. The pH dependence of the redox potential of PCb was studied too. It was found, that the value of E0' for PCb is constant in the pH range 6.5-9.5, but decreases in the range 4.8-6.5. On the whole, the dependence resembles that of PC from some well-known plant species, including poplar PCa. The changes of E0' in the pH-dependent region for poplar PCb, however, are smaller and are 13 mV per pH unit, whereas in the other well-known plant species the changes are about 50-60 mV per pH unit. It has been assumed that the weaker pH dependence of EO' of PCb accounts for some structural differences between PCa and PCb.

Plastocyanin microheterogeneity in Scenedesmus acutus MT8.

Two total plastocyanin (PC) fractions - loosely bound (lPC) and strongly bound (sPC) were extracted (84% and 16%, respectively) from the homogenate of Scenedesmus acutus MT8. Two-fold isolation-purification procedure including DE-52 chromatography separated IPC into a smaller oxidized [IPC (II)] and a larger reduced [IPC(I)] fractions, in contrast to sPC, where sPC(ll) greatly dominated over sPC(I). Analytical isoelectric focusing (IEF) separated IPC(II) into two main fractions only in the presence of 8 M urea, implying microheterogeneity. Preparative IEF in immobiline pH-gradient of 3.2-4.1 separated IPC(II) into two blue fractions - a more alkaline IPC(II) and a more acidic IPC"(II), which were probably stereoisomers. Their UV-Vis spectra exhibited rarely observed tryptophane (291.5 nm) and some differences at 270 and 287 nm. The exact molecular masses of apo-/holo-lPC (10131 Da/10194 Da) were determined by mass spectrometry. The number of -SH groups was determined from the mass difference between alkylated with 4-vinylpyridine (4-VP) and non-alkylated protein. Additionally, a simple procedure for simultaneous separation of both primary structure and stereoisomers of PC was developed.