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We evaluated S-100 levels in paired cerebrospinal fluid (CSF) and serum samples in a group of 135 patients referred to the German Creutzfeldt-Jakob disease (CJD) surveillance unit from June 1993 to May 1995. The patients were seen in a prospective case control study. The diagnosis of probable CJD during life was made in any patient presenting with rapidly progressive dementia of less than 2 years' duration, typical periodic sharp wave complexes (PSWCs) in the EEG and at least two of the following findings: myoclonus, visual/or cerebellar symptoms, pyramidal and/or extrapyramidal signs and/or akinetic mutism. Patients presenting with the above clinical signs and symptoms but without PSWCs were classified as possible, while those with a dementia of a duration exceeding 2 years and without PSWCs were classified as other. S-100 was determined in paired CSF and serum samples by a commercially available enzyme-linked immunosorbent assay. In a group of 76 patients with definite and probable CJD, S-100 concentration (median 25 ng/ml, range 2-117) in CSF was significantly higher (P < 0.0001) than in 32 patients diagnosed as other (median 4 ng/ml, range 1-19). Serum levels of S-100 were below 0.5 ng/ml in all groups. At a cut-off of 8 ng/ml an optimum sensitivity of 84.2% with a specificity of 90.6% for the diagnosis of CJD by the determination of S-100 in CSF is obtained. S-100 levels exceeding 8 ng/ml in CSF support the diagnosis of CJD in any patient presenting with rapidly progressive dementia.

A specific and sensitive two-side enzyme-linked immunosorbent assay (sandwich-ELISA) was established for the reliable quantification of human brain and placental choline acetyltransferase (ChAT). In contrast to the radiometric assay developed by Fonnum, which is widely used for the measurement of enzyme activity, the sandwich-ELISA particularly recognized inactivated forms of the antigen. In the assay, affinity-purified polyclonal synthetic peptide antibodies adsorbed to the polystyrene surface of the microtiter plate were employed as capture reagent. Based on standard peroxidase protocols, immobilized ChAT was detected using monoclonal antibodies raised against human placental ChAT. By use of this ELISA, ChAT was determined at various purification stages of the enzyme, in body fluids, during recovery experiments and in sera of patients with severe brain damage.

Synaptophysin (SYP I), an integral membrane protein, was purified on a large scale (0.55 - 2.7 mg) from isolated small synaptic vesicles (SSV) of porcine cortex. In order to achieve this, a conventional purification procedure which consists of size exlusion chromatography, hydrophobic interaction chromatography and chromatofocusing has been developed. This procedure was compared with purification of SYP I by immunoaffinity chromatography. The elution patterns of both procedures were monitored using sodium dodecylsulfate gel electrophoresis (SDS-PAGE) with subsequent Coomassie blue staining of proteins and simultaneous immunoblotting with SYP I-specific antibody. Contaminating proteins with relative molecular masses (M(r)) very similar to SYP I could be removed during the process of purification, demonstrating that the 38 kDa protein found after Triton X-100 lysis of enriched SSV does not exclusively represent SYP I. A specific antiserum was raised in rabbits using a highly purified preparation of SYP I. This antiserum was used in combination with a monoclonal antibody to establish a specific and sensitive enzyme-linked immunosorbent assay (ELISA) which allowed rapid and reliable quantification of this hydrophobic membrane protein in all purification steps, starting with Triton X-100-lysed brain homogenates. Using this ELISA, the concentration of SYP I in highly purified SSV was determined to be 5.8% of solubilized protein.

A two-sided enzyme-linked immunosorbent assay (ELISA) has been established for reliable, specific and sensitive determination of synaptophysin (SYN), an intrinsic membrane protein of the small synaptic vesicles. This ELISA used a highly specific monoclonal antibody (SY 38) as capture reagent and a specific SYN antiserum in combination with a secondary peroxidase-conjugated antibody for detection. Calibration was carried out with immunoaffinity-purified SYN from porcine cortex. The sensitivity was found to be improved substantially when the ELISA was compared with previously used dot-immunobinding assays. This ELISA allowed rapid and reliable determination of SYN from detergent lysed homogenates, partially and highly purified preparations of rat, porcine and human brain. SYN was determined in highly purified small synaptic vesicles, and it was calculated to be 5.8% of total detergent solubilized protein.

Expression of beta-trace protein (beta-trace), recently identified as glutathion-independent prostaglandin D synthase (prostaglandin-H2 D-isomerase; EC 5.3.99.2), was localized in paraffin sections of the human brain by in situ hybridization using digoxigenin-labeled antisense cRNA probes. The mRNA for beta-trace was predominantly found in the epithelial cells of the choroid plexus. Hybridization signals were also obtained in some oligodendrocytes, particularly in the white matter. In the leptomeninges, specific signals were found in meningeal macrophages and in single cells of the arachnoid barrier layer. The cells exhibiting hybridization signals with the antisense cRNA probes for beta-trace were identified by counterstaining with antibodies directed against specific cell markers. Additionally, beta-trace mRNA was localized in tubular epithelial and basal cells of the human epididymis and in different cell types within the seminiferous epithelium of the testis.

In the sera of patients with acute bactetial infections specific autoantibodies (sIAPa) of the imtnuno-globulin class G (IgG) were found which bind to intestinal alkahne phosphatase (IAP) through the Fab portion. This was demonstrated using immunoaffinity (IA) isolation of sIAPa from patients' sera (particularly bacterial meningitis and ventriculitis) digestion with pepsin, purification of F(ab')2 fragments on protein A and subsequently binding on IAP coupled to CNBr (cyanogen bromide)-activated Sepharose. Immunoblots using specific anti-Fc and anti-Fab antibodies showed that the bulk of F(ab')2 fragments had bound. Additionally, binding of native IAP to the F(ab')2 fragments was observed after separation of F(ab')2 fragments using isoelectric focusing (IEF), blotting onto nitro cellulose and incubation with IAP. Moreover, we have demonstrated the occurrence of natural anti-IAP autoantibodies (nIAPa) which were isolated from sera of healthy individuals using IA chromatography. Investigation of isotype distribution revealed that IgG but not IgM or IgA were predominant even among nIAPa. The nIAPa fraction exhibited lower binding efficiencies on IEF blots than the sIAPa fraction, however, in contrast to sIAPa, cross-reactions with other autoantigens were observed for nIAPa. NIAPa and sIAPa did not show subclass restriction. As revealed by IEF the spectrotypes of sIAPa were found to be patient-specific, poly- to oligoclonal and stable during longer periods.

In half of the sera from patients with acute bacterial infections and 15% of the sera from patients with acute viral infections glycoproteins were found that form a scalariform pattern in the cationic range upon isoelectric focusing. The cationic glycoproteins appeared with the clinical illness. After subsidence of the symptoms they disappeared within 4 to 6 weeks. The proteins were identified as immunoglobulin G (IgG) by determination of the NH2-terminal amino acid sequence. Remarkably, these IgG only contained light chains of the kappa type with high proportions of carbohydrates. Both, N-glycosidic- and O-glycosidic-bound glycans were present. The glycosylated light chains may render the cationic IgG multireactive. Thus, it may be part of an early nonspecific immune defense mechanism.

Choline acetyltransferase (ChAT) was purified by immunoaffinity chromatography using a covalently immobilized monoclonal antibody. In a two-step procedure, 10 kg porcine brain yielded 750 micrograms active enzyme of apparent homogeneity. This amount of ChAT was purified routinely. The purification factor was 18,000 and the yield of activity 4.3%. The affinity resin was stable under the experimental conditions applied and was used many times. The highly purified enzyme was subsequently employed to obtain a specific anti-ChAT antiserum of high titer.

Summary Activity of ethylene formation from 1-aminocyclopropane-1-carboxylic acid (ACC) was compared to peroxidase (POD) activity in crude extracts of etiolated pea shoots ( Pisum sativum L.) as well as in Sephadex and Concanavalin A-Sepharose (ConA) fractions of these extracts. In crude extracts the stability of POD activity was higher than that of ethylene formation. Mercaptoethanol and sodiumazide inhibited both activities, but other inhibitors of ethylene formation (hydroxylammoniumchloride, catalase, EDTA, KCN, CuSO 4 , CoCl 2 ) had a weak or no effect at all on POD activity. High-molecular weight fractions (FI) from Sephadex of pea crude extracts formed ethylene from ACC and exhibited POD activity. By mixing high molecular weight fractions with low molecular weight fractions from the Sephadex column ethylene formation was stimulated. A similar effect could be reached by substituting FI fractions with horse radish peroxidase (HRP), but ethylene production was very low. After ConA fractionation the highest ethylene formation was found to be in the POD containing eluate, but in addition we found ethylene formation in effluent fractions which showed no POD activity. Mn 2+ otherwise has a stimulating effect on ethylene production from Sephadex fractions as well as from HRP but not on the crude extract. It appears that peroxidase itself does not play a major role in decarboxylation or ring cleavage of ACC, but that it might be involved in a more complex way by providing radicals, H 2 O 2 or otherwise unknown factors.

Isoelectric focusing (IEF) of peroxidases of different organs and tissues of Nicotiana tabacum L. was performed on thin-layers of Sephadex and polyacrylamide. Isoelectric points (pI's) of peroxidase bands were measured by special electrodes. — The two types of layers showed very similar results. Reproductibility of pI's was better on polyacrylamide. This method is also easier to practise and requires less time than IEF on Sephadex (3 h versus 18). Thus for analytical purposes the acrylamide-technique is preferable, but if it is necessary to regain the separated enzymes it is better to perform IEF on Sephadex. — When IEF-patterns of peroxidase are compared with the disk electrophoresis (DE) patterns of the same tissue, important differences are observed. The 6 bands of GI (fast migrating, anodic group of DE) are reduced to 2 on the strongly acidic side of IEF (independent of the tissue studied). That means only 2 proteins in GI can be separated by pI's of the molecules. Maybe the heterogeneity of GI bands after DE indicates the presence of conformational isomers (conformers). — Because of the reduced number of bands in GI after IEF there is no difference in the patterns of many tissues (flowers, leaves, shoots, pith) as there is after disk electrophoresis. In the case of GII (slow migrating, anodic group of DE) on the other hand, there are always 4 different bands after isoelectric focusing in the lower acid region instead of 3 after disk electrophoresis. Disk electrophoresis of peroxidase-groups separated by isoelectric focusing shows the same patterns as direct disk electrophoresis of the extract. The methods produce no artifacts. —Comparison of these results with the peroxidase-patterns of tobacco found by other workers and by other techniques leads to the conclusion that there exist at least 4 “isoenzymes” of peroxidase corresponding to the 4 groups GI, GII, GIII, and GIV.

Summary The present communication describes the determination of apparent Michaelis-constants of three peroxidase-isoenzyme-groups for several artificial (chemicals which are active in the peroxidase catalysed reaction) and natural substrates (compounds which are found in plants and which possibly act as electrondonors for H 2 O 2 -reduction by peroxidases in vivo ). G I (cell wall located group) has lowest Michaelis-constants for artificial, G III (protoplast located group) for natural substrates. Scopoletin is no substrate for G III , but for G I particularly and to some extent for G II . Maximal oxidation rates of different flavan-derivatives are highest for G I or G II and lowest for G III . The in vitro polymerisation of cumaryland coniferylalcohol to lignin-like-substances is catalysed by G I and G II to a substantial rate but hardly by G III . All the isoenzyme groups exhibit indole-3-acetic oxidase but no phenol-oxidase-activity. The results are discussed in connection with several hypotheses of peroxidase-function and with the localisation of peroxidase-isoenzyme-groups in tissues of tobacco.

Vacuoles of tobacco mesophyll and of suspension-cultured cells were isolated in order to study the localization of peroxidase isoenzymes. Only basic peroxidases were detectable by electrophoretic separation of the vacuolar sap. Some of the basic peroxidases have formerly been described as an ionically bound cell-wall fraction. This fraction, however, was found to be an artifact produced by incomplete cell breakage. Reinvestigation of isolated cell walls confirmed that mainly acidic peroxidases are localized in the cell walls where they move freely or are bound. As a consequence of former and present results we think it probable that all of the peroxidase isoenzymes are secretory proteins because they have to be transported from the sites of synthesis in the cytoplasm to the sites of function, the extracytoplasmic spaces, cell wall (acidic peroxidases), and vacuole (basic peroxidases).

Mesophyll-protoplasts of tobacco show increasing peroxidase-activity immediately after isolation. This is due to an enhancement of activity of the constitutive isoenzymes of GIII (=slow migrating cathodic group) and to a new formation of GII-isoenzymes (=slow migrating anodic group). (GII is not present in intact leaves.) As both processes are inhibited by actinomycin and actidion it is assumed that there is a new synthesis of peroxidase enzymes —The peroxidase reaction is independent of the further development of the protoplasts, as was evidenced by culturing protoplasts in different media which regulate the development of the protoplasts. Peroxidase reaction is always the same whether or not there is cell-wall synthesis and cell division. This leads to the conclusion that peroxidases in this case have no relation to synthesis of primary cell-walls. On the other hand they could be related to the dedifferentiation processes that always take place in isolated protoplasts.

Peroxidase activity and isoenzyme pattern were studied during dedifferentiation of tobacco stem-sections leading to callus formation and during redifferentiation of tobacco callus leading to formation of shoots. These processes are both accompanied by an increase in total peroxidase activity and by characteristic changes in isoenzyme pattern. The isoenzyme pattern of tobacco callus differs from that of tobacco stem-tissue. The plantlets differentiated from the callus show the same pattern as seedlings do. During the differentiation process, before any buds are visible, the callus shows a peroxidase pattern that is determined by a reduction of fast-migrating anodic isoenzymes and by an increase of activity in all the other peroxidase isoenzymes. The formation of this pattern is independent of the growth regulators responsible for the differentiation: only the kind of differentiation itself determines the pattern. By artificial inhibition of callus growth it is possible to induce an isoenzyme pattern very similar to that of differentiation; the fast-migrating anodic enzymes are reduced in activity but the others are not increased as they are during differentiation. Therefore the question arises whether there are two independent processes taking place in differentiating callus. The one process, inhibition of growth in the cells that do not differentiate, is accompanied by a reduction of fast-migrating anodic isoenzymes. The other process, formation of meristemoids in the callus, is accompanied by a sharp rise in peroxidase activity of the other anodic and cathodic isoenzymes.

Isolation of cell wall-bound malate dyhydrogenase (MDH, EC 1.1.1.37) from tobacco was obtained by treating purified cell walls with pectinase and hemicellulase. By electrophoretic separation it was demonstrated that the wall-bound isoenzymes of MDH are different from the cellular ones.

Summary Separation and purification of three peroxidase-isoenzyme groups (GI, fast migrating anodic; GII, slow migrating anodic; GIII, slow migrating cathodic) was accomplished by ion-exchange chromatography. Chromatographic separation of the groups was controlled by disk-electrophoresis and by isoelectric focusing. Molecular weights were estimated by SDS-gelelectrophoresis and by thin layer chromatography. The results of both methods are similar. GI has a molecular weight of about 27,000, GII of 43,000 and GIII of 47,000. By affinity chromatography with Concanavalin A-sepharose it was shown that the enzymes of the three peroxidase groups are all glycoproteins. While heat inactivation of GI was measured at 50°C, inactivation of GII and GIII did not take place before 70°C. Absorption spectra of the three groups were typical for ferriperoxidases with the maximum of the Soret band at 401 nm.

Upon disk-electrophoresis with guaiacol as a substrate the peroxidase-isoenzymes of Nicotiana tabacum (L.) were localized on the gels in two anodic and two cathodic groups. By preparation of protoplasts and isolation of cell walls it was possible to show that only cathodic enzymes are located in the protoplasts in measurable amounts, whereas all the isoenzymes, anodic and cathodic, can be found associated with cell walls. The different groups of isoenzymes are bound to the cell wall in different ways as evidenced by differences in their extration. It seems possible that different biological functions are associated with the different groups of isoenzymes.The isoenzyme patterns of different organs and tissues of tobacco show qualitative differences only in the anodic (i.e. wall located) isoenzymes. It is suggested that the ontogenetic change in peroxidase-patterns is direct evidence of biochemical differences in the cell walls of the different tissues and organs.

H2O2 formed in the process of NADH-oxidation mediated by peroxidase (POD) and cofactors is measured by a newly developed enzymatic method. This method allows detection of very small quantities of H2O2 (10−5 M). Although this method was developed predominantly to study H2O2-formation in vitro it might also be useful for determining H2O2 content in plant saps and extracts. The amount of H2O2-formed in the course of NADH-oxidation is within certain limits proportional to the amount of available NADH and not dependent on the amount of enzyme and other cofactors. The stoichiometric relation of NADH/H2O2 was shown to be 0.5 which coincides with the expected theoretical values.

De-novo synthesis of acid and basic peroxidases has been studied in cell suspension cultures of tobacco by incorporation of (3)H- and (14)C-amino acids. Incorporation rates were found to be high for acid peroxidases and low for basic peroxidases. Synthesis of all peroxidases was inhibited by cycloheximide and actinomycin D. Subculturing of the cells increased the rates of radioactive amino-acid incorporation into all peroxidases within the first 24 h. This rise in peroxidase synthesis was correlated with the age of the transferred cells. The older the cells were the more pronounced was the effect. During the culture cycle the high rates of peroxidase synthesis at the second day dropped back to initial values. Peroxidase synthesis was thus inversely related to peroxidase accumulation which was very low at the beginning and increased continuously. By pulse-chase experiments it has been shown that newly synthesized acid peroxidases accumulated in the medium. This process was inhibited by monensin. Only the acid peroxidases were secreted into the cell wall and from there released. The basic peroxidases were not detectable in the medium.

Coniferyl alcohol is the primary substrate for peroxidase-mediated lignification, a process which depends on the generation of H 2 O 2 by NADH oxidation. We measured the concentrations of various phenols (synthetic and natural) at which maximal enhancement of NADH oxidation occurs. Coniferyl alcohol was found to stimulate NADH oxidation at a much lower concentration (0.01 mm) than other natural or synthetic phenols (1-100 mm). In addition, coniferyl alcohol prevented the conversion of active peroxidase into the inactive intermediate compound III—which is usually formed in the presence of NADH—at equally low concentrations. This conversion was found to be a prerequisite for stimulation of NADH-oxidation, but it was not necessarily connected to stimulation. The oxidation of NADH and coniferyl alcohol (or guaiacol) can occur simultaneously, but there is a strong competitive interaction between these two substrates. At pH 5, the presence of NADH at concentrations 30 to 60 times lower than the phenols completely prevents their oxidation. The results are discussed in relation to the role of cell wall peroxidases in conversion of coniferyl alcohol to lignin and in formation of H 2 O 2 .

The two peroxidase isoenzyme groups (G(I) and G(III)) localized in the cell walls of tobacco (Nicotiana tabacum L.) tissues were compared with respect to their capacity for NADH-dependent H(2)O(2) formation. Peroxidases of the G(III) group are slightly more active than those of the G(I) group when both are assayed under optimal conditions. This difference is probably not of major regulatory importance. NADH-dependent formation of H(2)O(2) required the presence of Mn(2+) and a phenol as cofactors. The addition of H(2)O(2) to the reaction mixture accelerated subsequent NADH-dependent H(2)O(2) formation. In the presence of both cofactors or Mn(2+) alone, catalase oxidized NADH. However, if the cofactors were absent or if only dichlorophenol was present, catalase inhibited NADH oxidation. No H(2)O(2) accumulation occurred in the presence of catalase. Superoxide dismutase inhibited NADH oxidation quite significantly indicating the involvement of the superoxide radical in the peroxidase reaction. These results are interpreted to mean that the reactions whereby tobacco cell wall peroxidases catalyze NADH-dependent H(2)O(2) formation are similar to those proposed for horseradish peroxidase (Halliwell 1978 Planta 140: 81-88).

Protein-body membranes (PBMs) were isolated from cotyledons of Phaseolus vulgaris L. by a procedure involving osmotic shock of purified protein bodies. The purified PBMs have a characteristic density of 1.16 g cm(-3). Treatment of the membranes with increasing concentrations of detergent (Triton X-100) or with a solution at pH 12.0 showed that the membranes contained a characteristic integral protein (IMP) with a relative molecular mass of 25,000. This IMP is not a glycoprotein. When developing cotyledons were labeled with (3)H-amino acids for 2-3 h, a radioactive polypeptide with the same mobility on denaturing polyacrylamide gels as IMP was found to be associated with the rough endoplasmic reticulum (ER). During a 24-h chase, a considerable portion of the radioactivity slowly transferred into the IMP associated with more rapidly sedimenting organelles, which sedimented in the same region of the sucrose gradients as the PBMs. Antibodies prepared against purified IMP crossreacted with an ER-associated protein which had the same mobility on denaturing acrylamide gels as authentic IMP. Synthesis of IMP occurred at all stages of cotyledon development examined, but not during seed germination. The results show that a newly synthesized protein of the PBM is associated with the rough ER, just like the soluble matrix proteins, phaseolin (R. Bollini, W. Van der Wilden and M.J. Chrispeels, 1983, J. Cell Biol. 96,999-1007) and phytohemagglutinin (M.J. Chrispeels and R. Bollini, 1982, Plant Physiol. 70, 1425-1428), but that the chase-out from the ER is much slower for IMP than for the matrix proteins.