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Summary A study was made of the proteins extracted from the acrosome of ejaculated spermatozoa of 44 bulls. The acrosomes were destroyed by acidic treatment. Cathodic (pH 4.3) and anodic (pH 8.9) electrophoretic systems were used for separation of acrosomal proteins. The application of the cathodic system revealed that the acrosomal proteins are polymorphic. Three phenotypes were observed, designated as type A, B and C; the three phenotypes occurred in 16, 17 and 11 of the bulls, respectively. Zusammenfassung Untersuchung der bovinen Akrosomproteine Aus ejakulierten Spermien von 44 Stieren wurden die Akrosomproteine extrahiert, wobei die Kopfkappen durch 2% Essigsaure zerstort wurden. Die Akrosomproteine wurden durch kathodische (pH 4,3) und anodische (pH 8,9) Elektrophorese aufgetrennt. Die kathodische Elektrophorese ergab das Vorliegen polymorpher Proteine. Drei Phanotypen bzw. Proteinmuster wurden gefunden, welche als Typ A, B und C bezeichnet wurden. Typ 1 wurde bei 16, Typ B bei 17 und Typ C bei 11 Stieren festgestellt. Resume Recherches sur les proteines de acrosomes bovins Les proteines d'acrosomes ont ete extraites a partir de spermatozoides ejacules de 44 taureaux. Les acrosomes furent detruits avec de l'acide acetique a 2%. Les proteines des acrosomes furent separees par electrophorese cathodique (pH 4,3) et anodique (pH 8,9). L'electrophorese cathodique a montre la presence de proteines polymorphes. On a trouve trois phenotypes, respectivement trois echantillons de proteines qui furent designes par les types A, B et C. Le type A a ete etabli chez 16 taureaux, le type B chez 17 et le type C chez 11. Resumen Investigacion sobre las proteinas en el acrosoma de los espermatozoides de toro Se cxtrajeron de los espermatozoides eyaculados de 44 toros las proteinas del acrosoma, destruyendose los capuchones cefalicos con un 2% de acido acetico. Las proteinas acrosomicas se separaron mediante sistemas electroforeticos catodicos (pH 4,3) y anodicos (pH 8,9). La aplicacion del sistema catodico revelo que las proteinas acrosomales son polimorfas. Se hallaron tres fenotipos resp. modelos proteicos, los cuales se designaron como tipo A, B y C. El tipo A se verifico en 16, el B en 17 y el C en 11 toros.

Summary Separation of LDH in bull seminal plasma was carried out by disc electrophoresis carried out simultaneously on polyacrylamide gel at concentrations of 5.5% and 7.5%. LDH isozyme separation was achieved more distinctly on gel with the 5.5% concentration. All five forms of LDH (LDH1, LDH2, LDH3, LDH4, LDH5) were found. Some variability in the number of LDH isozymes between different samples was observed. Zusammenfassung Discelektrophorese-Untersuchungen der LDH im Seminalplasma von Bullen Die LDH-Auftrennung erfolgte mittels simultaner Discelektrophorese in Polyacrylamidgel in Konzentrationen von 5,5% und 7,5%. Bei einer Gelkonzentration von 5,5% konnten die LDH-Isoenzyme besser separiert werden. Es wurden alle funf Isoenzyme (LDH1 bis LDH5) gefunden. Die Anzahl der Isoenzyme in den verschiedenen Proben schwankte etwas. Resume Determination de LDH dans le plasma du sperme de taureaux par l'electrophorese a disque La separation de LDH du plasma dans le sperme de taureaux a ete faite a l'aide de l'electrophorese a disque simultanement sur un gel de polyacrilamide en concentration de 5,5% et 7,5%. La separation des fractions d'isoenzymes fut meilleure avec le gel de concentration a 5,5%. On a trouve les 5 formes des LDH (LDH-1 a LDH-5). Certaines differences dans le nombre des isoenzymes ont ete constatees les divers echantillons. Resumen Estudios discoelectroforeticos sobre la LDH en el plasma seminal de toros Se llevo a cabo la separacion de la LDH mediante discoelectroforesis, simultaneamente sobre gelosa de poliacrilamida en concentraciones de 5,5% y 7,5%. Con la concentracion de gelosa de 5,5% se pudieron separar mejor las isoenzimas de la LDH. Se hallaron todas las cinco formas de la LDH (LDH1, LDH2, LDH3, LDH4 y LDH5). Variaba algo el numero de isonenzimas de la LDH entre las muestras diferentes.

The occurrence of one, or two, proteinase fractions was demonstrated for 5 strains of Vibrio comma using the zymogram technique for proteinases. Most of the proteinase fractions moved more or less rapidly towards the cathode, although some were shown to migrate in the opposite direction at pH 6.2. Serological cross reactions were observed between proteinase fractions produced by a strain of Aeromonas liquefaciens and 2 of the V. comma stvains examined. The proteinase A of Ae. liquefaciens was shown to be enzymoserologically identical, or closely related, to one of the proteinase fractions of V. comma, while no relationship was observed between the proteinase B of Ae. liquefaciens and any of the V. comma proteinases. In order to remove the naturally occurring proteinase inhibitors from the antiproteinase-containing γ-globulins in immune serum, precipitation with sodium sulphate was used, with success, as demonstrated by disc electrophoresis and the Casein Precipitation Inhibition test (CPI-test). The need for suitable separation techniques when using enzymes as a basis for the taxonomical differentiation of the corresponding organisms is emphasized.

Aeromonas liquefaciens and Aeromonas salmonicida were both found to produce considerable amounts of extracellular proteinases, when grown on semi-solid skim milk agar at 30° C. The proteinase A, produced by Ae. liquefaciens only, and the proteinase B, produced also by Ae. salmonicida were purified approximately 100 times by precipitation twice with (NH4)2SO4, batchwise treatment with DEAE-cellulose and, finally, gel filtration on Sepha-dex G-100. The pH-optimum was estimated to be 7.9 for proteinase A and 9.0 for proteinase B. Differences between the proteinases were also found with regard to thermoresistance, and to their behaviour in the presence of some naturally occurring proteinase inhibitors. The addition of iron and cobalt ions increased the activity of both proteinases. Molecular weights were estimated to be 22 100 for the proteinase A and 43 600 for proteinase B, on the basis of gel filtration on Sephadex G-100.

Enterotoxin from Clostridium perfringens was shown to retain its biological activity after labelling with 125I. When injected intravenously into mice and rats, most of the radioactivity in the organs was present in the form of intact toxin. Studies of the tissue distribution of labelled enterotoxin showed the largest amounts in the liver, where the activity reached a maximum 10--15 min after administration. The highest concentration per g tissue was found in liver and kidneys. The radioactivity was excreted in the urine as a mixture of intact labelled toxin and low molecular weight degradation products. In vitro studies with purified parenchymal liver cells showed rapid release of lactate dehydrogenase (LDH) during treatment with enterotoxin, thus indicating severe membrane damage.

Isoenzymes of lactate dehydrogenase (LDH) from human platelets exhibit formation of subbands when examined by disc acrylamide electrophoresis using a 13% acrylamide in separating gel with a running temperature within the column of 45° C. at pH 8.9. One subband is found close to LDH 1, two bands close to LDH 2, all exhibiting a slower electrophoretic mobility than the main band. Three bands are seen in the LDH 3 area. No subbands formation is seen in the LDH 4 area. According to macroscopic evaluation identical parameters are seen whether the platelets are taken from normal controls or from patients suffering from diseases combined with a change in H/M ratio and/or myeloproliferative disorders.

LDH isoenzymes in thrombocytes from 41 healthy subjects and 85 patients suffering from various diseases combined with changes in platelet function were studied. The isoenzymes were separated by disc acrylamide electrophoresis. In normal thrombocytes LDH 3 constituted around 41 per cent, LDH 2 around 32 per cent, LDH 1 around 12 per cent and LDH 4 around 14 per cent of the total amount, whereas only very little LDH 5 was found. The results have been evaluated statistically. No differences were found between younger and older healthy persons. In patients with hepatic cirrhosis and polycythaemia vera an increase in ratio between H and M units (increase in T-values) was demonstrated. Significant lower T-values were found in patients with cancer and acute phlebothrombosis. No relationship between isoenzyme parameters of thrombocytes and serum was found.

The seminal plasma proteins of patients before and after vasectomy as well as in different split ejaculate fractions were investigated using gradient polyacrylamide gel electrophoresis. Considerable interindividual variations of the protein patterns were observed. A distinct high molecular weight proteins band was found to disappear after vasectomy. This protein band could b4 attributed to the sperm-rich fraction of split ejaculates. It seems to be of testicular-epididymal origin and may be of interest for functional characterization of the testicular-epididymal fluid and for diagnosis of an occlusion of the excretory duct system.Ejaculates of 10 men were analyzed before vasectomy and 6 months later to study seminal plasma protein patterns, as well as the different split ejaculate fractions. The analysis was done using gradient polyacrylamide gel electrophoresis. Purpose of the analysis was to obtain information on the availability of a protein component within the testicular-epididymal secretions, which may be of potential interest as a specific marker of testicular and epididymal secretory functions. There were considerable individual variations of the protein pattern. The distinct high molecular weight protein band found to disappear after vasectomy could be attributed to the sperm-rich fraction of split ejaculates. This protein band could be of testicular-epididymal origin and may be of interest for functional characterization of the testicular-epididymal fluid and for diagnosis of an occlusion of the excretory duct system.

By discontinuous gradient centrifugation in two oily mixtures of n-dibutyl phthalate and Apiezon A oil of an accurately fixed specific gravity (1.0270 and 1.0405), platelets from 16 normal platelet pools were separated into extreme density platelet populations (large-heavy and small-light platelets) which were studied against unseparated control platelets for platelet volume and LDH isoenzymes. By the microhaematocrit method the mean platelet volume per platelet in large-heavy, small-light, and control populations was 11.6 μ3, 5.7 μ3, and 7.3 μ3. LDH isoenzyme determination, using disc polyacrylamide electrophoresis, revealed identical isoenzyme patterns in large-heavy, small-light, and control platelets. When expressed by the logarithm of the H/M ratio, the mean values for the three groups were 0.16, 0.15, and 0.15. Thus, the study adduced no evidence of a correlation between human platelet isoenzyme parameters and platelet volume.

A patient with widespread IgD-myelomatosis is described. The serum electrophoretic pattern exhibited at discovery of the disease two M-components: one of IgD-lambda type migrating in the γ1-region and one of Bence Jones protein lambda type in the β1-region. A Bence Jones protein of lambda type was also detected in the urine. During treatment with cyclophosphamide the IgD-lambda- and serum Bence Jones lambda-proteins disappeared and the urine Bence Jones lambda protein decreased. After 8 months' treatment another M-component was recognized in the slowest γ-region. This M-component was IgG, subclass IgG1 of kappa type. No signs of the IgGl component were seen when the first serum samples were retested with a specific antiserum raised against the IgG-kappa component. In the following samples also the IgG-kappa component decreased but traces were still demonstrable in all late samples. The patient improved during treatment. In the last obtained serum sample a reactivation of the IgD-lambda- and Bence Jones lambda-clones was seen.

Zusammenfassung Saure-extrahierte Glykoproteine im menschlichen Seminal-Coagulum Der Proteinanteil in dem intakten Coagulum des frischen menschlichen Ejakulates wurde mit 0,25 N HCl extrahiert, der Bedingung fur die Extraktion der basischen Spermaproteine. Etwa 75% der Total-Proteine des Coagulums wurden als saureextrahierbar und dementsprechend als sog. basische Coagulum-Proteine (CBP) befunden. CBP wurde glykoly-siert und war vollstandig zu unterscheiden von dem Sperma-Protamin in vielerlei Hinsicht. CBP bestand aus zwei Hauptkomponenten, analysiert mittels der Saure-Urea-Polyacryl-amid-Gelelektrophorese (PAGE). Natrium-dodecyl-sulfat-PAGE konnte CBP in drei Haupt-komponenten auflosen mit einem offenbaren Molekulargewicht von 52,000, 80,000 und 82,000 Dalton. Die Auflosung von CBP wahrend der in vitro-Verflussigung des frischen Ejakulates weist auf die mogliche Rolle des Glykoproteins in der Struktur des Coagulums hin.

The study of the structure of the peptides arising from native, oxidized, reduced and alkylated or citraconylated equine growth hormone on incubation with trypsin and chymotrypsin is reported. The data obtained, combined with results already published, support the assembly of a unique sequence of amino acids for the polypeptide chain of the protein.

The esterification of Ribonuclease-A in methanol/0.1 M hydrochloric acid has been studied by measuring the decrease in the number of titratable groups of the protein and estimating the amount of methanol incorporated. Esterification of nearly five of the 11 free carboxyl groups of the protein resulted in almost complete inactivation of the enzyme. The initial products of esterification have been chromatographed on Amberlite columns, and five partially active methyl ester derivatives of Ribonuclease-A have been isolated. The dimethyl ester, the initial product of esterification with reduced catalytic activity, has the carboxyl groups of Glu-49 and Asp-53 modified. Even in the non-aqueous solvent, as in the native structure of the protein in aqueous solution, these carboxyl groups are the fast reacting ones. Subsquently, the esterification reaction appears to proceed preferentially at the C-terminal region of the molecule. Comparison of the reactivities of carboxyl groups of Ribonuclease-A in acidic methanol to that known in aqueous solutions (with carbodiimides) suggests that the structure of Ribonuclease-A in the non-aqueous solvent resembles, at least in part, the structure in aqueous environment.

11S globulin, the major storage protein of soybean seeds, was isolated to be homogeneous in ultracentrifugation, disc electrophoresis and isoelectric focusing by a comparatively simple method using gel filtration on Sephadex G-100 and G-200 columns with a good yield. The protein had the molecular weights of 309,000–322,000 by three separate methods based on different principles. Subsequently, some physico-chemical properties of the protein were determined. For example, sedimentation coefficient, isoelectric point, intrinsic viscosity and absorptivity constant were 12.2S, pH 4.64, 0.0485 dl/g and 8.04, respectively. Optical rotatory dispersion studies of the protein gave the values of a0 = —246 and b0 = —33. The contents of α-helix and β structure calculated from these parameters were estimated to be 5.2% and 34.8%, respectively.

A study has been made on the changes in the enzymatic activity of Ribonuclease-A**-(RNase-A) exposed to highly acidic (pH less than 1) acqueous environment. Irreversible alterations of activity were observed when the protein was exposed to an acidic medium for a long period (20 to 60 h). Even prior to these changes in activity RNase-A was found to form intermediates which had very nearly the same activity as the native protein. The primary process in the acid denaturation of RNase-A was observed to be deamidation of the protein leading to the formation of active chromotographically distinct derivatives. The initial product of deamidation, a monodeamidated derivative, has been isolated by chromatography on Amberlite XE-64. This initial deamidation reaction proceeded with very high specificity. The subsequent deamidation reaction is comparatively slower, so that nearly 50% of the native protein could be converted to this derivative before any subsequent deamidation took place. This monodeamidated derivative has been designated RNase-Aa1. The conversion of RNase-A to RNase-Aa1 was not accompanied by any changes in the primary structure other than the observed deamidation. Apart from the differences in chromatographic and electrophoretic mobilities, RNase-Aa1 was found to have very nearly the same activity and physicochemical properties as the native enzyme. Significance of this specific and faster deamidation of RNase-A in this denaturing medium as well as the biological significance of such deamidation reactions of proteins are discussed.

1. The effects of various treatments on the extraction of acidic proteins and on enzymic activities in microsomal preparations of bovine cerebral cortex have been examined. After ultrasonic irradiation, density-gradient centrifugation demonstrated the association of proteins and the enzymes (Na+, K+)-ATPase and K+ -NPPase with particles of lower density than in the original membrane fraction. However, experiments involving ammonium sulphate precipitation, exclusion chromatography, and acrylamide-gel electrophoresis suggested that the material was still particulate. 2. Non-ionic detergents comprising homologous polyoxyethylene ethers (Lubrols) and Triton X-100, a cholate-deoxycho-late-dodecylsulphate mixture, and cetyltrimethylammonium bromide were selected for studies of surface-active agents. The anionic system released the most protein and K+ -NPPase activity after dialysis. 3. Despite the low recovery of enzymic activity, pentanol was found to release more acidic proteins than treatment with butanol, sodium iodide, or detergents. 4. Regardless of the method of preparation, extracted proteins had only a limited solubility at I – 0.1 below pH 5.5. Electrophoresis demonstrated the predominance of negatively-charged proteins in unprecipitated material after lowering the pH. 5. To elucidate the best conditions for the extraction with pentanol, the effects of the following variables were examined: pentanol concentration, pH and ionic strength of the medium, microsomal protein content, and time and type of mixing.

Purified rat liver aminopeptidase B preparations were found to contain two molecular forms of the enzyme which can be separated by preparative electrophoresis on poly-acrylamide columns. The one with higher electrophoretic mobility represents the actual liver enzyme and the one with lower electrophoretic mobility was seen to be derived most likely from hemolyzed red blood cells constantly present in liver homo-genates. This was proved by purifying aminopeptidase B directly from isolated rat erythrocytes with the same procedure earlier used for the liver enzyme. The enzymic properties of the three enzymes (two derived from livers and one derived from erythrocytes) were seen to be largely the same. The similarity was especially considerable in the kinetics of the enzyme-catalyzed reactions, where substrate inhibition was seen to be the most characteristic feature.

A method for the preparation of human STH* was developed on the basis of Wilhelm's technique from 1961 which was simplified, by for example, introducing gel filtration. The purity of the preparation was established by a large number of purity criteria: zone electrophoresis on starch, zone electrophoresis in a density gradient, determination of N- and C-terminal end groups, determination of the N-terminal amino acid sequence, quantitative amino-acid analysis, determination of molecular weight by gel filtration (mol. wt.: 2.2×104), etc. Oxidation of human STH causes polymerization. The alteration of the degree of polymerization of the oxidized product was studied at varying pH levels. The molecular weight of the preparation at the highest degree of polymerization was found to be 45×104.

Serum cholinesterase 2 (CHE2) was examined in a Danish material of normal families that has been tested earlier for 70-78 classical marker systems and 25 RFLP systems. DNA for RFLP typing was provided by transforming 16-year-old frozen lymphocytes. The frequency of allele CHE2*C5+ in the Danish population was found to be 0.0430. The highest lod score was between CHE2 and the gamma-crystallin gene cluster (CRYG) (zeta = 4.21 at theta = 0.00 in females). The scores were from a single family with 15 children. CHE2 may, accordingly, be assigned to the location of CRYG: chromosome 2, bands q33-q35.

Serum high density lipoproteins (HDL) from three patients with lecithin: cholesterol acyltransferase (LCAT) deficiency have been studied. HDL from the patients is composed of two components, one of high molecular weight (HM-HDL) and one of relatively low molecular weight (LM-HDL). The polypeptides of lipid-free HDL (apoHDL) from normal subjects were indistinguishable from those of apoHM-HDL from the patients as judged by treatment with 8 M urea and gel filtration on Sephadex G-200, immunological experiments, disc electrophoresis, and amino acid analysis. ApoLM-HDL contained mainly the apoA-I polypeptide with small amounts of apoA-II, whereas C-polypeptides were lacking. The results indicate that the presence of apparently “abnormal” HDL particles in serum of the patients is secondary to the lack of LCAT activity.

In the attempt to separate in a single gel run low- and high-molecular-weight proteins, we present here a multiphasic buffer system designed for this purpose. It avoids the continuous stacking of SDS as it occurs in the 'classical' SDS-PAGE. The system allows complete stacking and destacking of proteins in the 3.5-250 kDa range at acrylamide concentrations as low as 4.5% T (total acrylamide concentration in %) and 2.6% C (degree of cross-linking in %). Taurine is used as the trailing ion in the cathode buffer and in the resolving zone of the gel, and two different counterions (Tris and imidazole) in the stacking zone. The gel system is easy to prepare and, due to the very low acrylamide concentrations, it is ideal for analytical as well as for preparative tasks.

Summary Disc electrophoresis per se and in combination with immunodiffusion procedures facilitated the characterization of sialidase. Irrespective of the isolation procedures, sialidase of influenza virus (A2 Japan 305/57) appeared to be electrophoretically heterogeneous. This was indicated by disc electrophoresis and analysis of eluates in the “segmentation experiments” and by the “disc immunodiffusion” techniques. It was further noted that “purified” sialidase in phosphate buffer stored at 0 to 5°C. dissociated and simultaneously lost 90 per cent of its enzyme activity. The procedures described above proved valuable, particularly when analyzing limited quantities of material.

SUMMARY Crude human skin extracts having tissue-specific mitotic inhibitory activity (chalone), were studied by ethanol precipitation and disc gel electrophoresis. Precipitates obtained at various ethanol concentrations were dialyzed, lyophilized and tested for their mitotic inhibitory activity. It was found that the greatest inhibitory activity was concentrated in the precipitate formed in 81% ethanol, the active principle being concentrated about 200 times. Proteins in crude extracts were separated by disc gel electrophoresis, and at least eight bands were identified. Groups of bands were extracted from the electrophoretic gels and tested for their mitotic inhibitory activity. Fraction II, containing two bands, had the highest activity. Disc gel electrophoretic analysis of the 81% ethane precipitate revealed only two major bands, one of which could also be found in fraction II of the crude extract. This protein band, which is present in both of the mitotic inhibitory samples, may represent the human epidermal chalone.

The present review encompasses ca. 65 years of history of developments in electrokinetic separations, taking as a starting point the year 1937, i.e. the official launching of Tiselius’ moving boundary electrophoresis (MBE). The 1950s have been particularly rich in introducing novel methodologies in zone electrophoresis (ZE), thus bringing about the decline of MBE. Among them of extraordinary importance was the development of electrophoresis on agar gels coupled to immuno-diffusion at right angles, which brought a big revolution not only in biochemistry but also in clinical chemistry. Also the by now forgotten paper electrophoresis was a landmark in separation science, in that it implemented, in its “fingerprinting” version, the first genuine two-dimensional (2D) map, coupling orthogonally a charge to a hydrophobic scale separation, while permitting for the first time the detection of spot mutations, i.e. single amino acid replacements in a polypeptide chain, that paved the way to modern genetic analysis. Equally important was the introduction of starch-block electrophoresis, that brought about the notion of sieving and the first discontinuous buffers, refined, in the 1960s, by Ornstein and Davies with their classical papers combining multiphasic buffer systems to polyacrylamide gels, that went down to history as disc-electrophoresis. The 1960s also contributed with two fundamental techniques, isoelectric focusing (IEF) and sodium dodecyl sulphate–polyacrylamide gel electrophoresis (SDS–PAGE) that permitted to discriminate proteins solely on the basis of surface charge and molecular mass, respectively. The 1970s gave other fundamental contributions, such as isotachophoresis, the first example of a fully instrumental approach to electrophoresis, both in its analytical and preparative version (Tachophor and Tachofrac), 2D maps combining IEF to SDS–PAGE at right angles and silver staining techniques, that incremented sensitivity by 3 orders of magnitude. The 1980s generated immobilized pH gradients and capillary zone electrophoresis (CZE), two big players that dominated the electrokinetic horizon for all the 1990s and still in vigorous use in present days. The review terminates with a glimpse, in the third millennium, onto microchip technology and hyphenated techniques, notably direct interfacing of various electrophoretic separation methods with mass spectrometry (MS).

One of the peptide fragments obtained in a preceding study of the cyanogen bromide hydrolysate of S-sulfo-pepsin has been investigated in more detail. The peptide was purified by ion-exchange chromatography and obtained in homogeneous form. It contains 158 amino acid residues, among their number the only histidine residue of pepsin and two half-cystine residues. Evidence was obtained that the peptide is derived from the N-terminal region of the molecule of hog pepsin. The peptide was reduced by 2-mercaptoethanol and converted into its S-(β -amino-ethyl)-cysteinyl derivative. From the tryptic digest of this derivative a 105-residue peptide containing N-terminal isoleucine was isolated which represents the N-terminal segment of the pepsin molecule.

Mechanisms of riboflavin oversynthesis in a high flavinogenic mold, Eremothecium ashbyii, were examined in relation to growth, riboflavin formation and related synthases, and medium pH with increasing culture periods. Growth reached maximum at 1 d and then decreased, riboflavin formation proceeded rapidly up to 5 d and approached almost a plateau region. The medium pH reached minimum at 1 d and thereafter fairly rapidly increased until 3 d, then gradually increased to 7 d after cultivation. The crude enzyme solution from the mycelia at specified culture periods was run through a column of Sephadex G-200, indicating two riboflavin synthase activities on the chromatogram. The fluctuation of the growth and the specific activities of the two enzymes were examined with increasing culture periods, which showed that the heavy enzyme may be a constitutive one and that the light enzyme may be concerned with the oversynthesis of riboflavin in E. ashbyii. The heavy enzyme was then purified by 49-fold after dialysis of the ammonium sulfate precipitate by a series of column chromatographies with Sephadex G-200, hydroxyapatite, DEAE-Sepharose A-50 and DEAE-cellulose. The purified enzyme which was treated with weak alkaline solution was broken into the light enzyme, showing two bands on an acrylamide disc gel electrophoresis. The relation of the heavy and the light enzymes to the oversynthesis of riboflavin in E. ashbyii was discussed.

The development of a new approach to the diagnosis of infectious diarrhoea, caused by Escherichia coli heat-stable enterotoxin (ST), was preceded by a preliminary study. The purpose of the latter was to establish whether three preparations of ST produced by a human isolate of enterotoxigenic E. coli (STa), obtained at different steps of the purification procedure (involving Amberlite XAD2 resin chromatography (P3), a gel filtration chromatography on a Biogel P4 (P2) or a disc-gel electrophoresis (P1)), could be employed to titrate antisera to STa using an ST-biotin enzyme-linked immunosorbent assay (ELISA). The solid-phase STa was obtained by first coupling the toxin to biotinyl-N-hydroxysuccinimide and then binding this conjugate to avidin adsorbed to flat-bottomed polystyrene microtitre plates. Using these reagents, the assay conditions were examined. Checkerboard tests determined optimal biotin-P3, P2 or P1 toxin conjugate concentrations to be used as the immunosorbent for P3, P2 and P1 antiserum titration. The immunosorbent prepared with STa purified only on Amberlite XAD2 resin was unable to differentiate significantly between P3, P2 or P1 antisera. Immunosorbent prepared with P2 or P1 detected widely differing titres between the three antisera and gave more sensitive results. Only small but questionable differences were observed between P2 and P1 toxin preparations.

Summary A disc electrophoretic study was made of buffalo endometrial proteins. The endometrium of the luteal phase revealed nine protein bands, of which five were uterine-specific whereas in the follicular phase there were only five protein bands and only one was uterine-specific. Some of the soluble proteins of the luteal phase may play a role in the development of the conceptus. Zusammenfassung Endometriale Proteinmuster wahrend der Follikel- und Corpus-luteum-Phase des Buffels Mittels Elektrophorese (Polyacrylamidgel) wurden die endometrialen Proteine von Buffeln untersucht. Wahrend der Lutealphase zeigte das Elektropherogramm 9 Proteinbanden, von denen 5 uterusspezifisch waren, wahrend der Follikelreifungsphase enthielt das Pherogramm nur 5 Proteinbanden, davon war aber nur ein Band uterusspezifisch. Einige der loslichen Proteine der Lutealphase durften fur die Entwicklung des Embryos eine Rolle spielen. Resume Echantillon proteique de l'endometre pendant la phase folliculaire et du corps jaune chez le buffle Les proteines de l'endometre du buffle ont ete examines au moyen de l'electrophorese (gel de polyacrylamide). L'electropherogramme a montre 9 bandes de proteines pendant la phase du corps jaune dont 5 etaient specifiques de l'uterus alors que le pherogramme durant la phase de maturation du follicule a presente 5 bandes de proteines dont une seule etait specifique de l'uterus. Quelques proteines solubles de la phase du corps jaune devraient jouer un role dans le developpement de l'embryon. Resumen Modelo proteico del endometrio durante las fases folicular y luteal en la bufala Mediante electroforesis (gel de poliacrilamida) se examinaron las proteinas del endometrio de bufala. Durante la fase luteal mostraba el electroferograma 9 bandas de proteinas, 5 de las cuales eran especificas de utero, mientras que durante la fase de maduracion del foliculo solo contenia el ferograma 5 bandas de proteinas, mas nada mas una de ellas era especifica del utero. Algunas de las proteinas solubles de la fase luteal podrian jugar cierto papel en el desarrollo del embrion.

The presence of a unique population of Lagoa crispata , puss caterpillar, in western Oklahoma is reported. A detailed microscopic examination shows the structure of the L. crispata spines resemble the type 4 spines described by [Kawamoto, F., Kumada, N., 1984. Biology and venoms of lepidoptera. In: Tu, A.T. (Ed.), Handbook of Natural Toxins, Insect Poisons, Allergens and other invertebrate venoms, vol. 2, pp. 291–332 (ch. 9)]. The major food source of L. crispata are the leaves of oak (shin oak). The high tannin content of this food source results in spine extracts high in oak tannins. These extracts have activity but enzyme and toxin activity is lost with time. The gel filtration protein fractions are colored from brown to yellow and are inactive as enzymes or toxins. No hyaluronidase, protease or phosphohydrolase activity is detected in these protein fractions. The life cycle shows these caterpillars have 6 instars. Characterizations and annual emerging times of each instar are included.

Three cellobiases, here called cellobiase A, B, and C, from the culture filtrate of Aspergillus niger A20, were purified by precipitation with ammonium sulphate, gel filtration through Sephadex G-75, and column chromatography of DEAE-cellulose. The purified enzymes were homogeneous on polyacrylamide disk electrophoresis. The mol wt of the purified enzymes were estimated by SDS-gel electrophoresis to be 88,000, 80,000, and 71,000 for cellobiases A, B, and C, respectively. The enzymes were active at pH 4.5 and 55-60 degrees C. The pattern of their amino acid compositions showed high contents of aspartic acid, glutamic acid, threonine, serine, and glycine. The apparent K(m) values for cellobiose were 0.9, 1.63, and 1.0 mM for cellobiases A, B, and C, respectively. Calcium ions stimulated cellobiases B and C, and Co2+ and Mg2+ ions stimulated cellobiase A. The purified enzymes hydrolyzed cellobiose and aryl-beta-D-glucosides, but they had no action on sucrose, maltose, and cellulose. The three cellobiases catalyzed transglycosylase reaction, and the major product formed from cellobiose was tetramer of glucose.

Urine produced by normal human kidneys is almost always supersaturated with respect to calcium oxalate (CaOx), the most common constituent of human kidney stones. Crystallization, with risk of renal damage and kidney stones, appears to be affected by molecules in urine that retard nucleation, growth, aggregation, and renal cell adherence of CaOx. The repertoire of such molecules is incompletely known. We have purified a 28-kDa protein from urine using salt precipitation, preparative isoelectric focusing, and sizing chromatography. Amino acid composition and NH2-terminal amino sequence analysis showed complete homology to calgranulin. Calgranulin was found to be a potent inhibitor of CaOx crystal growth (44% of control) and aggregation (50% of control) in the nanomolar range. Calgranulin cDNA was cloned from a human kidney expression library. Western analysis of human and rat kidney homogenates and mRNA temporal expression from two independent renal epithelial cell lines showed that calgranulin is produced in the kidney. Given its urinary abundance and potency, calgranulin may contribute importantly to the normal urinary inhibition of crystal growth and aggregation and therefore to the renal defense against clinical stone disease.

A modified discontinuous electrophoretic method for the separation of standard and urinary glycosaminoglycans has been reported. The merits of the method are the simple and easy to handle apparatus, non-requirement of elaborate cooling system, sensitivity and high reproducibility of the results and applicability of the method for the preliminary grouping of the MPS patients to reduce the number of enzyme assays to be done.

[EN]: A study was made of the variations in the electrophoretic profile of myofibrillar proteins in the muscles of blue whiting (Micromesistius poutassou R.), horse mackerel (Trachurus trachurus L.) and mackerel (Scomber scombrus L.). It was shown that all species presented different deterioration patterns during frozen storage. The fish were caught at two separate times of the year (winter and summer) and were stored frozen at -18°C for 1 year. The results indicate that during frozen storage, electrophoretic patterns varied according to species. Comparison of myosin heavy chain/actin (MHC/A) ratios indicates that blue whiting is the species that undergoes most alteration, and that this is more intense in fish caught in the summer than in the winter. Alteration of the MHC/A ratio was similar in horse mackerel and mackerel caught in the winter, whereas in the summer horse mackerel proved to be the most stable species. In all cases, the reduction of the MHC/A ratio was due essentially to alteration of the MHC, an effect which was particularly marked in blue whiting. Tropomyosin remained stable throughout the storage period in all three species., [DE]: Es wurden die Veränderungen im elektrophoretischen Profil myofibrillärer Proteine in Muskeln von Blauem Wittling (Micromesistius poutassou R), Stöcker (Trachurus trachurus L) und Makrele (Scomber scombrus L) untersucht — Arten, deren Zustand sich während der Gefrierlagerung auf unterschiedliche Weise verschlechtert. Diese Fische wurden in zwei verschiedenen Jahreszeiten (Winter und Sommer) gefangen, in denen auch zu erwarten ist, daß sich der physiologische Zustand verändert und ein Jahr lang bei -18°C tiefgefroren gelagert. Die elektrophoretischen Muster änderten sich je nach Fischart während der Tiefkühllagerang. Aus dem Vergleich der MHC/A-Verhältnisse ist ersichtlich, daß Blauer Wittling die Art ist, die sich am meisten verändert und dies im Sommer intensiver als im Winter stattfindet. Bei Stöcker und Makrele ändert sich das MHC/A-Verhältnis auf ähnliche Weise im Winter, während sich Stöcker im Sommer als die stabilste Spezies zeigte. In allen Fällen lag die MHC/A-Verringerung grundsätzlich an der MHC-Veränderung und diese Wirkung war im Blauer Wittling besonders deutlich zu sehen. Bei allen untersuchten Arten blieb das Tropomyosin während der Lagerzeit stabil., Financial support for this study was provided by the CICyT (Project ALI91-0927-C02-01) and the EC (Project FAR UP 3/647).

Cell-free-antigen (CFA) vaccines of strain Y-1 (serotype 1), G-4 (serotype 2) and E-3 (serotype 5) of Actinobacillus pleuropneumoniae (A. pleuropneumoniae) were prepared by emulsifying concentrated culture supernatant with oil-adjuvant. Mice immunized with the CFA vaccine had a high survival rate (90-100%) against challenge with the homologous strain. They also had cross-protective activity against challenge with the heterogeneous strains but their survival rate was low (20-50%). On the other hand, mice immunized with whole cell vaccine showed serotype specific protection and only a little cross protection. The protective antigens of the CFA were investigated. MAbs were produced by the standard method using spleen cells of mice immunized with CFA. MAbs to Apx I, II, III and capsular antigen of serotype 5 were obtained. Only MAbs to Apx I showed hemolysin neutralization activity among them. The protective effect of these MAbs against A. pleuropneumoniae infection were examined by passive immunization. Administration of Apx I MAb to mice extended survival time after challenge with serotype 5. The mice showed partial cross-protection against challenge with serotype 1. Survival rate was considerably low after the challenge infection. None of the mice given MAbs to Apx II or III were protected against challenge with serotype 5. The mice given MAb to capsular antigen of serotype 5 had a high survivalrate (70%) against a challenge with a homologous serotype. Furthermore, mice given MAbs against Apx I and capsular antigen of serotype 5 were completely protected against a challenge with A. pleuropneufnoniae serotype 5. In this study, we concluded that capsular antigen and Apx I are identified as protective antigens and their antibody play an important role in protection against infection with A. Pleuropneumoniae serotype 5.

A scanning beam laser macroscope has been developed which scans an area of 7.5 x 7.5 cm in 5 s. This new imaging system is examined as a potential tool for scanning electrophoretic gels. A specially-designed telecentric, f* theta laser scan lens is used in the instrument to achieve a linear scan and a flat focal plane. The laser scan lens focuses the incoming beam from a laser to a 10 microns spot inside the gel. A raster scan is performed across the gel and the signal is detected with a photomultiplier, forming a 512 x 512 digital image stored as a computer file. Silver-stained protein polyacrylamide gels have been imaged in transmission and double-transmission, while DNA agarose gels (stained with ethidium bromide) have been imaged in fluorescence with better than 25 pg sensitivity. The macroscope has the advantage that it is not tied to the electrophoresis system as are end-of-line scanners, and the scan is rapid, so that several gels can be scanned in a very short time.

An improved Deriphat polyacrylamide gradient gel electrophoresis system was developed for the separation of chlorophyll-protein complexes. The relatively good resolution of the starting discontinuous gel system was further improved by using glycerol in gels and an acrylamide gradient with high acrylamide-to-N,N'-methylenebisacrylamide ratio in the separating gel. By applying mild but efficient glycosidic detergents for solubilization, and Deriphat to gels and buffers, the stability of complexes was increased, and only a low amount of pigment was removed. The advantage of our system is the better resolution of larger-size complexes, especially those of photosystem I. In addition, it makes possible an easier interpretation of results due to less overlapping of photosystem I and photosystem II bands when different plant species or the effects of different treatments are compared using whole thylakoid membranes.

Plasma alkaline phosphatase (ALP) activity is frequently measured in toxicity studies. In the present study, we assessed the usefulness of a commercially available polyacrylamide-gel (PAG) disk electrophoresis kit used in humans (AlkPhor System, Jokoh Co., Ltd., Tokyo, Japan) for identifying plasma ALP isoenzymes in mice of the Crlj:CD1 strain (ICR mice), which are commonly used in toxicity studies. We also examined age-related changes in plasma ALP isoenzymes in ICR mice. Electrophoresis was performed according to the manufacturer's instructions. In order to identify the origin of each ALP isoenzyme, in addition to plasma samples, tissue ALP extracts from the liver, bone and small intestine were treated with neuraminidase, anti-small intestinal ALP antibody, ALP inhibitor levamisole and/or wheat germ agglutinin (WGA). The kit revealed that main plasma ALP isoenzyme in intact ICR mice was bone-derived one, and it tended to decrease with age. On the other hand, liver-derived ALP isoenzyme greatly increased in plasma of cholestasis model mice induced by bile duct ligation. This model mouse had also a large molecular ALP detected in the stacking gel. This ALP was thought to be of intestinal origin because its activity remained even after levamisole inhibition. In addition, a minimum sample volume for sufficient resolution of plasma ALP isoenzymes was only 14µl. The results of this study suggest that the present method is a useful tool for detecting plasma ALP isoenzymes in mice and that pre-treatment of plasma with neuraminidase and concomitant levamisole inhibition with another gel is applicable for the evaluation of organ toxicity.

The glutathione (GSH) S-transferases are believed to have dual functions as hepatic detoxifying enzymes and intrahepatic binding proteins. Little is known about their alterations in human liver diseases. Therefore, we have studied the relationship between the enzyme activity and rose bengal (RB) binding in hepatic cytosol and plasma indocyanine green (ICG) kinetics in patients with various liver diseases. The enzyme activity was measured in samples of hepatic cytosol obtained from 52 patients. In addition, the content of cationic and neutral transferases was estimated in 17 biopsy samples by densitometry of Coomassie blue stained sodium dodecyl sulphate polyacrylamide gel electrophoretograms. RB binding studies also were performed on cytosol samples. ICG kinetic parameters were determined using the two-compartment open model in 17 patients who were given the dye (0.5 mg kg-1) intravenously. Correlations between the enzyme activity and liver function tests, content of the enzyme, RB binding and ICG kinetic parameters were evaluated. The following results were obtained. (1) The enzyme activities were high in alcoholic liver disease, fatty liver and Gilbert's syndrome, and low in cirrhosis. (2) The enzyme activities were positively correlated with serum cholinesterase activity, serum albumin level and hepaplastin test, and negatively correlated with ICG retention rate at 15 min. (3) The enzyme activity, its content and RB binding affinity of the cytosol were positively correlated with each other. (4) The enzyme activity was positively correlated with hepatic ICG distribution volume. These results are consistent with the role of the GSH S-transferases as ligandins in intracellular storage of dyes.

The synthesis of 23-kDa and 34-kDa proteins in mouse peritoneal macrophages was enhanced when they were incubated with oxidatively modified low-density lipoprotein (LDL). This response required the oxidation of the lipid of the LDL, because native LDL had little effect and acetylated LDL enhanced the synthesis of 34-kDa protein only slightly. Since macrophages can bind and sequester oxidized LDL, they are subjected to an oxidative stress. The results suggest that the 34-kDa protein, which is identical with heme oxygenase, and the 23-kDa protein serve as stress protein that might afford protection against the oxidative stress.

S 1 , a heterophile antigen present on human sarcoma cell lines in culture, has been previously defined by this laboratory [1,2]. This antigen is also present in guinea-pig kidney. Purification of the antigen to homogeneity has now been achieved by a combination of ammonium sulfate fractionation, DEAE-cellulose, sephadex, high pressure liquid chromotography and affinity chromotography. S 1 is a monomeric protein of 70 000 Da, as indicated by the presence of a single band on SDS-PAGE. Amino acid analysis demonstrates the prevalence of glycine, lysine and glutamic acid. Aspartic acid was found to be the N-terminal residue with further sequence of glycine-valine-alanine-glutamic acid (gly-val-ala-glut).

Two kallikreins were identified in a homogenate of bovine pancreas in terms of their differential elution from an anion-exchange chromatography column. The kallikreins were quantified by their ability to release kinin from a partially purified preparation of bovine kininogen. The second kallikrein, designated kallikrein B, was purified by a three-step procedure following anion-exchange chromatography consisting of affinity chromatography on a benzamidine-agarose resin, gel filtration and hydrophobic interaction chromatography. An overall purification factor of 556-fold was achieved with a 58% recovery of enzymatic activity. The final material was shown to be homogeneous by a number of electrophoretic analyses. The relative molecular mass of prokallikrein B was found to be 26,700 by gel filtration and that of kallikrein B to be 26,000 by SOS gel electrophoresis. Gel isoelectric focusing revealed the presence of several isoenzymic forms ranging in isoelectric point from pH 4.05 to 4.35.

For the identification of host species of blood-sucking parasites, the suitability of disc-electrophoresis of the stomach contents was tested. Mammalian blood in the stomach of the medicinal blood leech Hirudo medicinalis gave satisfactory results. In the case of mixed blood samples from H. medicinalis, the identification of the host according to the electrophoretic patterns of the stomach contents failed as compared with an immunological method such as the Ouchterlony test. Medicinal blood leeches (H. medicinalis) collected in Istria Croatia, or bought in a pharmacy contained blood from cattle, horses, or frogs in their stomachs. Specimens of H. medicinalis from Lake Neusiedl or from the Seewinkel Austria, had sucked blood from mallards or frogs. Blood of cattle, mallards, and frogs was found in the stomachs of H. medicinalis coming from the National Park Kiskunsag Hungary. For the first time, horses were established as hosts for free-living specimens of H. medicinalis. A comparison of the weights of H. medicinalis bought in a pharmacy revealed that specimens containing frog blood in their stomachs weighed significantly less than those containing horse blood. These results confirmed the reports from Ssynewa (1944) concerning the breeding experiments. Probably, there is a change in hosts from the frog to warm-blooded animals during the life cycle of H. medicinalis. There were also significant differences in the weights of leeches as revealed by a comparison of the population from the Neusiedlersee with the leeches bought in the pharmacy.

The polyacrylamide gel electrophoresis technique was used for comparison of common protein of body wall tissues in five species of Lumbricidae (Eisenia nordenskioldi, E.fetida, Lumbricus rubellus, Aporrectodea caliginosa, A. longa). The statistic processing of indexes of electrophoretic similarity revealed three levels of similarity: intraspecies, interspecies, and intergenus. It was discovered that the similarity of E. nordenskioldi and E.fetida proteins corresponded to the intergenus level. The use of this method for determination of the earthworm's taxonomic status is discussed.

Two extracellular keratinases of Scopulariopsis brevicaulis were purified and partially characterized. The enzymes were isolated by the techniques of gel filtration chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). These keratinases (K I & K II) were purified approximately 33 and 29 fold, respectively. SDS-PAGE of the products of gel filtration chromatography (K I & II) produced only one band each, suggesting homogeneity. The optimum pH for both keratinases was 7.8, while the optimum temperatures were 40 degrees C (K I) and 35 degrees C (K II). Estimated molecular weights were 40-45 KDa and 24-29 KDa for K I & K II respectively. Both keratinases were inhibited by phenylmethylsulfonyl fluoride which suggests a serine residue at or near an active site.