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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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

[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.

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.

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.

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.

When mammalian protamine is dissolved in aqueous buffers at neutral or alkaline pH, both ends of the protein fold inward toward the center of the molecule and form disulfide crosslinks that stabilize several different structures. In the absence of reducing agents, these folded forms of protamine may be visualized and quantitated by gel electrophoresis. Using this technique, we have examined the formation of bull protamine disulfides in solution and describe a variety of factors that affect this process. At pH 8, disulfide-stabilized folded forms of protamine appear within minutes after solublization of the fully reduced protein. Five different monomers are detected by electrophoresis. Each of these monomers is stabilized by at least one disulfide crosslink and migrates with a distinct mobility, ahead of the fully reduced and extended protein. Under certain conditions, dimers of these folded structures crosslinked by interprotamine disulfides are also formed. The appearance of these disulfide-crosslinked forms of protamine is effected by air oxidation, accelerated at alkaline pH, inhibited upon lowering the pH below pH 7 and eliminated by modifying the protein's cysteine residues. Similar intramolecular disulfides are also produced after the protamine molecule binds to DNA. These results suggest that only those cysteines located within the amino- and carboxyterminal ends of the protein appear to participate in forming intramolecular disulfides in vitro.

Homogenates of Sarcocystis muris merozoites (cyst form) and the subcellular fraction of dense granules were assayed for protease activity with substrate-impregnated SDS-polyacrylamide gels. Four acidic and several basic proteases were detected in the merozoites. One of the basic proteases was further characterized as a thiol protease (EC 3.4.22). The activity of this protease was enriched in the dense granule fraction.

Steroid sulfatase was purified approximately 170-fold from normal human placental microsomes and properties of the enzyme were investigated. The major steps in the purification procedure included solubilization with Triton X-100, column chromatofocusing, and hydrophobic interaction chromatography on phenylsepharose CL-4B. The purified sulfatase showed a molecular weight of 500-600 kDa on HPLC gel filtration, whereas the enzyme migrated as a molecular mass of 73 kDa on sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric point of steroid sulfatase was estimated to be 6.7 by isoelectric focusing in polyacrylamide gel in the presence of 2% Triton X-100. The addition of phosphatidylcholine did not enhance the enzyme activity in the placental microsomes obtained from two patients with placental sulfatase deficiency (PSD) after solubilization and chromatofocusing. This result indicates that PSD is the result of a defect in the enzyme rather than a defect in the membrane-enzyme structure. Amino acid analysis revealed that the purified human placental sulfatase did not contain cysteine residue. The Km and Vmax values of the steroid sulfatase for dehydroepiandrosterone sulfate (DHA-S) were 7.8 microM and 0.56 nmol/min, while those for estrone sulfate (E1-S) were 50.6 microM and 0.33 nmol/min, respectively. The results of the kinetic study suggest the substrate specificity of the purified enzyme, but further studies should be done with different substrates and inhibitors.

Hepatocellular carcinoma (HCC) is one of the deadliest cancers with few treatment options. It is a hypervascular tumor in which angiogenesis plays a critical role in its progression. Tumor capillary endothelial cells (TECs) in HCC are known to originate from liver sinusoid endothelial cells, which then go through a capillarization process to become morphologically as well as functionally different TECs. In this work, we investigated proteins differentially expressed between freshly isolated TECs and sinusoid endothelial cells from well-formed rat HCC using 2-D DIGE coupled with MALDI-TOF/TOF MS. Thirty-eight unique proteins were identified to be differentially expressed more than twofold between the two endothelial cell types. Amongst the differentially expressed proteins, two novel endothelial markers, EH domain-containing protein 3 and galectin-3, were confirmed by Western blot and immunohistochemistry in both rat and human HCC samples. We showed that EH domain-containing protein 3 is significantly down-regulated in TECs, but galectin-3 is up-regulated. We propose possible roles of these two proteins in tumor vessel development in HCC.

Volume 42 number 3 FEBS LETTERS June 1974 HYBRIDIZATION STUDIES WITH RNA AND DNA ISOLATED FROM EUGLENA GRACILIS CHLOROPLASTS AND MITOCHONDRIA Edwin J. CROUSE*, Jobst P. VANDREY** and Erhard STUTZ* Department of Biological Sciences, Northwestern University, Evanston, H 1. 60201, USA Received 26 March 1974 1. Introduction Euglena gracilis when grown in light contains chlor- oplasts and mitochondria. Both types of organelles contain their own set of duplex DNA [1-3] which re- plicate at a different pace than the nuclear DNA [4,5] does. These organelles also are equipped with functio- nal 70 S ribosomes containing 23 S, 16 S rRNA [6]. Organellar rRNA is considered to be a transcript of the respective organellar DNA. Euglena chloroplast DNA (chDNA) was shown to code for 23 S, 16 S rRNA [7-9]. The number of cistrons per chromosome, how- ever, is still in doubt and could be between one and three [9-11]. We have reported that the hybridization capacity of chDNA for 23 S, 16 S chloroplast RNA va- ries with the amount of a 'heavy' chDNA component, isolated from highly purified chloroplasts. This 'heavy' component, depending on the average fragment size, has a buoyant density of 1.692 to 1.701 g/cc (peak densi- ties) [12]. Since Euglena mitochondrial DNA (mtDNA) was reported to have densities in the range of 1.690 to 1.692 g/cc [1-3], the 'heavy' chDNA might be, at least partly, of mitochondrial origin. In order to clari- fy this point, we found it necessary to isolate and cha racterize mtDNA from Euglena gracilis and in particu- lar to measure the affinity of chloroplast rRNA for this DNA. Further, it seemed necessary to measure as well the hybridization capacity of mtDNA towards its own major RNA components since such an experi- ment was still lacking. Mitochondrial rRNA hybridizes with mtDNA to 3.7%, while only to 0.16% with chDNA under identi- cal incubation conditions. Ctdoroplast rRNA hybridi- zes with total chDNA, containing 30% of 1.692 g/cc DNA to 2.9%, while only 0,12% with mtDNA. We conclude that the 1.692 g/cc DNA found in chloro- plast DNA preparations is not of mitochondrial origin. 2. Materials and methods 2.1. Buffers Buffer I: 0.05 M Tris-HC1 (pH 7.9), 0.I KC1, 0.01 M MgC12, 5 mM 2-mercaptoethanol. Buffer II: 0.01 M Tris-HC1 (pH 7.9), 0.1 M KC1, 0.01 M MgC12. Buffer II1:0.1 M Tris-HC1 (pH 7.9), 2.5% sodium do decyl sulfate, 0.01 MEDTA. Buffer IV: 0.1 M NaC1, 0.05 M Na2 HPO4,0.05 M NaH2PO4,0.1 mM EDTA (pH 6.8). Buffer V: 0.04 M Tris-acetate (pH 7.9), 0.1 M sodium acetate, 1 mM magnesium acetate. New addresses. * Laboratoire de physiologie v~g6table et biochimie, Istitut de Botanique, Universite de Neuchfitel: rue de Chantemerle 18, CH-2000 NEUCHATEL, Suisse. ** Department of Pathology St. Louis University School of Medicine, St. Louis, Mo. 63104, USA. 2.2. Isolation and purification of mitochondrial RNA For the isolation of mitochondria, we used the aplastidic strain W3BUL (a gift from J. A. Schiff, Brandeis University). Cells were grown in a hetero- trophic medium [13] modified by using the trace ele- ment combination of Cramer and Meyers [ 14]. W3 BUI_ 262 North-Holland Publishing Company - Amsterdam

A new enzyme, L-tartrate decarboxylase, was found in cells of Pseudomonas sp. group Ve-2. The enzyme was purified to homogeneity and characterized. The enzyme requires K+, Mg2+, and NAD+ for L-tartrate decarboxylation. The dependence of the enzymatic decarboxylation on NAD+ suggests that the decarboxylation involves redox reactions of the substrate. The enzyme catalyzes NAD(+)-linked oxidative decarboxylation of D-malate as well. The enzyme is composed of four subunits with identical molecular weight (Mr 40,000). The apparent Michaelis constants for L-tartrate and NAD+ are 1.1 mM, respectively. The cofactor requirements and the physical properties of the enzyme were similar to those of L-tartrate dehydrogenase-D-malate dehydrogenase from Rhodopseudomonas sphaeroides, and tartrate dehydrogenase from P. putida.

A simple method for dissociating photoreceptor units from Rhodocyclus gelatinosus is described. Incubation of a chromatophore extract (Agalidis, I., Rivas, E. and Reiss-Husson, F. (1990) Photosynth. Res. 23, 249-255) at 4 degrees C with decyltetraoxyethylene and octyl-beta-D-thioglucopyranoside, followed by temperature-induced phase separation at 20 degrees C, led to the formation of three phases: a pellet composed of pure B875 antenna; an oily layer containing cytochrome c and other proteins; a detergent-poor supernatant containing crude reaction centers. This method provided a first step towards further purification of reaction centers and B875 antenna, respectively.

1. After a brief survey of the basic affinity electrophoresis concepts, the usual ways for preparing affinity electrophoresis ligands are examined. 2. Then results obtained on cholinesterases are reviewed. This section includes (a) structural and functional investigations on anionic sites, i.e., study of ligand-induced conformational change, organophosphate-induced “aging,” genetic variants, and active-site topology; and (b) characterization of cholinesterase conjugates (hybrid proteins) and glycoinositol phospholipid-anchored cholinesterases. 3. The future prospects of affinity electrophoresis, e.g., investigations on the esteratic site and exploration of the carbohydrate moiety, are emphasized in the concluding section.

A proteolytic enzyme, curcain, has been extracted from the latex of Jatropha curcas Linn. The enzyme was purified by chromatography on carboxymethyl cellulose and gel filtration on Sephadex G-200. The homogeneity of protein associated with curcain was established by non-denatured polyacrylamide gel electrophoresis using a discontinuous buffer system. The molecular weight of curcain was estimated by Sephadex G-100 gel filtration using a calibration curve of standard proteins to be around 22000 daltons.

The expression of the protein IIIa gene from human adenovirus type 2 (Ad2) in Escherichia coli has been described previously (M. Cuillel, M. Milleville, and J. C. D'Halluin, 1987, Gene 55, 295-301). The same construct has now been used to express a protein IIIa gene from an Ad2 mutant ts 112 whose functional mutation occurs in this gene. The mutant virus is defective at nonpermissive temperatures in the latest stage of virus maturation. Both the wild-type and ts 112 recombinant proteins are produced in E. coli in an insoluble form, but are readily solubilized in urea. They have the same molecular weight in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), they sediment as a monomeric species in sucrose gradient centrifugation, and proteolytic digestion reveals a similar pattern for both proteins. Hydrodynamic studies and electron microscopy show that both proteins have an elongated shape, which can be approximated to a cylinder of 20 nm in length and 2.8 nm in diameter. The only well-established difference between the mutant and the wild-type recombinant protein is the higher solubility of the mutant.

Twenty-five (25) electrophoretic bands with esterase activity were distinguished in supernatants of cauda epididymidis of DBA/2J mice. Twenty (20) of these were assigned to 10 genetically defined esterases (ES-1, ES-2, ES-3, ES-6, ES-7, ES-11, ES-14, ES-17, ES-19, ES-22) which were already known from investigations of other mouse tissues. Furthermore, ES-10 was identified in cauda supernatants after isoelectric focussing. A hitherto genetically undefined esterase was assigned to locus Es-28 which was expressed solely in the epididymis. Three phenotypes were distinguished: ES-28A was present in the majority of the inbred strains examined. ES-28B was observed in AKR/Han mice and ES-28C was found in SEG/1 mice.

Male Sprague-Dawley rats were given single i.p. injections of 1,3-bis(2-chloroethyl)-1-Nitrosourea (BCNU) to investigate changes in hepatic microsomal cytochrome P-450 content and metabolic activity. On day 14 after treatment (20 mg/kg), cytochrome P-450 content had decreased by approximately 25% and ethylmorphine N-demethylase activity (nmol product/nmol P-450/min) had decreased by 36%. In contrast, ethylmorphine O-deethylase and 7-ethoxycoumarin O-deethylase activities were not significantly decreased by BCNU treatment. Hepatic delta-aminolevulinic acid synthetase activity was only 60% of control values, and microsomal heme oxygenase activity was slightly but not statistically elevated. Cytochrome P-450 content in control and BCNU-treated rats increased in a similar manner after phenobarbital or beta-naphthoflavone induction. Electrophoretic analysis of cytochrome P-450 proteins isolated from hepatic endoplasmic reticular membranes of treated and control rats suggested that alterations in these proteins occurred in BCNU-treated rats. These changes in cytochrome P-450 content and activity are very similar to those reported in isolated systems exposed to bile acids or in rats with experimentally produced cholestasis. BCNU has been shown to produce cholestasis, which precedes its effects on microsomal mixed-function oxygenase activity. Thus, the delayed effects of BCNU on microsomal drug metabolism are probably secondary to its interference with bile formation.

A simple and practical 6.8-cm-diameter (36.30-cm2 cross-sectional-area) preparative disk gel electrophoresis device, based on the design of M. Hayakawa et al. (Anal. Biochem. 288 (2001) 168), in which the elution buffer is driven by an electroosmotic buffer flow through the membrane into the elution chamber from the anode chamber was constructed. We have found that the dialysis membranes employed provide suitable flow rates for the elution buffer, similar to those of an earlier 3.6-cm-diameter device, resulting in the prevention of excess eluate dilution. The efficiency of this device was demonstrated by the fractionation of a bovine serum albumin (BSA) Cohn V fraction into monomer, dimer, and oligomer components using nondenaturing polyacrylamide gel electrophoresis (native-PAGE). The maximum protein concentration of the eluate achieved was 133 mg/ml of BSA monomer, which required a dilution of the eluate for subsequent analytical PAGE performance. As a practical example, the two-dimensional fractionation of soluble dipeptidyl peptidase IV (sDPP IV) from 50 ml fetal bovine serum (3.20 g protein) per gel is presented. The sDPP IV enzyme protein was recovered in a relatively short time, utilizing a 6.5% T native-PAGE and subsequential sodium dodecyl sulfate–PAGE system. This device enhances the possibility of continuous electrophoretic fractionation of complex protein mixtures on a preparative scale.

A d -galactose binding lectin (SLL-2) was isolated from Sinularia lochmodes, an octocoral, by a combination of affinity chromatography on acid-treated agarose and FPLC on Superdex 200. SLL-2 agglutinated rabbit and horse erythrocytes while SLL-1, a minor component, reacted only with rabbit erythrocytes. SLL-2 is a glycoprotein with a molecular mass of 122 kDa and is composed of eight identical subunits (15 kDa). The sequence of the amino terminal region of SLL-2 did not show any apparent homology to the sequences of other animal and plant lectins. d -Galactose, N-acetyl- d -galactosamine, lactose, and melibiose were moderate inhibitors to the agglutination of rabbit erythrocytes. In contrast, horse erythrocytes were much more susceptible to agglutination by SLL-2, which was inhibited by sugars and glycoproteins such as d -galactose, N-acetyl- d -galactosamine, lactose, melibiose, and porcine stomach mucin. SLL-2 showed considerable tolerance to heating and kept its activity after heating at 80°C for 60 min. In immuno-histochemical studies using an anti-SLL-2 antiserum and protein A gold conjugate, SLL-2 was found to be present in high amounts in the nematocysts. SLL-2 was also detected on the surface of symbiotic dinoflagellate, Symbiodinium sp. cells irrespective whether they were surrounded with or without host cells. These observations suggest the presence of lectin-mediated interaction between symbiotic dinoflagellates and S. lochmodes.

On polyacrylamide gel (PAG) disc electrophoresis of serum lipoproteins, the band(s) which migrates between pre beta- and beta-lipoproteins was more frequently observed in hyperlipidemics with diabetes mellitus (73%), than in those without diabetes mellitus (37%) (P0.01). Those bands were seen at three positions between pre beta- and beta-lipoproteins. A higher incidence of coronary artery disease (CAD) was observed in patients with midband as a shoulder of beta-lipoproteins (44%), than in those without midband (11%) (P0.05) after the matching of other risk factors. These results suggest that midband as a shoulder of beta-lipoproteins may be a risk factor for CAD in diabetes mellitus with hyperlipidemia.

Here we report that soluble CD14 isolated from the urine of nephrotic patients (uCD14) contains a potent cytokine inducing activity. CD14 derived from urine appeared to consist of two major polypeptides of about 54 and 48 kDa. In uCD14 isolated from three different nephrotic patients the cytokine-inducing activity appeared to co-migrate with the 48-kDa polypeptide which upon sequencing had the same N-terminal sequence as native CD14. Treatment of human monocytes and the human astrocytoma cell line U373 with uCD14 resulted in a strong secretion of tumor necrosis factor (TNF) and interleukin-6, respectively. The cytokine-inducing activity of the uCD14 preparations was unaffected by the absence of serum. This is in contrast to the activation of human monocytes and U373 cells by lipopolysaccharide (LPS) which is highly dependent on the presence of serum. The cytokine-inducing activity was not affected by LPS-binding protein (LBP) or polyclonal rabbit antibodies against LBP. The TNF-inducing activity of uCD14 was also heat labile in contrast to the cytokine-inducing activity of LPS, which was relatively heat resistant. The results suggest that CD14 may exist in at least two forms of which one is involved in cytokine induction.

Purified membrane vesicles were prepared from Chinese Hamster lung fibroblasts expressing high amounts of P-glycoprotein (P-gp), which is responsible for the multidrug resistance. P-gp ATPase activity, characterized in the presence or absence of verapamil, had a Michaelian behavior for its MgATP dependence. Thus only one MgATP molecule should be sufficient for the catalytic cycle. With increasing verapamil concentrations, a bell-shape curve was observed for ATPase activity, with half-activation and -inhibition concentrations of 1.2 μM and 490 μM, respectively. No cooperativity for verapamil was detected. These results strongly suggest that P-gp functions as an active transporter, with a coupling stoichiometry of one MgATP molecule hydrolysed for one verapamil molecule transported.

Maleylacetate reductase (EC 1.3.1.32) plays a major role in the degradation of chloroaromatic compounds by channeling maleylacetate and some of its substituted derivatives into the 3-oxoadipate pathway. The enzyme was purified to apparent homogeneity from an extract of 2,4-dichlorophenoxyacetate (2,4-D)-grown cells of Alcaligenes eutrophus JMP134. Maleylacetate reductase appears to be a dimer of two identical subunits of 35 kDa. The pI was determined to be at pH 5.4. There was no indication of a flavin prosthetic group. The enzyme was inactivated by p-chloromercuribenzoate but not by EDTA, 1,10-phenanthroline, or dithiothreitol. Maleylacetate and 2-chloromaleylacetate were converted with similar efficiencies (with NADH as cosubstrate, Km = 31 microM for each substrate and kcat = 8,785 and 7,280/min, respectively). NADH was preferred to NADPH as the cosubstrate. Upon reduction of 2-chloramaleylacetate by the purified enzyme, chloride was liberated and the resulting maleylacetate was further reduced by a second NADH. These results and the kinetic parameters suggest that the maleylacetate reductase is sufficient to channel the 2,4-D degradation intermediate 2-chloromaleylacetate into the 3-oxoadipate pathway. In a data base search the NH2-terminal sequence of maleylacetate reductase was found to be most similar to that of TfdF, a pJP4-encoded protein of as-yet-unknown function in 2,4-D degradation.

A capillary permeability-increasing enzyme-2 was purified from the venom of A. caliginosus by ion-exchange chromatography and gel filtration on Sephadex G-100. By this procedure, 3.1 mg of purified enzyme was obtained from 4 g of the venom. The mol. wt of the purified enzyme was estimated to be approximately 44,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme hydrolyzed N-alpha-tosyl-L-arginine methylester with a specific activity of 56.5 units/mg of protein, and did not show any caseinolytic, clotting or bradykinin-releasing activity. When 13.9 micrograms of the enzyme was injected into the depilated skin on the back of a rabbit, capillary permeability was increased.

To detect von Willebrand factor multimers in plasma samples and factor VIII concentrates, a vertical discontinuous SDS electrophoresis was developed. A vacuum blotting system allowed to improve the transfer to the nitrocellulose membrane. The visualization of the separated multimers was sensitized by applying an alkaline phosphatase anti-alkaline phosphatase staining technique. The reported method clearly shows structural abnormalities of von Willebrand factor and deficiency of high multimers, the vacuum transfer is efficient and the sensitivity of the staining system is very high.

The uptake of serum albumin by maturation-stage rodent enamel and the resulting effects on the growth of enamel crystallites were investigated in vitro. Albumin uptake was demonstrated by means of gel electrophoresis and confirmed by Western blotting with use of monoclonal antibodies. Measurement of crystal size was carried out by direct TEM measurement of enamel crystallite outlines after incubations in metastable solutions of calcium phosphate. The ability of endogenous enamel enzymes to degrade albumin was investigated by substrate-specific zymography. The results showed that albumin could be taken up by maturation-stage enamel and produce inhibition of crystallite growth. There was no detectable proteolytic activity in the enamel against albumin substrate, which suggests that albumin entering enamel by extravasation in vivo may produce incomplete tissue maturation, resulting in a white, opaque appearance on eruption.

SummaryDiscontinuous PAGE at alkaline and acid pH, polyacrylamide gel isoelectric focusing, two dimensional electrophoresis and immunoblotting have been used to study the heterogeneity of sheep caseins. Three main phenotypes were selected either because of mobility at alkaline and acid pH of the individual αs components or because of their relative intensity. On electrophoresis at alkaline pH, one phenotype showed two distinct bands of lower electrophoretic mobility than β,- and β2-caseins. A detailed study of these components using immunospecific detection with β-casein antiserum showed that these minor components of ovine casein may have a polypeptide chain similar to that of β1- and β2-caseins. Complete electrophoretic patterns of the casein components in some individual milks are also presented.