Louis Kanarek

N-terminal sequences of the α and β subunits of the lectin from the garden pea (Pisum sativum)

Lectins constitute a group of proteins mainly found in the seeds of a wide variety of plants and in certain invertebrates. These proteins have proven to be useful tools for the study of protein-carbohydrate interactions and the topography of cell surfaces. #en immobilised on Sepharose, lectins may be used for the fractionation of cells and for the isolation of glycoproteins and cell receptors. Certain lectins are able to induce mitogenesis in lymphocytes a phenomenon analogous to the stimulation of immune cells by a specific antigen [l-3] . Although lectins have been extensively studied for their biological properties, few structural data are available to relate their activity to their structure, except for Concanavalin A [4]. In this paper we describe the subunit structure and N-terminal sequences of the oand &subunits of the phytohemagglutinin of the garden pea (Pisum sutivum), a protein with sugar binding properties similar to those of concanavalin A.

Thiourea: The antioxidant of choice for the purification of proteins from phenol-rich plant tissues☆

Metabisulfite, diethyldithiocarbamate, and thiourea are potent phenoloxidase inhibitors commonly used during the extraction of plant proteins. Their effects on several amino acid derivatives and peptides are reported. Important and pH-dependent changes are induced by metabisulfite in the uv-absorption spectrum of N-acetyltryptophanamide but not of N-acetyltyrosinamide. Similar spectral changes are also induced in tryptophanyl-containing peptides. Neither diethyldithiocarbamate nor thiourea modified the spectral properties of tryptophan or tyrosine. Cysteinyl groups are rapidly modified by diethyldithiocarbamate, especially in the lower pH range, but not by metabisulfite or thiourea. Diethyldithiocarbamate as well as metabisulfite interfere with cysteinyl groups. It is concluded that thiourea is the most appropriate reagent for use as a phenoloxidase inhibitor.

The immuno-histochemical localization of lectin in pea seeds (Pisum sativum L.).

The lectin from the garden pea (Pisum sativum L.) has been localized at the ultrastructural level by the unlabeled peroxidase-antiperoxidase procedure of L.A. Sternberger et al. (1970, J. Histochem. Cytochem 18, 315–333) in 24 h imbibed seeds. Upon examination by light microscopy and transmission electron microscopy, the lectin was only found in the protein bodies of cotyledons and embryo axis. Cell walls as well as membraneous fractions were completely devoid of lectin. These results are discussed in relation to the possible physiological function of seed lectins.

Epinephrine and norepinephrine stimulation of adenylate cyclase in bovine retina homogenate: evidence for interaction with the dopamine D1 receptor

Dopamine, epinephrine and norepinephrine provoke dose-dependent stimulation of adenylate cyclase activity in bovine retina homogenates. The stimulatory effect of all three compounds is inhibited by the D1 antagonist SCH 23390 and, in a stereoselective manner by the cis-isomer of flupenthixol. The D2 antagonist haloperidol and alpha-and beta-adrenergic antagonists failed to block the catecholamine stimulation. These results evidence that, in bovine retina, not only dopamine but also epinephrine and norepinephrine interact with dopamine D1 receptors that are stimulatory coupled to an adenylate cyclase system.

Chicken heart fumarase: Its purification and physico-chemical charcterization. A comparison with the enzyme from pig heart

Abstract 1. 1. A simple method is presented for the preparation of large amounts of chicken heart fumarase in high yields. It is based on affinity chromatography. 2. 2. Physico-chemical properties of this enzyme are described and a comparison is made with the already more extensively studied pig heart fumarase.

The purification and physicochemical characterization of maize (Zea mays L.) isocitrate lyase.

A purification scheme is described for the glyoxylate cycle enzyme isocitrate lyase from maize scutella. Purification involves an acetone precipitation and a heat denaturation step, followed by ammonium sulfate precipitation and chromatography on DEAE-cellulose and on blue-Sepharose. The latter step results in the removal of the remaining malate dehydrogenase activity, and of a high molecular mass (62 kDa) but inactive degradation product of isocitrate lyase. Catalase can be completely removed by performing the DEAE-cellulose chromatography in the presence of Triton X-100. Pure isocitrate lyase can be stored without appreciable loss of activity at -70 degrees C in 5 mM triethanolamine buffer containing 6 mM MgCl2, 7 mM 2-mercaptoethanol, and 50% (v/v) glycerol, pH 7.6. Maize isocitrate lyase is a tetrameric protein with a subunit molecular mass of 64 kDa. Purity of the enzyme preparation was demonstrated by polyacrylamide gel electrophoresis in the presence of dodecylsulfate, in acid (pH 3.2) urea and by isoelectric focusing (pI = 5.1). Maize isocitrate lyase is devoid of covalently linked sugar residues. From circular dichroism measurements we estimate that its structure comprises 30% alpha-helical and 15% beta-pleated sheet segments. The enzyme requires Mg2+ ions for activity, and only Mn2+ apparently is able to replace this cation to a certain extent. The kinetics of the isocitrate lyase-catalyzed cleavage reaction were investigated, and the amino acid composition of the maize enzyme was determined. Finally the occurrence of an association between maize isocitrate lyase and catalase was observed. Such a multienzyme complex may be postulated to play a protective role in vivo.

Tumor growth inhibition mediated by trypsin inhibitor or urokinase inhibitors

Abstract When testing in vitro, we often observed that erythrocytes taken from Ehrlich ascites tumor bearing mice displayed enhanced agglutinability by the lectin Concanavalin A, suggesting that protease activity operates in vivo. In several studies, we were able to inhibit Ehrlich ascites tumor growth by the repeated administration of soya bean trypsin inhibitor. Studies of dosages and schedules of treatment showed that for 20,000 initially grafted cells, treatment resulted in 0–70% of long-term survivors and induced a significant increase in mean survival time of treated mice over control mice. For 200,000 grafted tumor cells, 0–40% of long-term survivors were recorded. In a comparative study, we found that different inhibitors of urokinase displayed a similar chemotherapeutic effect against 200,000 inoculated cells. Our results corroborate the idea that a plasminogen activator monitored chain of fibrinolytic and proteolytic activity controls tumor growth and metastasis enzymatically.

The subunit structure and N-terminal sequences of the α- and gb-subunits of the lentil lectin (Lens culinaris)

Lectins are proteins or glycoproteins present in many plants and are usually recognized by their ability to agglutinate erythrocytes. Agglutination of cells is in many cases inhibited by specific sugars, suggesting that the binding is to sugar residues on the cell surface. Very little is known of the mechanism by which the lectin leads to agglutination or cellsurface alteration. In order to understand the relationship between the activity and the structure of lectins, we have undertaken the determination of the amino acid sequence of several of these proteins. The lectin studied in this work is present in seeds of the lentil, Lens culinaris [ 1,2] . The protein has a molecular weight of approximately 49 000 and is composed of two types of subunits with molecular weights of 18 000 and 8000 [3] . The isolation and characterization of this hemagglutinin was described in our previous communication [4]. We report here the N-terminal sequences of the (Yand P-subunits of the lentil lectin. This protein has the same sugar-binding specificity as two other mitogenic lectins, Concanavalin A [5] and pea lectin [6], for which sequence studies were reported previously [7,8]. The comparison of these three proteins reveals surprising homologies.

Antitumour immunoprotection by an immunobacterial lectin approach

Abstract By in vivo screening against lymphoid leukemia L1210 in BDF1 mice and Ehrlich ascites carcinoma in BALB/c mice, we found that a vaccination with a synthetic antigen, built up by the cell wall of Micrococcus lysodeikticus (ATCC 4698) on which we coupled oligosaccharides of poly-N-acetyl-D-glucosamine, was able to protect to a fairly good extent against both tumours in the case of L1210 leukemia, hyperimmunization induced an increase in life span of 75% over controls and long term survivals of 25%; in the case of Ehrlich carcinoma, it induced an increase in life span of 104% and long term survivals of 25%. Under the present conditions, we observed that antibodies to the less immunogenic N-acetyl-D-glucosamine polymer were ineffective. Different antigens, antigen doses and immunization schemes were investigated. This system can be considered as an immunobacterial equivalent of both the lectins wheat germ agglutinin and concanavalin A. Although bacterial vaccines are widely used now as non-specific immunoadjuvants that stimulate the T helper cell function and non-specific macrophage immunity, we present evidence that subpopulations of antibacterial antibodies may be tumour specific.

Successful immunotherapy with micrococcus, BCG or related polysaccharides on L1210 leukaemia after BCNU chemotherapy.

The experiments aimed at evaluating the optimal parameters in the chemo-immunotherapeutic treatment of the L1210 lymphoid leukaemia grafted to [female BALB/c (H2d) X male DBA/2 (H2d)]F1 hybrid mice, hereafter referred to as CDF1 mice. in vitro irradiation of leukaemic ascites cells by X- or gamma-rays and subsequent inoculation in mice showed that optimum immunogenicity is radiation dose-dependent. Grafting mice with 10(7) leukaemic ascites cells irradiated at optimum dose (80 GyX- or gamma-rays) delays mortality of the animals when challenged later with untreated L1210 cells, but is unable to cure mice. By contrast, specific immunoprophylaxis induced by Micrococcus, complement-triggering polysaccharides or BCG and irradiated leukaemic cells was able to protect mice against grafts of 10(4) L1210 cells. The i.p. route was notably superior to the i.v. route. When mice bearing advanced L1210 tumour were treated by chemotherapy (12 mg/kg of BCNU) on Day 6.5 after grafting 10(4) L1210 cells and subsequently treated by immunotherapy, a very high percentage (up to 90%) of mice with 10(8) leukaemic cells could be cured by repeated 1mg injections of bacterium or polysaccharide, and challenge with irradiated leukaemic cells was unnecessary. Because of the high cure rate obtained, the very regular response pattern and the non-pathogenicity, the bacterium Micrococcus lysodeikticus would seem a promising new candidate for chemo-immunotherapeutic antitumour strategies.