The Chemistry of Allergens. VIII. Isolation and Properties of an Active Proteinpolysaccharidic Fraction, CB-1A, from Castor Beans<sup>1a, b</sup>

Spies, Joseph R.; Coulson, E. J.

doi:10.1021/ja01249a020

Cited by 50 publications

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“…After electrophoresis in SDS-polyacrylamide gels of high density (15%), the proteins of the isolated protein bodies of castor bean were resolved into two major groups (Fig. la) (9,10). The isolated 2S albumins and the CB-IA had similar electrophoretic patterns although the amount of the lower band was reduced in the CB-IA (Fig.…”

Section: Resultsmentioning

confidence: 99%

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Youle¹,

Huang²

1978

Plant Physiol.

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The well characterized castor bean (Rhscm is L*) allergens were Identdfied as the low molecular weight albmin storae (12,14). A careful literature survey of the known properties of the castor bean allergens and of the albumin storage proteins revealed some similarities, such as the amino acid composition, the low mol wt, and the solubility in water. In this communication, we present evidence that the castor bean allergens are indeed the albumin storage proteins in the protein bodies of castor bean. MATERIALS AND METHODSPreparation of Protein Fractios. The matrix proteins, the lectins (4-6S albumins), the I IS globulins, the 2S4 albumins, and the total proteins of castor bean (Ricinus communis var. Hale) were purified as described (14, 15 at room temperature for 30 min, or at 100 C for 2 min in 0.01 M Tris-HCl (pH 6.8) and 1% SDS. Mol wt standards were used as described (14).Immun_okglcal Techniques. Each 2.27-kg New Zealand White rabbit was injected in the footpads and the back subcutaneously with 2 mg of CB-IA mixed in complete Freund's adjuvant. After I week, they were injected in the back subcutaneously with 2 mg of CB-IA mixed in incomplete Freund's adjuvant. They were bled 10 days after the second injection and the -y-globulins were purified by the procedure of Kendall (3). The y-globulins were dialyzed against 0.01 M Na-phosphate buffer (pH 7.5), 0.05'M NaCl, and after centrifugation at 10,000g for 30 min, the supernatant fraction was retained. y-Globulins were also prepared by an identical procedure from untreated rabbits as controls. Double diffusion tests were performed according to Ouchterlony (6). Equal amounts of protein samples to be tested were applied to each well except that twice the amount of the organelle matrix proteins and the total castor bean proteins were used.In the electrophoretic analysis of the antigenicity of the 2S albumins, 0.08 mg of the 2S albumins were mixed with 200 ,ul of 10 mm Na-phosphate buffer (pH 7.5) and 15 mm NaCl, and 200id of either anti-CB-lA y-globulins or unsensitized y-globulins.The solution was incubated at 23 C for 1 hr and then at 4 C for 16 hr. After centrifugation at 10,000g for 30 min, the supernatant fraction was lyophilized to dryness. The lyophilized powder was dissolved in 100 ,ul of 10 mm Na-phosphate buffer (pH 7.5) and 15 mm NaCl and 100 p1 of similar y-globulins. After similar incubation and centrifugation, the supernatant fractions were analyzed by SDS-polyacrylamide gel electrophoresis. RESULTSThe properties of the well characterized castor bean allergens, the CB-IA fraction, and our purified 2S albumin storage proteins were compared. SDS-Polyacrylamlde Gel Electropboresis and Mol Wt. After electrophoresis in SDS-polyacrylamide gels of high density (15%), the proteins of the isolated protein bodies of castor bean were resolved into two major groups (Fig. la) (Fig. la) and their estimated sedimentation value (15) are similar to those of the castor bean allergens reported earlier (9, 10). The isolated 2S albumins and the CB-IA had similar ...

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Section: Resultsmentioning

confidence: 99%

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Youle¹,

Huang²

1978

Plant Physiol.

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“…Nevertheless, we do not rule out the possibility that ricinosomes may be sites of storage for protein, or protein complex, peculiar to castor bean. Castor beans contain a unique protein called ricin (17,24,25), which is present to some extent in all portions of the plant (17). So far as we can determine, nothing has been published regarding the intracellular location of ricin even though it may be present in relatively large amounts in some tissues.…”

Section: Discussionmentioning

confidence: 99%

Studies on Seeds

Mollenhauer

Totten

1970

Plant Physiol.

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Glyoxysomes, a form of microbody, are present in castor bean endosperm during the first 8 days of seed germination.They have a "typical" microbody form and are shown histochemically to contain catalase. The catalase label is distributed throughout the microbody and is not an exclusive feature of the crystalline or amorphous core.Castor bean endosperm contain a second cytosome, only slightly larger than the glyoxysomes, which is bound by a rough-surfaced membrane and which does not label for catalase. We have not observed these cytosomes in other tissues, suggesting that they may have a specific cellular function characteristic of castor bean endosperm.Glyoxysomes are a morphologically and biochemically unique component of many cells (3,4,12,13). They are a form of microbody containing enzymes of the glyoxylate cycle. Like peroxisomes and other types of microbodies, they also contain catalase and glycolic acid oxidase (4).Microbodies are present in castor bean endosperm throughout seed development, dormancy, and germination, but enzymes of the glyoxylate cycle do not reach maximal activity until the 4th or 5th day of seed germination (5, 12). There is no indication from ultrastructural studies that microbodies disappear, reform, or change significantly in number, in relation to the appearance of enzymes of the glyoxylate cycle. It is assumed, therefore, that glyoxysomes are somehow evolved from microbodies by enzyme activation or by gradual replacement of existing microbodies with those of the glyoxysome type.We have isolated microbodies, as well as glyoxysomes, from a number of seeds and have found that the glyoxysome fractions from castor bean endosperm sometimes contain a contaminating particle that is similar in size to glyoxysomes. Studies in vivo of germinating castor bean confirm that a microbody-like particle does exist in the endosperm and is most easily recognized when enzymes of the glyoxylate cycle are active. We thought initially that these new particles might be related, or equivalent, to glyoxysomes and, therefore, initiated these studies. This report describes these new particles and compares them structurally and histochemically with microbodies. 2"Studies on Seeds, I through IV" will appear in the Journal of Cell Biology. MATERIALS AND METHODSCastor bean (Hale variety and Baker variety 296 obtained from The Baker Castor Oil Co., La Mesa, Calif.) seeds were scarified and the seeds were soaked in water for 2 to 24 hr. The seeds were germinated for 3 to 8 days in the dark at 28 C in vermiculite moistened with distilled water.Slices of endosperm were prefixed in a mixture of 2% glutaraldehyde-2 % paraformaldehyde in 0.05 M collidine buffer containing 0.06 M sucrose, pH 7.3 to 7.4 for 2 hr, cold. After prefixation, the tissues were rinsed in six 5-to 10-min changes of buffer. Unless otherwise indicated, the buffer used was 0.05 M collidine with 0.06 M sucrose, pH 7.3 to 7.4. The tissues were either postfixed (see below) or allowed to warm to room temperature in the last buffer rinse and then ...

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“…This small heat-stable protein appeared to be similar or related to castor bean allergens. Crude allergens were prepared by extracting the castor beans with tris and phosphate buffers and the CB-1A classical allergen was prepared by the method of Spies and Coulson (12). The CB-lA allergen is essentially a water extract of castor beans with a series of alcohol precipitations and a lead acetate precipitation step.…”

Section: Separation Of Two Cofactorsmentioning

confidence: 99%

Acid lipase of the castor bean

Ory

1969

Lipids

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The acid lipase of the castor bean is present in the dormant seed. It is extracted from the fat pad obtained by centrifuging a macerate of the seed in pH 7.0 buffer containing cysteine and ethylene diaminetetraacetic acid. The pH optimum of the enzyme is 4.2; it is rather heat‐stable, and is inhibited by mercurials and sulfhydryl reagents. Maximum hydrolysis of saturated triglycerides occurs with fatty acids of chain length C4 to C8; unsaturated C18 triglycerides are hydrolyzed at a slightly lower rate. This lipase is a three‐component system consisting of the apoenzyme, a lipid cofactor (a cyclic tetramer of ricinoleic acid), and a protein activator (a small, heat‐stable glycoprotein which appears to be related to some of the castor allergens). Maximum lipolysis requires all three components. Lipase activity is associated with the spherosomes, the subcellular site of oil storage in the endosperm.

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The Chemistry of Allergens. VIII. Isolation and Properties of an Active Proteinpolysaccharidic Fraction, CB-1A, from Castor Beans^{1a, b}

Cited by 50 publications

References 0 publications

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Studies on Seeds

Acid lipase of the castor bean

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The Chemistry of Allergens. VIII. Isolation and Properties of an Active Proteinpolysaccharidic Fraction, CB-1A, from Castor Beans1a, b

Cited by 50 publications

References 0 publications

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Evidence That the Castor Bean Allergens Are the Albumin Storage Proteins in the Protein Bodies of Castor Bean

Studies on Seeds

Acid lipase of the castor bean

Contact Info

Product

Resources

About

The Chemistry of Allergens. VIII. Isolation and Properties of an Active Proteinpolysaccharidic Fraction, CB-1A, from Castor Beans^{1a, b}