Accessibility of tryptophan residues in immunoglobulin M as an index of its conformational changeability.

Demonstrated herein is the possibility of using the accessibility of tryptophan (Trp) residues in immunoglobulin M (IgM) upon modification with Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) as an index of the conformational changeability of IgM. Of fourteen Trp's in the native IgM (per HL-region) only one appeared to be most accessible, evidently Trp312 in the mu-chain. Irreversible acidic and thermal conformational transitions in IgM increase the number of accessible Trp's approximately two-fold. Following partial enzymatic deglycosylation of IgM, deep scission of mannose in particular, all Trp's become inaccessible. Modification of the most accessible Trp increases 2-3 fold the number of tyrosine residues readily accessible upon nitration with tetranitromethane. Modification of four trp's using spin-label method data causes a sharp reduction of the mobility of the C mu 3 domain and a simultaneous decrease in the solubility of modified IgM.

[Accessibility of tryptophan residues in immunoglobulin M molecule as an indicator of its conformational variability]. / Dostupnost' ostatkov triptofana v immunoglobuline M kak pokazatel' ego konformatsionnoi izmenchivosti.

The accessibility of tryptophan residues in immunoglobulin M to modification with the Koshland reagent (2-hydroxy-5-nitrobenzyl bromide) was used as an indicator of its conformational variability. Of 14 tryptophan residues (per HL-fragment) in the native IgM, only one (presumably Trp312 in the mu-chain) was the most accessible. Irreversible acid- or temperature-induced conformational changes of IgM increased almost 2-fold the number of accessible tryptophan residues. After partial enzymatic deglycosylation of IgM (especially by an intense splitting of mannose), all tryptophan residues became inaccessible. Modification of the most accessible tryptophan residue increased 2- to 3-fold the number of tyrosine residues accessible to nitration with tetranitromethane. Using the spin label method, it was demonstrated that modification of four tryptophan residues in IgM considerably decreased the mobility of the Cmu 3 domain together with an essential drop in. the solubility of the modified IgM.

[Various research results on the role of carbohydrate groups in macroimmunoglobulins]. / Nekotorye itogi issledovaniia roli uglevodnykh grupp v makroimmunoglobulinakh.

Evidence on the role of carbohydrates in the structure and function of macroimmunoglobulin (IgM) was obtained. The principal functions are the increase of hydrophilicity and preservation of a definite conformation of the protein molecule which, in its turn, is necessary for the implementation of its biological function. The protection of tryptophan residues at the molecule surface against the contacts with the surrounding water is conditioned by the above properties. The functions of each of the five carbohydrate groups in the heavy chains of IgM are reviewed.

[Changes in the immunoglobulin M conformation in media with different degrees of acidity]. / Izmenenie prostranstvennoi konformatsii makroimmunoglobulinov pod vliianiem razlichnoi kislotnosti sredy.

The pH-dependent conformational changes in immunoglobulin M were studied by differential spectrophotometry. It was found that the state of chromophores (tryptophan and tyrosine) which reflects conformational changes of the structure alters stepwise in the course of acidification. The native structure is not restored by neutralization. The recovery of the native structure was obtained only at pH approximately 6.5 of the IgM solution. A possible explanation of concrete conformational transitions during the pH change is proposed. These changes were shown to be similar for IgM and IgG.

[Isolation of thyroglobulin from the bovine thyroid free of endogenous proteolytic activity]. / Vydelenie svobodnogo ot éndogennoi proteoliticheskoi aktivnosti tireoglobulina shchitovidnoi zhelezy krupnogo rogatogo skota.

Thyroglobulin was isolated from the cattle thyroid gland by chromatography on Sephadex G-200 and Sepharose 4B. It was found homogeneous according to disc electrophoresis (pH 8.6) and analytical ultracentrifugation. However, gel filtration of thyroglobulin incubated at 37 degrees through Sepharose 6B revealed that it contained proteinases of the serine type. The conditions for the proteinases' inhibition were found. The possibility of obtaining thyroglobulin free of the proteinases by means of affinity chromatography is demonstrated.

[Modification of tryptophan residues in immunoglobulin M by 2-hydroxy-5-nitrobenzyl bromide]. / Modifikatsiia ostatkov triptofana v immunoglobuline M 2-oksi-5-nitrobenzilbromidom.

The modification of tryptophan residues in monoclonal immunoglobulin M (IgM) by 2-hydroxy-5-nitrobenzyl bromide (RK) was studied at pH 2.0-2.85 and 7.0 and a RK to tryptophan molar ratio (K) from 1 to 40. At pH 2.85, the number of RK residues bound to IgM (N) in account to one HL-fragments does not exceed 10 (the HL-fragment of IgM contains 14 tryptophan residues); the plot of N vs K reaches a plateau at K greater than 20. When the pH is lowered to approximately 2, N rises to approximately 15, but the plateau is not reached. At pH 7.0, the modified IgM with N greater than 1 gives a sediment, while the product with N approximately equal to 1 remains in solution. Evidently, the latter contains the most accessible tryptophan residue (calculated per one HL-fragment). This residue was found to be one of the three residues localized in the C mu 2-domain and the adjoining N-terminal part. The possibility of multiple modification of tryptophan residues during the RK interaction with IgM in acid medium at high values of K is discussed.

[The role of carbohydrate groups in IgM. VIII. The effect of treatment of IgM with acids on the subsequent action of glycosidases and endogenous proteinases]. / Rol' uglevodnykh grupp v immunoglobulinakh M. VIII. Vliianie kislotnoi obrabotki IgM na posleduiushchee deistvie na nego glikozidaz i éndogennykh proteinaz.

Exposure of IgM to acidic medium (pH approximately 3, 30 min, at 20 degrees) and subsequent returning to neutral conditions leads to irreversible changes in the state of the molecule. This results in the loss of IgM accessibility for the action of glycosidases and, at the same time, makes it more susceptible to the action of proteinases. Both effects are thought to be due to an irreversible conformational rearrangement of IgM in acidic medium.

[Study of conformational changes in immunoglobulin M using the method of differential spectroscopy]. / Issledovanie konformatsionnykh izmenenii immunoglobulinov M metodami differentsial'noi spektroskopii.

Using differential and solvent-perturbation spectrophotometry, the nature of conformational changes in immunoglobulin M (IgM) in different regimens was investigated. The quantities of tryptophan and tyrosine chromophores exposed on the surface of the molecule and screened, were evaluated. The changes in pH (7.8----2.0) of the surrounding medium and splitting of carbohydrate groups from IgM were shown to cause opposite effects, i. e., a "blue shift" of the spectrum and exposure of new chromophores by acidification, and a "red shift" and screening of chromophores by splitting of carbohydrate groups. The experimental results agree well with the previously made assumption on the differences in the spatial conformational changes in the IgM molecule under effects of pH of the surrounding medium and the loss of carbohydrate groups. Analysis of the spectral characteristics of some free Fab- and (Fc)5-fragments derived from the IgM molecules allowed a specification of the changes occurring in different parts of the whole molecule. The main conformational changes after acidification occur in the (Fc)5-fragment responsible for the effector function of IgM.

[The role of thyroglobulin carbohydrates in its hormone-liberating function. Proteolysis of deglycosylated thyroglobulin]. / Rol' uglevodov tireoglobulina v ego gormonoobrazuiushchei funktsii. Proteinoliz deglikolizirovannogo tireoglobulina.

The proteolysis of thyroglobulin was performed after partial cleavage of its carbohydrate moiety. It was demonstrated that enzymatic removal of some thyroglobulin sialic acids results in increasing its resistance to proteolysis. Apparently, sialic acids are essential for maintaining an optimal thyroglobulin conformation for the action of proteases at hormonal biosynthesis from the reserve form.

[Immunogenicity and thymus-dependence of the polymerized alpha-toxoid of Clostridium perfringens]. / Immunogennost' i timuszavisimost' polimerizovannogo alpha-anatoksina Clostridium perfringens.

Polymeric alpha-toxoid with a molecular weight of 450 000--600 000 was obtained by condensation of alpha-toxoid of Cl. perfringens, type A, with glutaric aldehyde. Experiments on guinea pigs showed that in the adsorbed preparations the immunogenic properties of both monomeric and polymeric alpha-toxoids are practically identical. The primary immune response after the immunization with nonadsorbed antigens was 3 times greater than that induced by the monomer. Polymerization of alpha-toxoid failed to change its thymus dependency.

[Isolation and characterization of beta-D-galactosidase from gastric juice]. / Vydelenie i kharakteristika beta-D-galaktozidazy iz kishechnogo soka krupnogo rogatogo skota.

A method for beta-D-galactosidase isolation from cattle gastric juice has been developed. Gastric juice mucus was removed by and addition of equimolar amounts of Na2HPO4 and CaCl2. The removal of proteases and other proteins was achieved by the treatment with resins KB-51X2 and AN-22. The resulting preparation had specific activity of 0.14 units per mg of protein. Gel filtration on Sepharose 4B led to an increase of specific activity of the preparation up to 0,4 units per mg of protein. Some properties of the beta-D-galactosidase obtained were compared to relation of lactose and o-nitrophenyl-beta-D-galactoside (pH optima, temperature optimym and thermostability).

[Anionic center of porcine pancreatic phospholipase A2]. / Anionnyi tsentr pankreaticheskoi fosfolipazy A2 svin'i

The interaction between porcine pancreatic phospholipase A2 and low-molecular fragments of its substrate -- lecithine was studied using gel-diffusion of the enzyme in lecithin-agarose plates. When the inhibitor was added, a decrease in the magnitude of cleared areas (l/l0) around the depots filled with enzyme solution was observed. A marked decrease in l/l0 in the presence of alpha- and beta-glycerophosphates supported the statement that the cathionic center is a part of the enzyme active site SII. The potent inhibition of phospholipase activity in the presence of phosphocholine, choline, acetylcholine, thiocholine and acylthiocholines suggests the existence of an anionic center SIII in the active site. This suggestion is supported by intensive inhibition of phospholipase activity by certain, aliphatic amines. It was shown that the center is spaced in the direction of the cathionic center. SII. The main contribution to the binding of the cathionic lecithin part ("head") with the anionic center SIII is probably provided by the ion-ionic interactions.

[Study of the composition of killer whale meat as a source of balanced proteins]. / Issledovanie sostava miasa kosatok kak istochnika polnotsennykh belkov

Investigations have shown the proteins of the Orca meat to consist largely of complete water-salt and alkaline-soluble fractions and of an insignificant amount of connective-tissue proteins. The proteins of the Orca meat contain all the amino acids, including essential ones. The lysine and histidine content there in is 1.5 times as high in the proteins of beef. The completeness of the amino acids composition of Orca meat proteins enables it to utilize this raw material for obtaining valuable food products in the form of concentrates or hydrolysates.

[Selectivity of lysozyme oxidation by singlet oxygen]. / O selektivnosti okisleniia lizotsima singletnym kislorodom.

Peptide analysis of tryptic hydrolysates of two lysozyme forms derived from oxidation of lysozyme with singlet oxygen shows that Trp-62, located at the active site, is destroyed. This is confirmed by the protective effect of the substrate (chitin), whose presense practically prevents the oxidation. A possibility of oxidating different tryptophan residues is discussed from the view-point of their availability to the reagent.

[Nature of tryptophan photooxidation products in lysozyme in the presence of methylene blue]. / O kharatere produktov fotookisleniia triptofana v lizotsime v prisutstvii metilenovoi sini

One out of six trytophan residues in two lysozyme modification, obtained under lysozyme photooxidation in the presence of methylene blue, is found to be oxidized to N'-formylkinurenine (in one modification) and to kinurenine (in the other modification). The transition of one modification into another via detaching of N'-formyl group by soft acid hydrolysis has shown that one and the same tryptophan residue is oxidized in both products, Possible mechanism of tryptophan oxidation to the products mentioned is discu-sed on the basis of the hypothesis on signlet mechanism of lysozyme photooxidation in the presence of methylene blue.