[A simple method of isolating monoclonal immunoglobulin M, possessing rheumatoid activity]. / Prostoi metod vydeleniia monoklonal'nogo immunobloulina M, obladaiushchego revmatoidnoi aktivnost'iu.

A simple procedure for obtaining highly purified preparations of native monoclonal (Waldenström's disease) immunoglobulin M possessing a rheumatoid activity (IgM-RF) has been developed. The method is based on the use of affinity chromatography with a new readily available adsorbent (immunoglobulin G-porous glass) and 3 M LiCl in Tris-buffer pH 8.3-8.4 able to induce the dissociation of the IgM-RF-IgG complex. The IgM-RF preparation thus obtained was characterized in terms of amino acid composition (relative to conventional monoclonal IgM), carbohydrate composition and structure of oligosaccharide moieties of a complex type. It was shown that some dissociation conditions for the IgM-RF-IgG complex routinely used to isolate IgM-RF provoke irreversible denaturation of IgM-RF when applied to a preliminarily purified complex.

[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.

[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.

[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 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.

[Carbohydrate component of Penicillium funiculosum dextranase]. / Uglevodnyi komponent dekstranazy Penicillium funiculosum.

The carbohydrate composition of dextranase from Penicillium funiculosum 15, as well as the composition of products of dextran deep hydrolysis by the enzyme were studied. The products are normally used to stabilize the enzyme during its purification. Using the methods available, it was possible to identify only part of strongly bound (adsorbed) carbohydrates. It was found that dextranase from Pen. funiculosum 15 contained two types of carbohydrates strongly bound with protein: adsorbed and covalently bound carbohydrates. A procedure allowing a complete separation of adsorbed carbohydrates was developed. The procedure is based on the use of stabilizing additives of readily separable carbohydrates. The enzyme, which is shown by polyacrylamide gel electrophoresis in the presence of Na-dodecyl sulfate and beta-mercaptoethanol to be homogeneous, consists of 313 amino acid residues, 3 glucosamine residues and residues of mannose, galactose and fucose in the ratio 6:2:1.