Proteolysis of homologous and heterologous proteins by rabbit spleen extracts

Abstract

The function of antibodies in a biochemical way is not known, though phenomena of proteolysis consecutive to an­tigen antibody reaction in vivo have pointed out, like the intervention of these phenomena in the release of pharmacologic mediators (1), the possible proteolytic proenzime nature of complement components, wich appear to be activated by the union with the antigen antibody complex (2), and the activation by this complex of other proteolytic enzymes (4, 11, 12). We don’t have enough data about the intesity of intracellular pro­ teolysis of a given protein and its relation with its antigenic capacity. So, we were interested in the study of the pro­ teolysis by intracellular proteinases of homologous and heterologous proteins, in order to verify, if there is a difference or not among them. In this paper we present the first results obtained with aqueous rabbit spleen extracts, in connection with its autoproteolytic activity and its proteolytic activity in front of human and rabbit gamma globulin, and human albumin.

The autoproteolysis of rabbit spleen extracts investigated is active from 3 to 6, with a wide optimum of activity (/’H 3,5 to 5). Autoproteolysis at p~EL 6,5 is very small and inappreciable at pH 7 or higher. This curve of autopro­ teolytic activity is shown in fig. 1, which corresponds to the average of 9 duplicate experiments. As we have not seen autoproteolytic activity beyond pH 7, this means that it is produced by the acid proteinases or catepsins, and probably contributing various of them, in account of the wide zone of activity.
In fig. 2, we present the mean curves, corresponding each one to 6 duplicate experiments of the proteolysis by aqueous rabbit spleen extracts of human albumin and human and rabbit gamma globulins. In all three, a sharp increase of proteolysis at 2,5 is observed, with a well delimited optimum of activity at pH 3,0-3,5, which also decreases rapidly, reaching low valúes at p~H. 5. No sig­nificative difference exists between hu­man and gamma globulins (p 0,20-0,30).

That fact favours the idea that no difference exists between the proteolysis homologous and heterologous protein, at least for'gamma globulins. The curve of albumin proteolysis: presents the same shape with a significative difference (p<o,ooi) in its intensity; this may be interpreted as a consequence of a greater or more rapid proteolysis for albumin than for gamma globulin. This observation should be in line with that of McMaster and Kruse (8), which verified that azo-albumin persists intracellularly much less than azo-gamma globulin, and also with the known difference of catabolization in vivo of both proteins, which corresponds to a T% lower for albumin than for gamma globulina; nevertheless this observation must be extended to other albumins, as for example rabbit and bovine albumins in front of the rab­ bit spleen extract.

The data in this paper give us also an indication in the sense that the anti­gen antibody complex probably plays no role in intracellular proteolysis, at least for the proteins investigated and in the conditions of the experiments, in which it take place at an optimal pH of 3,5; at this pH, antigen antibody com­plexes do not form and if they were previously constituted, they dissociate (5, 6).