Proteolysis of Mitotic Chromosomes Induces Gradual and Anisotropic Decondensation Correlated with a Reduction of Elastic Modulus and Structural Sensitivity to Rarely Cutting Restriction Enzymes

The effect of nonspecific proteolysis on the structure of single isolated mitotic newt chromosomes was studied using chromosome elastic response as an assay. Exposure to either trypsin or proteinase K gradually decondensed and softened chromosomes but without entirely eliminating their elastic response. Analysis of chromosome morphology revealed anisotropic decondensation upon digestion, with length increasing more than width. Prolonged protease treatment resulted only in further swelling of the chromosome without complete dissolution. Mild trypsinization induced sensitivity of chromosome elasticity to five- and six-base-specific restriction enzymes. These results, combined with previous studies of effects of nucleases on mitotic chromosome structure, indicate that mild proteolysis gradually reduces the density of chromatin-constraining elements in the mitotic chromosome, providing evidence consistent with an anisotropically folded “chromatin network” model of mitotic chromosome architecture.

INTERACTION OF PLASMIN WITH ALPHA-2 MACROGLOBULIN (α2 M): EFFECT OF ANTIFIBRINOLYTIC AGENTS

Harpel (Harpel, P.C. (1981) J. Clin. Invest 68, 46-55) reported that levels of α2M-plasmin complexes are elevated in patients receiving urokinase. He found that the distribution of plasmin between the two inhibitors, α2M and α2-plasmin inhibitor (α2PI) is dependent upon whether plasmin is added directly to plasma, or whether plasminogen in plasma is activated to plasmin by urokinase. In order to investigate possible mechanisms regulating the distribution of plasmin between these two inhibitors, a study was initiated to examine the effects of antifibrinolytic agents on the reaction of plasmin with α2M. The kinetics of the reaction were measured by monitoring conformational changes in the inhibitor resulting from exomplex formation. In order to minimize nonspecific proteolysis of the inhibitor by plasmin, the reaction was performed under conditions where the concentration of α2M was greater than that of the enzyme. The reaction between Lys77-plasmin and α2M followed second order kinetics with a rate constant of 1.8 X 105M-1 s-1. This rate was not affected 1 mM EACA or by 10 uM histidine rich glycoprotein (HRG). Further, it was found that the rate of Val442-plasmin was essentially the same as that found for Lys77-plasmin. Therefore, the binding of these ligands to the lysine binding sites of plasmin do not affect the association rate between plasmin and α2M. This is in contrast to the reaction of plasmin with α2-PI, where the binding of ligands to the lysine binding sites of plasmin reduce the rate of the reaction (Petersen & Clerrmensen (1981) Biochem. J. 199, 121-127). The kinetic constants measured predict that under conditions when the lysine binding sites of plasmin are occupied, α2M will effectively compete with α2PI in inhibiting plasmin. Further, these studies inplicate HRG as a molecule capable of regulating the distribution of plasmin between these two inhibitors.