Extracellular Protein Profile of Bacillus cereus Strains

Extracellular protein production by 16 pathogenic and nonpathogenic strains of Bacillus cereus was determined by ion exchange chromatography, isoelectric focusing, and HPLC through a DEAE column. Most of the bands obtained by electrofocusing appeared at pI from 4.70 to 5.30. The profiles could not be related to pathogenicity. With HPLC, 81% of the strains showed three major proteins at retention time between 2.51 and 4.40 min. This feature is speculated to relate the strains to each other and their pathogenicity.

Enkephalinases

Enkephalins can be degraded by a variety of peptidases. We have chara­terized several membrane-associated brain peptidases in an effort to determine which if any are concerned with the physiological inactivation of synaptically released enkephalin. We have distinguished two carboxyldirected dipeptidylpeptidases, designated enkephalinase A 1 and A 2 , that give rise to the Tyr-Gly-Gly fragment. Both enzymes are physically separable from angiotensin converting enzyme. Regional variations in enkephalinase A 1 activity and opiate receptors are similar. A novel amino-terminal-directed dipeptidylpeptidase, enkephalinase B, which generates Tyr-Gly, has been identified. All of these enzymes as well as aminopeptidase have been solubilized from brain membranes by detergent treatment and have been mutually resolved by DEAE column chromatography. Enkephalinase A 1 has been purified 1500-fold, to appar­ent homogeneity.

Activation of Factor X

Summary1. Methods have been developed for the preparation of factor VII free of prothrombin and factor X, and of factor X with only a very low contamination with factor VII.2. Factors VII and X could be found with one stage methods in purified prothrombin prepared according to Seegers.3. Purified prothrombin was chromatographed on DE AE-cellulose. Two active fractions could be eluted, the first with the characteristics of Seegers’ DE AE-Prothrombin, the second with the characteristics of factor X.4. It was confirmed that DE AE-Prothrombin does not activate to thrombin in 25% citrate, and does not generate autoprothrombin C.5. The combination of both clotting active fractions coming from the DEAE-column restores the properties of non-chromatographed prothrombin.6. DE AE-cellulose chromatography of prothrombin does not induce a molecular change of prothrombin, but separates factor X from prothrombin.7. Factor X can be activated with tissue thromboplastin-factor VII or with high concentrated neutral salt solutions to a substance with the biological characteristics of autoprothrombin C.8. TAMe activity is generated in factor X preparations after incubation with tissue thromboplastin and calcium.9. Factor VII cannot be transformed into autoprothrombin C under the conditions tested.10. Factor VII is necessary for the activation of factor X with tissue thromboplastin but not with RVV.11. The amount of factor X available determines the amount of autoprothrombin C formed, whereas thromboplastin and factor VII influence the rate of the reaction.12. The final conclusion is that activated factor X is similar to or identical with autoprothrombin C.

PURIFICATION OF THE GAMMA GLOBULIN CHARACTERISTIC OF CEREBROSPINAL FLUID

Experiments are described which have led to the standardization of a procedure for the purification of the γc globulin characteristic of cerebrospinal fluid (CSF). The procedure involves the stepwise elution of three chromatographic fractions from a DEAE column by TRIS–HCl buffers at pH 8.5. The composition of the fractions will vary with the relative concentration of the γc globulin in the CSF applied to the column. From an average CSF pool the first fraction, F1, eluted by 0.002 M TRIS–HCl will usually contain only the γc globulin. The second fraction F2, eluted by 0.02 M TRIS-HCl, will contain a mixture of the γc, γs, and βc globulins. The third fraction F3, eluted by 0.08 M TRIS–HCl, will contain chiefly γs globulin as well as traces of the γc and βc globulins. Most of the γc globulin from pooled CSF may then be obtained in purified form by rechromatographing the F2's from several runs. Almost all of the γc globulin applied will be found in F1. The γs and βc globulins will be eluted in the other two fractions.

PURIFICATION OF THE GAMMA GLOBULIN CHARACTERISTIC OF CEREBROSPINAL FLUID

Experiments are described which have led to the standardization of a procedure for the purification of the γc globulin characteristic of cerebrospinal fluid (CSF). The procedure involves the stepwise elution of three chromatographic fractions from a DEAE column by TRIS–HCl buffers at pH 8.5. The composition of the fractions will vary with the relative concentration of the γc globulin in the CSF applied to the column. From an average CSF pool the first fraction, F1, eluted by 0.002 M TRIS–HCl will usually contain only the γc globulin. The second fraction F2, eluted by 0.02 M TRIS-HCl, will contain a mixture of the γc, γs, and βc globulins. The third fraction F3, eluted by 0.08 M TRIS–HCl, will contain chiefly γs globulin as well as traces of the γc and βc globulins. Most of the γc globulin from pooled CSF may then be obtained in purified form by rechromatographing the F2's from several runs. Almost all of the γc globulin applied will be found in F1. The γs and βc globulins will be eluted in the other two fractions.