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Purification and characterization of an acid protease and vasopeptide kinins from Murphy-Sturm lymphosarcoma
Abstract:
An acid protease from the rat Murphy-Sturm lymphosarcoma (MSLS) tumor was isolated, purified and characterized. The protease hydrolyzed bovine hemoglobin and formed vasopeptide kinins when incubated with purified rat plasma kininogen. The purification was carried out in seven steps involving homogenization of the tumor tissue, acid precipitation with glacial acetic acid, ammonium sulfate precipitation to 60% saturation, DEAE-Sephadex batch treatment, chromatography on a QAE-Sephadex column, gel filtration on Sephadex G-200 and finally chromatography on CM-32 cellulose. The purification scheme resulted in a 640-fold purification and a homogeneous preparation as confirmed by disc gel electrophoresis and Ouchterlony immunodifussion. Sephadex G-200 elution profiles indicated two different protease fractions containing protease activity which showed a single band with identical relative mobility on polyacrylamide gel electrophoresis in the presence and absence of SDS, and also showed immunological cross-reactivity against anti-acid protease antiserum raised in the rabbit against one of the fractions. The acid protease is presumed to be a single protease which has a tendency to aggregate. An apparent molecular weight of 39,500 was estimated by gel filtration on Sephadex G-200, and 41,000 by polyacrylamide gel electrophoresis. SDS-polyacrylamide gel electrophoresis showed that the acid protease was comprised of a major band with a molecular weight of 4,000 and two fainter bands with molecular weights of 27,000 and 12,000. The purified enzyme showed three major isozymic forms (alpha, beta, gamma) which had isoelectric points (pI) of 5.2, 5.5 and 5.8 respectively, and had near identical amino acid compositions. The carbohydrate moiety contained 2 mol of N-acetylglucosamine and 8 mol of mannose per mol enzyme. The pH optimum for the digestion of bovine hemoglobin was approximately 3.0. The protease activity was very stable above pH 3.4. The acid protease released kinin from purified rat plasma kininogen at an initial rapid rate which plateaued at 460 ng bradykinin equivalents per mg substrate after 2 hr incubation at 37 degrees. The vasopeptide was purified to electrophoretic homogeneity by gel filtration on Sephadex G-50 and chromatography on a column of CM-Sephadex. The amino acid composition of vasopeptide was Ser2, Gly1, Pro4, Ile1, Leu1, Phe2, Arg2.
Insights
This study isolated and purified an acid protease from rat lymphosarcoma, demonstrating its ability to hydrolyze hemoglobin and produce vasopeptide kinins. The enzyme exhibits stability and specific activity, offering insights into tumor-associated proteases.
Area of Science:
- Biochemistry
- Enzymology
- Cancer Research
Background:
- Tumor-associated proteases play crucial roles in cancer progression.
- Understanding the specific properties of proteases from lymphosarcoma is essential for targeted therapeutic strategies.
Purpose of the Study:
- To isolate, purify, and characterize an acid protease from rat Murphy-Sturm lymphosarcoma (MSLS).
- To investigate the enzymatic activity of the purified protease, including its substrate specificity and kinin-releasing capability.
Main Methods:
- A multi-step purification process involving homogenization, precipitation, and various chromatography techniques (DEAE-Sephadex, QAE-Sephadex, Sephadex G-200, CM-32 cellulose).
- Enzyme characterization using disc gel electrophoresis, Ouchterlony immunodiffusion, SDS-polyacrylamide gel electrophoresis, and isoelectric focusing.
- Assays for protease activity, hemoglobin hydrolysis, and kinin release from rat plasma kininogen.
Main Results:
- A 640-fold purification of a homogeneous acid protease from MSLS tumor tissue.
- The enzyme exhibits optimal activity at pH 3.0, is stable above pH 3.4, and releases kinins from rat plasma kininogen.
- Estimated molecular weights varied between 39,500-41,000 Da, with SDS-PAGE revealing subunits of 4,000, 12,000, and 27,000 Da. Three isozymic forms (pI 5.2, 5.5, 5.8) were identified.
Conclusions:
- The purified acid protease from MSLS is a distinct enzyme with significant hydrolytic and kinin-releasing activities.
- The enzyme's properties suggest a potential role in tumor pathophysiology and provide a basis for further functional studies.