Publisher Summary This chapter describes the conditions and procedures that affect the levels and activities of the vacuolar proteases and present both genetic and biochemical assaying methods for coping with protease problems. Protease B activity in colonies can be assayed using an overlay test. It is based on the principle that states that protease B, which is freed from cells by lysis and from its inhibitor by the SDS present in the overlay, solubilizes the particles of Hide Powder Azure in the overlay, uncovering the colony and surrounding it with a clear halo. Mutant colonies remain covered. Carboxypeptidase Y activity in colonies can be assessed by using an overlay test that relies on the esterolytic activity of the enzyme. The substrate is N-acetyl-DL-phenylalanine β-naphthyl ester, cleavage of which, in colonies anyway, is catalyzed only by CpY. In biochemical analyses, commonly used assay for protease A activity measures the release of tyrosine-containing acid-soluble peptides from aciddenatured hemoglobin. Protease A is apparently the only protease to catalyze the reaction at acid pH. It is reported the use of a protease-deficient strain can ease protease problems. Strains bearing the pleiotropic pep4-3 mutation have greatly reduced, but not zero, levels of PrA, PrB CpY, and ApI. Strains such as EJ101 that carry prb1-1122 have also been used successfully in some purifications. Use of a double mutant is recommended that carries mutations both in the PEP4 gene and the PRB1 gene.

Tackling the protease problem in Saccharomyces cerevisiae.

Isoelectric points and molecular weights of proteins.

Carboxypeptidases are proteolytic enzymes which only cleave the C-terminal peptide bond in polypeptides. Those characterized until now can, dependent on their catalytic mechanism, be classified as either metallo carboxypeptidases or as serine carboxypeptidases. Enzymes from the latter group are found in the vacuoles of higher plants and fungi and in the lysosomes of animal cells. Many fungi, in addition, excrete serine carboxypeptidases. Apparently, bacteria do not employ this group of enzymes.

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Serine carboxypeptidases. A review

Isoelectric points and molecular weights of proteins : A new table

Proteinases, proteolysis and biological control in the yeast Saccharomyces cerevisiae.

Inhibitors for yeast proteinase A have been purified from boiled extracts from baker's yeast. Upon SP-Sephadex C-25 column chromatography, four different peaks of proteinase A inhibitory activity appear. The inhibitors IA2 and IA3 have been further purified by QAE-Sephadex chromatography and preparative disc electrophoresis. Both inhibitors are homogeneous in dodecylsulfate-gel electrophoresis. From the electrophoretic mobilities in dodecylsulfate-polyacrylamide gels, a molecular weight of 6100 ± 200 was estimated for both inhibitors. In the absence of dodecylsulfate, both inhibitors showed one predominant band and a second faint, diffuse band. As a result of Sephadex G-75 gel filtration, a molecular weight of 23000 ± 2000 was obtained for both inhibitors, indicating an aggregation of the monomers to a tetramer. Upon electrofocusing in acrylamide gels the isoelectric points of the inhibitors were found to be 5.7 for IA2 and 6.3 for IA3. Both inhibitors inhibited proteinase A. Neither proteinases B and C from yeast nor pepsin were inhibited. Both inhibitors were inactivated after incubation with purified yeast proteinase B, but not with the yeast proteinases A or C. The inhibition of proteinase A at high inhibitor/proteinase ratios was more pronounced at pH 5 and 7 as compared to pH 3. An autocatalytic activation of a mixture of inhibited proteinases A, B and C by addition or liberation of traces of active proteinase A or B is discussed.

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1111/j.1432-1033.1974.tb03697.x

Yoshiko Matsuda

Papers

Purification and Characterization of Macromolecular Inhibitors of Proteinase A from Yeast