The proteolytic system of Methylophilus methylotrophus

Abstract

This thesis describes studies on the proteolytic system of Methylophllus methylotrophus. Proteinases have been partially purified and characterized from Its cytoplasmic, periplasmic and membrane fractions. These enzymes are serine- and metalloproteinases maximally active in the pH range 7.3-8.2 at 37 C. They hydrolyze a broad range of substrates and several are dependent on metal-ions for maximum activity and/or stability.

The levels of proteolytic activity towards both endogenous and exogenous protein substrates are much lower in extracts from cells grown exponentially than In those from the stationary phase of growth.

Storage of cell extracts results in the breakdown of most intracellular proteins and an Increased ability to degrade exogenous protein substrates. PMSF and EDTA reduce proteolysis in both cases.

A non-dlalyzable heat-stable factor Isolated from soluble extracts partly inactivates trypsin and chymotrypsin in addition to certain endogenous proteolytic enzymes. The inhibitory factor(s) responsible for proteinase inactivation have not been purified.

A sudden Increase In temperature during exponential growth results in (1) a decrease in the synthesis of the majority of cellular proteins and (2) the induction of a number of unique proteins called ‘heat-shock proteins’ (hsps). These hsps have apparent molecular weights of 83, 78, 63, 60, 27, 20, 16 and 14 kD. A similar although not identical response is observed when cells are treated with methanol or ethanol. Hsps are induced that are unique to the type of ‘stress-shock’ treatment. Simultaneous exposure to heat-shock and either methanol or ethanol results in the synthesis of both sets of hsps. Hsps are found in all three subcellular fractions.

Stress-shock of growing cells also results in the specific induction and repression of proteolytic enzymes with a net increase In cellular proteolysis. A comparable response Is observed in Escherichia coli cells.

Overall this thesis provides Information on the basic features of the proteolytic system of the obligate methylotroph, Methylophilus methylotrophus.