Energetics of Leucyl-Leucine Hydrolysis in Streptococcus cremoris Wg ₂

Abstract

The hydrolysis of the dipeptide leucyl-leucine by whole cells of Streptococcus cremoris Wg2 was dependent on the presence of the energy source lactose. Incubation of cells with uncouplers or ATPase inhibitors prevented the increase of peptidase activity upon the addition of lactose. Incubation with the ionophore nigericin resulted in decreased peptide hydrolysis activity, while incubation with valinomycin led to increased hydrolysis activity. In the presence of nigericin the ApH component of the proton motive force was decreased, while the electrical potential was increased. With valinomycin, the electrical potential was collapsed and the ApH was increased. When the external pH was decreased from 8 to 5, the rate of peptide hydrolyzing activity by whole cells increased with increasing ApH component. In contrast, the peptide hydrolyzing activity in the cell extract decreased with decreasing external pH. These results indicate that the ApH component of the proton motive force determines the leucyl-leucine hydrolyzing activity in S. cremoris Wg2.

Purification and Characterization of an Aminopeptidase from Lactococcus lactis subsp. cremoris Wg2

“…A dipeptidase from L. Iactis subsp. cremoris Wg2, which only hydrolyzed dipeptides, has already been purified by van Boven et al (28,31). A peptidase that was able to hydrolyze the synthetic substrate leucyl-p-nitroanilide was also reported for this strain.…”

A Phosphate-Bond-Driven Dipeptide Transport System in Streptococcus cremoris Is Regulated by the Internal pH

“…3). At a lower external pH, the ApH is larger (30) and the rate of leucyl-['4C]leucine uptake in cells of S. cremoris E8 is higher.…”

“…Energy requirement of peptide uptake. In a previous study (30), it was shown that the 'rate of hydrolysis of leucylleucine by S. cremoris depends on the presence of an energy source. This was confirmed by determining the uptake of leucyl-["'C]leucine by S. cremoris E8 in the prese'nce and absence of an energy source (Fig.…”

“…So far, accumulation of peptides across the cytoplasmic membrane of streptococci could not be demonstrated directly because of the very high intracellular peptidase activities. Indirect evidence for energy-dependent peptide uptake by streptococci has been obtained by measuring the uptake of 14C-labeled peptides, the amount of peptides in the supernatant using fluorescamine (29), or both or by measuring the rate of amino acids released from the cells (30). Peptide uptake was abolished when the cells were incubated with uncouplers, indicating an active role of the proton motive force in the uptake of peptides.…”

Proteolytic enzymes of dairy starter cultures

“…Distinct systems exist for amino acid, di-and oligopeptide transport in lactic streptococci [55,82,83; see review 53]. A recent study [84] involving S. cremoris Wg2 confirmed the energy-dependent utilization of dipeptides [83] and showed that the hydrolysis of leucyl-leucine was dependent on a pH gradient across the cell membrane. The rate of hydrolysis was apparently limited by the activity of the uptake system since hydrolysis was markedly accelerated on disruption of the permeability barrier but the results leave open the question as to whether the transport and hydrolysis steps are quite independent or are coupled via a membrane-bound dipeptidase system.…”