Summary: Phosphoenolpyruvate (PEP) carboxykinase (EC 184.108.40.206) from the obligate anaerobe Anaerobiospirillum succiniciproducens was purified 18-fold. The enzyme was monomeric, with an M r of 57000 ± 2000. The enzyme was oxygen stable, had a pH optimum of 6.5-7.1, and was stable from pH 5.0 to 9.0. The enzyme displayed Michaelis-Menten kinetics for the substrate PEP and the cosubstrates bicarbonate and ADP with a K m of 0.54 mM, 17 mM and 0.42 mM, respectively. The enzyme required Mn2+ or Co2+ in addition to Mg2+ to exhibit maximum activity. p-Chloromercuribenzoate inhibited activity and phosphoenolpyruvate protected the enzyme against inactivation, suggesting that an essential cysteine may be in the active site.
Summary: The phosphate-irrepressible alkaline phosphatase of the ethanol-producing bacterium Zymomonas mobilis was solubilized from membranes by the cationic detergent N-cetyl-N,N,N-trimethylammonium bromide (CTAB) and purified by fast protein liquid chromatography. The purified enzyme was a monomeric protein of molecular mass 54 kDa, highly resistant to heat and to ionic strength. The alkaline phosphatase of growing Z. mobilis cells (ZAPase), remained active in the presence of an ethanol concentration as high as 100 g I−1. However, in vitro, the stability of the purified ZAPase was severely affected at low ethanol concentration (7.8 g I−1), showing the importance of the membrane environment in vivo. ZAPase differed from other bacterial alkaline phosphatases by having a higher affinity for the substrate 4-nitrophenylphosphate, a higher K i for phosphate, only a partial reactivation by Zn2+ after EDTA inhibition, and a higher specific activity.
Summary: Analytical and preparative isoelectric focusing were used to separate extracellular isoenzymes of aminopeptidase (pI 4.51, M r 45000, pH optimum 7.0) and prolyl-dipeptidylpeptidase (pI 4.01, M r 74000, pH optimum 8.0) produced by the entomopathogenic fungus Metarhizium anisopliae during growth on locust cuticle. Production of both activities is repressed by readily utilized nitrogen sources, but unlike the aminopeptidase, the dipeptidylpeptidase was also excreted at high levels during growth on casein. Casein-grown cultures contained additional isoenzymes with activity against lysyl-alanyl-4-methoxy-2-naphthylamine indicating M. anisopliae possesses multiple peptidases as an adaptation to different nutrient conditions. The aminopeptidase hydrolysed alanyl-leucyl-alanine and showed a broad specificity versus monoaminoacyl β-naphthylamine (βNA) substrates with alanine βNA being the most rapidly hydrolysed. Inhibition by both bestatin and amastatin indicated similarities to the class of alanyl aminopeptidases (aminopeptidase M). Metal complexing agents also inhibited the aminopeptidase indicating a metal ion requirement. A specific inhibitor for serine proteases [diisopropyl fluorophosphate (DFP)] was without effect. The dipeptidylpeptidase showed a strong preference for substrates having a penultimate proline residue including alanyl-prolyl-glycine and aa-prolyl-βNA substrates. The enzyme showed a broad specificity at the N-terminal amino acid. Inhibition by diprotin A indicates similarities with mammalian prolyl-dipeptidylpeptidases. The enzyme was also inhibited by DFP, implying involvement of a serine residue in catalysis. The results are discussed in the context of cuticle degradation and the participation of exopeptidases as mediators in releasing amino acids necessary for pathogen growth.
Summary: The amino acid sequence of the so-called 70 kDa (actually 64 kDa) serine protease secreted by the Gram-negative fish pathogen Aeromonas salmonicida has been determined. It shows a high degree of homology with the complete sequence of other bacterial serine proteases which, with molecular masses of approximately 30 kDa, are less than half its size. This homology is particularly marked in regions adjacent to the catalytic triad Asp32, His64 and Ser221 of subtilisin BPN'. Significant features of the A. salmonicida enzyme, a new member of the group of cysteine-containing subtilisin-type serine proteases, are the presence of six cysteine residues in the mature enzyme, a 37 amino acid extension at the N-terminus and 215 amino acids at the C-terminus when compared with subtilisin BPN'. In addition to a number of smaller peptide insertions there is a non-aligned 32 amino acid sequence in a position corresponding to its introduction between Lys213 and Tyr214 of subtilisin BPN'. This sequence is highly hydrophilic, with Asp/Asn accounting for 10 of the 32 amino acids. Further, the possession of two Cys residues separated by 24 amino acids provides the capacity for stabilizing the peptide as an externalized loop.
Summary: Dark-grown resting (non-dividing) cells of Euglena gracilis var. bacillaris and mutants W3BUL (with a proplastid remnant) and W10BSmL (lacking plastids) incubated with 35SO2- 4 form a series of labelled lipids which are low or absent in dividing cells. These lipids all release labelled taurine on mild acid-hydrolysis. Treatment of the labelled lipids with 2,4-dinitrofluorobenzene (DNFB) followed by acid hydrolysis does not yield labelled dinitrophenyltaurine (DNP-taurine), but treatment with DNFB after hydrolysis readily forms labelled DNP-taurine, indicating that taurine is linked to the lipids by at least the amino group. Illumination increases the labelling of these taurolipids in plastid-containing cells (wild-type and W3BUL) but has little effect in cells lacking plastids (W10BSmL); labelling is highest in W10 cells irrespective of illumination. This indicates that the presence of a plastid may exert a negative control on taurolipid formation which is relieved by light. The same series of labelled lipids is found in isolated purified mitochondria from mutant W10, indicating that this organelle is a site for taurolipid deposition. The formation of taurolipids under non-dividing conditions may be a response to nutritional stress and these negatively charged constituents (as well as the thylakoid sulpholipid) may serve to protect membranes by repelling deleterious negatively charged oxygen species.