The coupling of diazotized aryl amines with proteins in alkaline solution yield chromophoric protein derivatives. Digestion of a solution of such azoproteins with proteolytic enzymes results in the formation of colored components soluble in trichloroacetic acid. The intensity of the color in the trichloroacetic acid filtrate of the digested substrate is a function of the proteolytic activity of...

The seven antigenically distinct serotypes of Clostridium botulinum neurotoxins cleave specific soluble N-ethylmaleimide-sensitive factor attachment protein receptor complex proteins and block the release of neurotransmitters that cause flaccid paralysis and are considered potential bioweapons. Botulinum neurotoxin type A is the most potent among the clostridial neurotoxins, and to date there i...

We have developed a sensitive and highly efficient whole-cell methodology for quantitative analysis and screening of protease activity in vivo. The method is based on the ability of a genetically encoded protease to rescue a coexpressed short-lived fluorescent substrate reporter from cytoplasmic degradation and thereby confer increased whole-cell fluorescence in proportion to the protease's app...

Substrate inhibition is a common phenomenon in enzyme kinetics. We report here for the first time its study by a combination of the electrophoretically mediated microanalysis (EMMA) methodology with a partial filling technique. In this setup, the part of capillary is filled with the buffer best for the enzymatic reaction whereas, the rest of the capillary is filled with the background electroly...

For membrane-bound enzymes that act on substrates that partition between the membrane and aqueous phases, it is possible to imagine two fundamentally different mechanisms. Interfacial enzymes must access their substrate from the membrane phase, in other words substrate in the membrane binds directly to the active site of the enzyme at the membrane without mixing with substrate molecules in the ...

The epoxide hydrolase activities of the 100,000 g pellet (microsomal) and 100,00 g soluble (cystosolic) fractions of mouse, rat, and guinea pig liver were measured with three closely related compounds used as substrates. Differences between the species in the distribution of the cytosolic and microsomal hydrolases and in their substrate specificities and pH optima demonstrate why epoxide hydrol...

BACKGROUND Plasmodium falciparum, the causative agent of human malaria, expresses two aminopeptidases, PfM1AAP and PfM17LAP, critical to generating a free amino acid pool used by the intraerythrocytic stage of the parasite for proteins synthesis, growth and development. These exopeptidases are potential targets for the development of a new class of anti-malaria drugs. METHODOLOGY/PRINCIPAL FI...

CMP-N-acetylneuraminate:lactosylceramide alpha-2,3-sialyltransferase is tightly associated with the luminal side of the Golgi membrane as is its lipid substrate, lactosylceramide. In order to understand the kinetics, properties, and regulation of this enzyme, it is necessary to alter the amount and type of substrate in the membrane while minimizing changes in the membrane environment or in the ...

A novel inhibitor of angiotensin I converting enzyme (ACE), designated K-26, was isolated from the broth filtrate of an actiomycete K-26. K-26 is a water soluble, acidic peptide composed of an equal mol of L-isoleucine, L-tyrosine and 1(R)-1-amino-2-(4-hydroxyphenyl)-ethylphosphonic acid. The IC50 of K-26 for ACE inhibition was 6.7 ng/ml when hippuryl-L-histidyl-L-leucine was used as a substrat...

The effect of several nutritional and environmental parameters on Penicillium purpurogenum growth and sacharogenic amylase production was analyzed. High enzyme levels (68.2 U mg(-1)) were obtained with Khanna medium at initial pH 6.0, incubated at 30°C for 144 hours. The optimum pH and temperature activities were 5.0 and 65°C, respectively. The enzyme presented a half-life (t50) of 60 min, at 6...