Formyl peptide chemotaxis receptors on the rat neutrophil: Experimental evidence for negative cooperativity

To examine the existence of negative cooperativity among formyl peptide chemotaxis receptors, steady-state binding of f Met-Leu-[ 3 H]Phe to viable rat neutrophils and their purified plasma membranes was measured and the data were subjected to statistical analysis and to computer curve fitting using the NONLIN computer program. Curvilinear, concave upward Scatchard plots were obtained. NONLIN and statistical analyses of the binding data indicated that a two-saturable-sites model was preferable to a one-saturable-site model and statistically valid by the F-test (P < 0.1). In addition, Hill coefficients of 0.80 ± 0.02 were obtained. Kinetic dissociation experiments using purified plasma membranes showed evidence of site-site interactions of the destabilizing type (negative cooperativity). Thus, unlabeled f Met-Leu-Phe accelerated the dissociation of f Met-Leu-[ 3 H]Phe under conditions where no rebinding of radioligand occurred. The rate of dissociation of f Met-Leu-[ 3 H]Phe from the plasma membranes was dependent on the fold excess of unlabeled f Met-Leu-Phe used in the dilution medium; at the highest concentration tested (10,000-fold excess), the dissociation rate was more than double the dissociation rate seen with dilution alone, In addition, occupancy-dependent affinity was ascertained directly by studying the effect of increasing fractional receptor saturation with labeled ligand on the dissociation rate of the receptor-bound labeled ligand. These data showed that the f Met-Leu-[ 3 H]Phe dissociation rate was dependent on the degree of binding site occupancy over the entire biologically relevant range of formyl peptide concentrations. Furthermore, monitoring of the time course of dissociation of the receptor/f Met-Leu-[ 3 H]Phe complex as a function of receptor occupancy revealed that receptor affinity for f Met-Leu-Phe remained occupancy-dependent during the entire time of dissociation examined (up to 10 min). Finally, the average affinity profile of the equilibrium binding data demonstrated a 60% decrease in receptor affinity in changing from the high affinity to the low affinity conformation.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/38446/1/240270406_ftp.pd

Studies on a peptidase acting on a synthetic collagenase substrate developmental pattern in rat granuloma tissue and distribution of enzyme in the tissues of various animals

The developmental pattern in experimental rat granuloma tissue and the distribution in the tissues of a few animals (monkey, rabbit, guinea pig anrat) of a peptidase acting on a synthetic collagenase substrate, 4-phenylazobenzyloxycarbonyl-L-Pro-L-Leu-Gly-L-Pro-D-Arg (Pz-peptide) has been studied. Maximum enzyme activity was found in 4-month-old rats and on the fourth day of implantation of the cotton wick. Pz-peptidase appears to have a ubiquitous distribution in animal tissues; the highest enzyme activity was generally found in liver, intestine and kidney of the animals. The total activity in other organs (spleen, heart, lungs and brain) was much less compared to that of liver, intestine or kidney

Purification and properties of a peptidase acting on a synthetic collagenase substrate from experimental granuloma tissue in the rat

An enzyme which acts on a synthetic collagenase substrate, 4-phenylazobenzyl-oxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-d-Arg (Pz-peptide) has been purified 250-fold from soluble extracts of experimental granuloma tissue of the rat and its properties studied. It is optimally active around pH 7.2. Its apparent Km value for Pz-peptide is 0.01 mM and V is 100 nmoles/mg protein per min. It is reversibly inhibited by p-hydroxymercuribenzoate (PHMB) and HgCl2, whereas iodoacetamide does not affect the enzyme activity. Heavy metals like Cu2+, Cd2+, Ag+, Ni2+ and Zn2+ completely inhibit the enzyme activity while the inhibition by Co2+ was only partial. Fe2+, Ba2+, Mn2+, Pb2+ and Ca2+ did not exert any affect on the activity. Chelating agents like EDTA, sodium diethyl dithiocarbamate and &#945;,&#945;'-dipyridyl do not affect the enzyme activity. Approximate molecular weight of the purified enzyme was estimated to be 56000

Purification and properties of a peptidase acting on a synthetic substrate for collagenase from monkey kidney

A peptidase cleaving a synthetic substrate for collagenase, 4-phenylazobenzyloxycarbonyl-l-Pro-l-Leu-Gly-l-Pro-d-Arg (designated as Pz-peptide) has been purified extensively (about 5200-fold) from a soluble extract of monkey kidney with a view to carrying out studies on its possible physiological role. The purified Pz-peptidase appeared essentially free of collagenase, nonspecific protease and di- and tri-peptidase activities. The properties of the purified Pz-peptidase resemble very much the granuloma enzyme. It is optimally active around pH 7.0. Its apparent Km value for Pz-peptide is 0.72 mM and V is 10.1 &#956;mol/mg protein/min. It is reversibly inhibited by p-hydroxymercuribenzoate and HgCGl2, whereas iodoacetamide does not affect the enzyme activity. N-Ethylmaleimide inhibited the enzyme partially (50%). Heavy metals like Cu2+, Cd2+, Ag+, Pb2+, Ni2+ and Zn2+ completely inhibited the enzyme activity, while the inhibition by Co2+ was only partial. Fe2+ did not exert any effect on the activity. The enzyme activity was completely inhibited by EDTA and was restored almost to the original value by metal ions like Mn2+, Mg2+, Ca2+ and Ba2+. The approximate molecular weight of the purified enzyme was estimated to be 56 000

Optimising the Heuristics in Latent Semantic Indexing for Effective Information Retrieval

Latent Semantic Indexing (LSI) is a famous Information Retrieval (IR) technique that tries to overcome the problems of lexical matching using conceptual indexing. LSI is a variant of vector space model and proved to be 30% more effective. Many studies have reported that good retrieval performance is related to the use of various retrieval heuristics. In this paper, we focus on optimising two LSI retrieval heuristics: term weighting and rank approximation. The results obtained demonstrate that the LSI performance improves significantly with the combination of optimised term weighting and rank approximation.Information retrieval, Latent Semantic Indexing, rank approximation, term weighting, vector space method

Latent Semantic Indexing based Intelligent Information Retrieval System for Digital Libraries

To the information retrieval research community, a digital library can be viewed as an extended information retrieval system. The primary goal of an information retrieval system is to retrieve all the relevant documents, which are relevant to the user query. Disparities between the vocabulary of the system’s authors and that of their users pose difficulties when information is processed without human intervention. In this paper, we present a novel approach to enhance the efficiency of the information retrieval system based on Latent Semantic Indexing, using intelligent information processing technique. Experiments carried out are giving most encouraging results