ABSTRACT
The degradation of N-Ac-Ser-Asp-Lys-Pro (AcSDKP), a negative regulator controlling the proliferation of the haematopoietic stem cell, by enzymes present in human plasma, has been investigated. Radiolabelled AcSD[4-3H]KP ([3H]AcSDKP, 1 mM) was completely metabolized in human plasma with a half-life of 80 min, leading exclusively to the formation of radiolabelled lysine. The cleavage of AcSDKP was insensitive to classical proteinase inhibitors including leupeptin, but sensitive to metalloprotease inhibitors. The degradation was completely blocked by specific inhibitors of angiotensin I-converting enzyme (ACE; kininase II; peptidyldipeptide hydrolase, EC 3.4.15.1), showing that the first step of the hydrolysis was indeed due to ACE. In dialysed plasma, the hydrolysis proceeded at only 17% of the maximal rate, whereas addition of 20 mM NaCl led to the recovery of the initial rate observed with normal plasma. Hydrolysis of AcSDKP by commercial rabbit lung ACE generated the C-terminal dipeptide Lys-Pro. Thus, ACE cleaves AcSDKP by a dipeptidyl carboxypeptidase activity. In fact the formation of Lys-Pro was observed when AcSDKP was incubated in human plasma in the presence of HgCl2. These results suggest that ACE is involved in the first limiting step of AcSDKP degradation in human plasma. The second step seems to be under the control of a leupeptin- and E-64-insensitive, HgCl2-sensitive plasmatic enzyme.
Subject(s)
Oligopeptides/metabolism , Peptidyl-Dipeptidase A/blood , Amino Acid Sequence , Chromatography, High Pressure Liquid , Dipeptides/analysis , Half-Life , Humans , Kinetics , Molecular Sequence Data , Oligopeptides/chemical synthesis , Protease Inhibitors/pharmacology , Substrate Specificity , TritiumABSTRACT
An original procedure which preserves the structure of the sugar ring is described to link a plant hormone as N6-isopent-2-enyladenosine [( 9R]iP) onto a protein carrier to prepare a more specific immunogen. This cytokinin is bound to bovine serum albumin (BSA) and ovalbumin by a five-step procedure. These [9R]iP-protein conjugates have a maximal absorption at 269 nm and show molar ratios of hormone bound to proteins in the range of 12:1 and 18:1 for BSA and ovalbumin, respectively. Polyclonal antibodies were raised in rabbits against [9R]iP-BSA and were purified by affinity chromatography. Titers and specificity of the antisera and purified antibodies were determined by ELISA and RIA. These antibodies are highly specific for [9R]iP and do not cross-react with zeatin and ribosylzeatin. An immunoaffinity matrix was prepared with a capacity of 1 microgram of [9R]iP/mL of gel.
Subject(s)
Antibodies/immunology , Cytokinins/immunology , Isopentenyladenosine/immunology , Ovalbumin/immunology , Serum Albumin, Bovine/immunology , Animals , Antibodies/isolation & purification , Antibody Specificity , Chromatography, Affinity , Cross Reactions , Cytokinins/chemistry , Enzyme-Linked Immunosorbent Assay , Isopentenyladenosine/chemistry , Ovalbumin/chemistry , Rabbits , Serum Albumin, Bovine/chemistry , Spectrophotometry, UltravioletABSTRACT
Alpha- and beta-D-galactosidases were characterized from a hydroalcoholic extract of wheat germ (Triticum vulgare). Kinetic constants (Vmax and KM) and the optimal pHs for the hydrolysis of p-nitrophenyl galactopyranosides by both enzymes were determined. These enzymes presented a high stability in hydroalcoholic medium and were inhibited by iodoacetamide and sodium p-hydroxy-mercuribenzoate.
Subject(s)
Galactosidases/analysis , Acetates/chemistry , Acetic Acid , Ethanol/chemistry , Hydrogen-Ion Concentration , Temperature , TriticumABSTRACT
Analogues of NAcSerAspLysPro (AcSDKP), a natural regulator of hematopoiesis isolated from fetal calf bone marrow, were synthesized. The biological activity of these molecules were evaluated in vitro in the rosette assay, which measures the interaction between human Jurkat T-cells and sheep red blood cells. In this test, the tripeptide SerAspLys was the most efficient. Inhibitory activity was detected at the concentration 10(-14) M for the analogue and at 10(-9) M for the parent tetrapeptide. The dipeptide NAcSerAsp still showed activity but at much higher doses (10(-6) M). Substitution of polar amino acids led mostly to inactive molecules. Thus, replacement of Ser by Ala, or Lys by Orn yielded completely inactive compounds and replacement of Asp by Glu decreased the activity (10(-6) M). The present study gives an insight into the role of individual amino acids of AcSDKP in the inhibition of the rosette formation which implicates interactions with T-cell CD2 glycoprotein.
