Arieh Yaron

Proline-Dependent Structural and Biological Properties of Peptides and Proteins

Proline residues confer unique structural constraints on peptide chains and markedly influence the susceptibility of proximal peptide bonds to protease activity. This review presents a critical analysis of peptidases involved in the cleavage of proline-containing peptide bonds, with particular attention to the role of proline peptidases in the regulation of the lifetime of biologically active peptides. Peptidases discussed include aminopeptidase P, prolidase, dipeptidyl peptidase IV, prolyl endopeptidase, and prolyl iminopeptidase. Attention is also given to HIV-1 protease, because this key enzyme processes an Xaa-Pro peptide bond. Analysis of the above enzymes reveals that they may function as key pacemakers in the control of the activity of many peptide hormones and that they are involved in a variety of immunological processes, including T-cell-mediated immune response. The novel occurrence of cis-trans isomerization about Xaa-Pro bonds and the biological function of peptidyl-prolyl cis-trans isomerases (immunophilins) are reviewed.

Distribution of proline-specific aminopeptidases in human tissues and body fluids.

The proline-specific peptidases, aminopeptidase P (EC 3.4.11.9) and dipeptidyl peptidase IV (EC 3.4.14.5), were measured in human tissue homogenates and physiological fluids. All tissues examined contained measurable aminopeptidase P and dipeptidyl peptidase IV activities. High specific activities for both enzymes under study were found in benign prostatic hypertrophy. Normal prostate and prostatic adenocarcinoma had a much lower activity. This difference, however, is not reflected in the serum values of the patients. The most striking finding is the extremely high activity of dipeptidyl peptidase IV in prostatosomes, prostate-derived organelles, which occur freely in human seminal plasma, and which are important for enhancement of sperm forward motility.

Characterization of dipeptidyl peptidase IV (CD26) from human lymphocytes

The membrane-bound dipeptidyl peptidase IV (DPP IV, EC 3.4.14.5) has been purified 5,400-fold from human peripheral blood mononuclear cells. The purification procedure included detergent solubilization and successive chromatography on DEAE Sepharose Fast Flow, Con A Sepharose, Cu2+ loaded metal-chelating Sepharose, Sephacryl S-300 High Resolution and Q Sepharose Hiload. The molecular mass of the native, detergent solubilized enzyme estimated by gel filtration was 264.kDa. Chromatofocusing indicated a pI of approximately 5.0. The pI optimum was 8.7. The enzymatic activity of the purified preparation was irreversibly inhibited by N-(H-Phe-Pro)-O-(4-nitrobenzoyl)hydroxylamine hydrochloride in the micromolar range. The binding of purified DPP IV to CD26 monoclonal antibodies confirmed the identity between CD26 and dipeptidyl peptidase IV. The purification and characterization of lymphocytic dipeptidyl peptidase IV is of great value for the identification of its natural substrates and for the study of its physiological significance in the T-lymphocyte function.

Kininase activity in human platelets: cleavage of the Arg1-Pro2 bond of bradykinin by aminopeptidase P.

A proline-specific peptidase aminopeptidase P (APP, EC 3.4.11.9) that cleaves the Arg1-Pro2 bond of bradykinin was isolated from human platelets by liquid chromatography. The enzyme was purified 557 times. The native molecule has a M(r) of 223,000. Human platelet APP exists as a trimer with a subunit M(r) of 71,000. The apparent Km of platelet APP is 66 mumol/L for bradykinin and 47 mumol/L for the internally quenched fluorogenic substrate Lys (2,4-dinitrophenyl)-Pro-Pro-NH-CH2-CH2-NH-2-aminobenzoyl. 2HCl which is used for the routine determination of the enzyme activity. The optimum pH for hydrolysis of the fluorogenic substrate is 8.0, and the optimum temperature is 43 degrees. Platelet APP is inhibited by 1,10-phenanthroline and activated by Mn2+, thus confirming its metalloprotease nature. Cu2+, Zn2+ and Hg2+ are strongly inhibitory. Inhibition by cysteine protease inhibitors suggests the presence of a thiol group essential for enzymatic activity. Serine protease inhibitors do not affect the enzyme activity.

Fusion of phospholipid vesicles containing a trypsin-sensitive fluorogenic substrate and trypsin: A new method to study membrane fusion activity in a model system

It is well established that fusion of membranes is involved in several biological processes [l-4]. Numerous attempts have been made to elucidate the molecular mechanisms underlying membrane fusion, and various model membrane systems have been applied for this purpose [5-l 11. A number of those studies rely on transfer of spinlabelled lipids between vesicles [ 121 or on changes in NMR spectra [ 13-151 as criteria for fusion, leaving considerable uncertainty concerning the question whether the change in membrane composition observed is entirely due to fusion or partly to molecular exchange. The ambiguity of such observations calls for cautious interpretation of experimental data. Despite these complications substantial evidence has been obtained during recent years for the occurrence of fusion as a result of vesicle-vesicle interaction [12,16,17]. Thus far, however, scanty attempts have been made to demonstrate the coalescence of aqueous intravesicular compartments as a result of fusion events. The methods published’s0 far to demonstrate fusion on the basis of this criterion have shown more or less serious shortcomings. Even recent work [ 18,191 involving the use of the luciferase/ ATP system leads to ambiguous results as indicated

A fluorimetric assay for angiotensin-I converting enzyme in human serum

A fluorimetric method is described for a simple, sensitive and reproducible assay for angiotensin-I converting enzyme in human and guinea pig sera. The very weak fluorescent substrate o-aminobenzoylglycyl-p-nitro-L-phenylalanyl-L-proline is enzymatically hydrolyzed, producing the highly fluorescent o-aminobenzoylglycine that is quantitatively determined by spectrofluorimetry. Dependence of activity on substrate concentration, amount of serum, time of incubation and pH were investigated. The KM value for the substrate is 0.1 and 0.032 mM for the human and guinea pig serum enzyme, respectively. The mean value of serum angiotensin-I converting enzyme for 16 normal adult persons was 2.56 +/- 0.10 (S.E.) with a standard deviation of 0.81 nmol/min/ml serum.

A novel aminopeptidase from clostridium histolyticum

Abstract An aminopeptidase was found in the culture filtrate of Cl. histolyticum and purified to homogeneity (130 times) in a two-step procedure. All types of N-terminal amino acids, including proline and hydroxyproline are cleaved by the enzyme from small peptides and from polypeptides. A low rate of hydrolysis was observed for β-naphthylamides and for alanine amide; p-nitroanilides were not hydrolyzed. Kinetic parameters (K m and V max ) for several tripeptides and the tetrapeptide Pro-Gly-Pro-Pro were determined. The enzyme has a pH optimum at 8.6. The presence of either Mn ++ or Co ++ is essential for its activity. Only slight activation was observed with Ni ++ and Cd ++ , while Zn ++ and Cu ++ were inhibitory. The molecular weight of the native enzyme is about 340,000, and a molecular weight of about 60,000 was determined for the reduced and denatured enzyme by gel electrophoresis in sodium dodecyl sulfate (SDS). The culture filtrate of Cl. histolyticum has been shown to contain various proteolytic enzymes, in addition to collagenase 1–5 . In a search for enzymes acting on proline-rich peptides, we tested the crude filtrate with (Pro-Gly-Pro) n , (Pro-Gly-Pro) n -OMe, α,DNP-(Pro-Gly-Pro) n and poly-L-proline as substrates. Proline was formed only from (Pro-Gly-Pro) n and its methyl ester. This showed the presence in Cl. histolyticum filtrate of an aminopeptidase which cleaves N-terminal proline from polypeptides but not from polyproline. The purification and some of the properties of this clostridial aminopeptidase (CAP) are described in this communication.

Aminopeptidase P and dipeptidyl peptidase IV activity in human leukocytes and in stimulated lymphocytes

Human white blood cells were shown to contain high aminopeptidase P activity. The specific activities found in the high-speed supernatant of the extracts of granulocytes, lymphocytes and monocytes ranged from 30 to 70 units per mg protein. Culturing lymphocytes during 7 days in the presence of phytohaemagglutinin resulted in a 70-200% increase in the specific aminopeptidase P activity and a 200% increase in the specific activity of dipeptidyl peptidase IV. The time-course of the activity of both aminopeptidase P and dipeptidyl peptidase IV during the stimulation of human T-lymphocytes by phytohaemagglutinin indicates an involvement of these two enzymes in the proliferative process of these immunocompetent cells. Due to their substrate specificity their potential substrates must have the N-terminal Xaa-Pro sequence known to be present in several immunologically important polypeptides.

Multi-chain polyamino acids containing glutamic acid, aspartic acid and proline

Abstract 1. 1. A number of multi-chain polyamino acids were synthesized by reacting N-carboxyamino acid anhydrides with multi-functional amines such as polylysine or preformed multi-chain polyamino acids. The purpose of this synthesis was to obtain a family of compounds which could serve as protein models in the study of hydrodynamic, electrochemical and conformational properties. 2. 2. It was found that in this method of synthesis linear polymers may be formed as by-products and methods were worked out for the purification of the multi-chain fraction. Molecular weights of the materials obtained ranged from 50 000 to 1 500 000. There was good agreement between molecular weights as determined by physical methods and those calculated from the degree of polymerization of the polylysine “backbone” and total amino acid analysis. 3. 3. Applying the “equivalent ellipsoid” approach to hydrodynamic measurements reasonable values were calculated for the length of the resulting molecules, but it was concluded that a more refined theory is necessary to describe the overall shape of the multi-chain compounds. 4. 4. Electrochemical studies showed that various ionizable groups had dissociation constants similar to those observed for the corresponding groups in proteins. There seems to be evidence that the dissociation constant are influenced more by next-neighbour effects than by the general charge of the molecule. 5. 5. The side chains of the multi-chain polymers are able to assume helical conformation if long enough. Solvent-induced helix-coil transitions were observed with poly-β-benzyl- L -aspartate and poly-γ-benzyl- L -glutamate side chains. Poly- L -proline side chains were shown to be able to assume the poly- L -proline I or poly- L -proline II conformation (according to the solvent employed) if the average chain length was larger than six.

Exopeptidases in human platelets: an indication for proteolytic modulation of biologically active peptides

The determination in human platelets of four exopeptidases--aminopeptidase P, dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme--by means of fluorometric or liquid chromatography techniques was carried out. The results obtained show that the specific activities of dipeptidyl peptidase IV, carboxypeptidase N, and angiotensin converting enzyme in intact and disrupted platelets are small compared to their specific activities in serum. However, for aminopeptidase P the specific activity of this enzyme is much higher in platelets than in serum. This suggests that circulating platelets may have a significant role as scavengers for circulating peptides containing bonds susceptible for aminopeptidase P.