New class of sensitive and selective fluorogenic substrates for serine proteinases. Amino acid and dipeptide derivatives of rhodamine.

A series of dipeptide derivatives of Rhodamine, each containing an arginine residue in the P1 position and one of ten representative benzyloxycarbonyl (Cbz)-blocked amino acids in the P2 position, has been synthesized, purified and characterized as substrates for serine proteinases. These substrates are easily prepared with high yields. Cleavage of a single amide bond converts the non-fluorescent bisamide substrate into a highly fluorescent monoamide product. Macroscopic kinetic constants for the interaction of these substrates with bovine trypsin, human and dog plasmin, and human thrombin are reported. Certain of these substrates exhibit extremely large specificity constants. For example, the kcat./Km for bovine trypsin with bis-(N-benzyloxycarbonylglycyl-argininamido)-Rhodamine [(Cbz-Gly-Arg-NH)2-Rhodamine] is 1 670 000 M-1 X S-1. Certain of these substrates are also highly selective. For example, the most specific substrate for human plasmin, (Cbz-Phe-Arg-NH2)-Rhodamine, is not hydrolysed by human thrombin, and the most specific substrate for human thrombin, (Cbz-Pro-Arg-NH)2-Rhodamine, is one of the least specific substrates for human plasmin. Comparison of the kinetic constants for hydrolysis of the dipeptide substrates with that of the single amino acid derivative, (Cbz-Arg-NH)2-Rhodamine, indicates that selection of the proper amino acid residue in the P2 position can effect large increases in substrate specificity. This occurs primarily as a result of an increase in kcat. as opposed to a decrease in Km and, in certain cases, is accompanied by a large increase in selectivity. Because of their high degree of sensitivity and selectivity, these Rhodamine-based dipeptide compounds should be extremely useful substrates for studying serine proteinases.     

Conformation of Lys-plasminogen and the kringle 1-3 fragment of plasminogen analyzed by small-angle neutron scattering

Native human Glu-plasminogen (Glu1-Asn791) was previously shown to have a radius of gyration of 39 A and a shape best described by a prolate ellipsoid [Mangel, W. F., Lin, B., & Ramakrishnan, V. (1990) Science 248, 69-73]. Upon occupation of a weak lysine-binding site, the shape reversibly changes to that best described by a Debye random coil with a radius of gyration of 56 A. Conversion from the closed to the open form is not accompanied by any change in secondary structure, hence the closed conformation is formed by interaction between domains, the five kringles and the protease domain, and this is abolished upon conversion to the open form. Here we analyzed by small-angle neutron scattering the conformations of human Lys-plasminogen (Lys78-Asn791) and the fragment K1-3 that contains the first three kringles of plasminogen (Tyr80-Val338 or Tyr80-Val354). The shape of Lys-plasminogen was best described by a Debye random coil with a radius of gyration of 51 A, and occupation of its lysine-binding sites by 6-aminohexanoic acid did not dramatically alter its conformation. Thus Lys-plasminogen was in the open form, similar to that of Glu-plasminogen with its lysine-binding sites occupied. The fragment K1-3 in the absence or presence of 6-aminohexanoic acid had a shape best described equally either by an elongated prolate ellipsoid or by a Debye random coil, with a radius of gyration of 29 A. Our model for the two forms of plasminogen is that, in the closed form, domain interaction generates a compact, almost globular, structure.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation of the Plasminogen Activator Activity of a Transformed Cell Line by Cell Density

The effects of variations in cell density on the expression of the plasminogen activator activity of a tumorigenic rat cell line were analyzed. At low cell densities, the plasminogen activator activity per cell was high and independent of cell density. As the cell density increased, the plasminogen activator activity per cell decreased until it eventually became inversely proportional to cell density. Inhibition of the plasminogen activator activity per cell by increases in cell density was not the result of the presence of a soluble inhibitor but seemed to require cell-to-cell contact. The V_max per cell for the activation of plasminogen changed at high cell densities, but the K_m did not change. This change in the V_max per cell was in part the result of a change in the catalytic rate constant for the conversion of plasminogen to plasmin. This was inferred from studies on the kinetics of inhibition of plasminogen activator activity by diisopropyl fluorophosphate as a function of cell density. For cells growing at high densities, the rate of inhibition was constant, exhibiting a second-order rate constant of 2.6 × 10⁻²M⁻¹ s⁻¹. For cells growing at low densities, the plasminogen activator activity was inhibited at two different rates, one exhibiting a second-order rate constant of 2.6 × 10⁻²M⁻¹ s⁻¹ and the other exhibiting a second-order rate constant of 9.4 × 10⁻²M⁻¹ s⁻¹. We discuss the importance of cell density in assays of the plasminogen activator activity of cells, the use of this cell line to study the biochemical basis of the density dependence of plasminogen activator activity, and the density-dependent role of plasminogen activator activity in tumor formation and metastasis.     

Behavioral stabilization of host-parasite population dynamics

We demonstrate that individual behavior can stabilize classical (Nicholson-Bailey) host-parasite population dynamics. Our model assumes that hosts can be divided into at least two phenotypes and that parasites either do not attack one of the phenotypes or attack them facultatively. The former case corresponds to a behavioral refuge (Hassell 1978) and it is known that other kinds of refuges lead to stability of population dynamics. Behavioral refuges can stabilize the population dynamics in the same way that spatial refuges do. When parasites attack hosts facultatively within the year, strange attractors may arise in the year-to-year population dynamics, in response to the nonlinear nature of the facultative response to the distribution of host densities.     

Dependence of photosensitivity of bileaflet lipid membranes upon the chlorophyll and carotenoid content.

Bileaflet lipid membranes were formed from solutions containing lecithin, chlorophyll and carotene in various concentrations. If all the above components were present at sufficient concentrations the membranes were photosensitive; i.e., a photocurrent was produced if a redox potential gradient was present across the membranes. The presence of chlorophyll and carotene were essential for the photosensitivity of the membranes. Photoresponse could be elicited by illuminating the membrane with light which did not excite carotene. On the other hand, elimination of the part of the light spectrum which excites chlorophyll led to the abolition of the photoresponse. The findings of this study are consistent with the assumption that the excited chlorophyll chromophores allow electron exchange at the membrane-water interface while the presence of carotene allows electron movement across the "bulk" lipid membrane.     

The movement of ion pairs across bilayer lipid membranes.

If a polyhalide concentration gradient exists across a bilayer lipid membrane (BLM), ion pair movement occurs. The term ion pair indicates a lipid soluble complex of cation and anion with stoichiometry dictated by the respective charges. In a mixture of metal halide (MX_n, X = I, Cl, Br) and iodine, the ion pair is of the form M(I₂X)_n. The flux of ion pairs was monitored by measuring the flow of metal ions or polyhalide ions across the BLM. The flux of ion pairs across the BLM depended on cation crystal radius, fluidity of the membrane, strength of the ion pair complex and on the osmotic gradient (i.e., there exists a coupling between water and ion pair fluxes). The relationship between ion pairing and the electrical conductivity of BLM is briefly discussed.     

Further inhibition studies on guanidinobenzoatase, a trypsin-like enzyme associated with tumour cells

Guanidinobenzoatase is a proteolytic enzyme capable of degrading fibronectin and is a tumour associated enzyme. Guanidinobenzoatase has been shown to be an arginine selective protease and is distinct from trypsin, plasminogen activator, plasmin, thrombin and a newly described tumour associated enzyme specific for guanidino phenylalanine residues. These conclusions have been derived from inhibition studies employing 4-methyl-p-guanidinobenzoate as substrate. Three active site titrants for trypsin have been shown to be good substrates for guanidinobenzoatase. A new active site titrant for trypsin, rhodamine bisguanidinobenzoate, can also be used to assay guanidinobenzoatase in a stoichiometric manner. This active site titrant can be employed to label guanidinobenzoate on the surface of leukaemia cells.     

EGTA induced fast potentials in rat small intestine.

Rat small intestine exhibits spontaneous slow-waves and spikes in normal solution. When treated with 0.3 – 0.7 mM EGTA, in calcium free solution, normal rhythmicity disappears and fast rhythmic potentials of duration intermediate to slow-waves and spikes appear. These induced fast potentials are absent in sodium free solution and are eliminated by verapamil, a calcium channel blocker. Application of EGTA to cat, guinea pig, mouse, and toad intestine did not yield fast potentials. The fast potentials appear to result from sodium entering through channels usually used by calcium. The fast potentials may be a phenomena exclusive to rat small intestine.