Margaret M. Griffin

Aggregation of fibrinogen molecules by metal ions

The ability of metal ions to cause physical aggregation of neutral solutions of bovine fibrinogen has been studied. Three categories were found: (a) ions (such as Ca2+, Mg2+ and Mn2+) which did not cause aggregation even when present in 1–100 mm concentrations: (b) ions (such as Fe2+, Cu2+ and Ni2+) which caused aggregation in the 0–10 mm concentration range, (c) ions (such as Hg2+, Zn2+, Cr3+, La3+) which caused aggregation in the 0–1000 μm concentration range. Aggregation occurs immediately the metal ion is brought into contact with the fibrinogen, and product formation reaches a steady state within 5 min. With the exception of Zn2+, all the ions that caused aggregation exhibited a threshold concentration below which no observable aggregation took place. The threshold concentration for Hg2+, the most effective ion studied, was 6 μm. Addition of excess EDTA caused resolubilization of the aggregated fibrinogen due to removal of the metal ions. Aggregation is thus thought to be a physical process initiated by binding of metal ions to those carboxyl groups in fibrinogen responsible for keeping the monomers apart in solution. The aggregation does not involve prior proteolytic degradation of the fibrinogen.

The role of inhibitors in the fluorescent staining of benign naevus and malignant melanoma cells with 9-amino acridine and acridine orange.

Guanidinobenzoatase is a trypsin-like protease capable of degrading fibronectin. An inactive form of guanidinobenzoatase is present on the surface of benign naevus cells and these cells stain very weakly with 9-aminoacridine, a known competitive inhibitor of guanidinobenzoatase. Malignant melanoma and metastatic malignant melanoma cells exhibit strong surface staining with 9-aminoacridine and also exhibit strong staining of cytoplasmic RNA with acridine orange. These simple fluorescent techniques have been used to distinguish benign naevus cells from malignant melanoma cells in human skin sections. This difference in cell surface staining with 9-aminoacridine has been demonstrated to be caused by the presence or absence of an inhibitor. The inhibitor can be displaced from the cell surface enzyme and then replaced by an affinity purified inhibitor obtained from fresh liver homogenates. It is proposed that the inhibition or control of cell surface guanidinobenzoatase may be one of the regulatory mechanisms by which benign naevus cells are prevented from developing into malignant melanoma cells.

EVIDENCE FOR CARRIAGE OF SILVER BY SULPHADIMIDINE: HAEMOLYSIS OF HUMAN ERYTHROCYTES

1 Human erythrocytes suspended in isotonic saline haemolyse in the presence of both Ag+ ions and sulphadimidine. 2 Neither Ag+ ions nor sulphadimidine on their own will haemolyse erythrocytes suspended in isotonic saline. 3 At constant Ag+ ion concentration the degree of haemolysis of saline‐suspended erythrocytes depends upon the concentration of sulphadimidine. 4 Human erythrocytes suspended in isotonic sucrose (chloride‐free) haemolyse in the presence of Ag+ ions. 5 Sulphadimidine in chloride‐free sucrose competes with erythrocytes for Ag+ ions resulting in stoichiometric protection of the erythrocytes from the haemolytic action of Ag+ ions. 6 Haemolysis occurs when each erythrocyte receives approximately 1.2 × 109 Ag+ ions whether suspended in saline or sucrose. 7 Sulphadimidine acts as a carrier for Ag+ ions and so prevents their precipitation as AgCl when erythrocytes are suspended in saline.

Evidence for inhibitors of the cell surface protease guanidinobenzoatase.

Guanidinobenzoatase is a protease present on the surface of tumour cells. The present study describes the isolation of a protein inhibitor of guanidinobenzoatase obtained from extracts of liver and pancreas and purified by affinity techniques. Pancreatic acinar cells have been shown to possess a latent form of guanidinobenzoatase and this latency is due to complex formation with the inhibitor. A fluorescent marker has been employed to demonstrate the presence or absence of the inhibitor on sections of pancreatic tissue. The inhibitor has been shown to be exchangeable with liver and pancreatic inhibitors obtained from different species. It is postulated that these inhibitors may play a role in enzyme control.

Biphasic kinetics of metal ion reactivation of trypsin-thiol complexes

This report describes biphasic kinetic data obtained when trypsin was inhibited by a thiol-containing inhibitor present in Ehrlich ascites tumour cells and then subjected to addition of Hg2+, Cu2+ or Ag+. This resulted in an initial re-activation of the trypsin, followed by inhibition of the enzyme with the addition of higher concentrations of these ions. The significance of these observations is 2-fold: (i) help to elucidate the mechanism of metal ion activation of latent enzymes, and (ii) also indicate that, in certain circumstances, the concentration of added metal ion determines whether the metal acts as an activator or an inhibitor of enzyme activity.

Inhibition properties of Sepharose-bound trypsin and a protease on the surface of Ehrlich ascites tumour cells.

Ehrlich ascites cells have been shown to possess a protease with beta-naphthylamidase activity located on the surface of these cells. This enzyme is protected from the inhibitory action of protein inhibitors of trypsin (EC 3.4.21.4) in free solution, but is inhibited by high concentrations of active site-directed inhibitors of trypsin. We believe the protection against inhibition is provided by the location of this protease on the cell surface. We employed a model system of trypsin coupled to Sepharose to demonstrate the protective action of an inert surface, resulting in a marked reduction in inhibition of trypsin-Sepharose, compared to trypsin in free solution, when exposed to both high and low molecular weight inhibitors. This cell protease has been shown to play a role in activation of the zymogen of collagenase exported by tumour cells. This role may have important implications for tumour cell invasion of the intercellular matrix.

Inhibition of trypsin and papain by sodium aurothiomalate mediated by exchange reactions.

1 Sodium aurothiomalate has been shown to participate in exchange reactions leading to the inhibition of trypsin; for this exchange to take place it was necessary to include in the test system a suitable thiol, such as N‐acetyl‐cysteine. 2 Neither N‐acetyl‐cysteine nor aurothiomalate on their own had any inhibitory action on trypsin. 3 The results indicate that aurothiomalate dissociates in the presence of a carrier to form thiosuccinate and gold. 4 The gold is responsible for trypsin inhibition since independent experiments demonstrated that the total concentration of thiosuccinate was insufficient to cause the observed inhibition of trypsin. 5 Bovine serum albumin was shown to act as a carrier in place of N‐acetyl‐cysteine. 6 It is known that histidine in the active centre of trypsin binds heavy metal ions with consequent inhibition of the enzyme. In this study, imidazole was shown to act as a carrier for gold from aurothiomalate to trypsin resulting in inhibition. This inhibition by gold was reversed when higher concentrations of imidazole were added to the test system due to competition for the trypsin‐bound gold by imidazole. 7 Conversely, the thiol enzyme papain was re‐activated in the presence of low concentrations of sodium aurothiomalate and inhibited by higher concentrations of this reagent in a biphasic manner. This observation will be discussed in relation to the dissociation of sodium aurothiomalate. 8 These observations can also be explained in terms of exchange reactions involving thiols and free metal ions.

Evidence for exchange of inhibitors which bind to the active site of trypsin: Displacement of one inhibitor with a competitive inhibitor

Two classes of inhibitors of trypsin (ED 3.4.21.4) have been studied, viz. active site-directed agents such as ovomucoid and active site titrants such as 4-methylumbelliferyl-4-guanidinobenzoate. The kinetics of beta-naphthyl-amidase inhibition by an active site-directed agent were markedly different from simultaneous assays of the availability of the active site towards active site titrants in the presence of the active site-directed agents. Analysis of these data indicated an exchange of active site-directed agent by subsequent addition of active site titrant. One class of trypsin inhibitor could be displaced by another from the trypsin active centre. Competitive chase experiments were designed to measure this exchange in which the active site-directed agent was first equilibrated with trypsin, then partially displaced by incremental additions of an active site titrant; the degree of active sites occupied by these two agents was then determined by active site titration with a second reagent.

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.

Design of fluorescent probes for an enzyme on the surface of tumour cells

The proteolytic enzyme guanidinobenzoatase is specific for arginyl peptide bonds and is capable of degrading fibronectin. This enzyme is associated with the cell surface of tumour cells in formaldehyde-fixed wax-embedded sections of human pathological tissue. We have designed fluorescent probes for the active site of guanidinobenzoatase: these probes act as competitive inhibitors and can be used to locate cells possessing guanidinobenzoatase. The processes of designing probes, testing their potential as inhibitors, and applying these probes to tumour cell location, all depend upon affinity principles.