Specific binding of angiogenin (ANG) to calf pulmonary artery endothelial cells was demonstrated. Cellular binding at 4TC of 125-Ilabeled human recombinant ANG was time and concentration dependent, reversible, and saturable in the presence of increasing amounts of the unlabeled molecules. The interaction was shown to be specific since a large excess of unlabeled ANG reduced labeled ANG binding by >80%, whereas similar doses of RNase A, a structurally related protein, had no effect. Scatchard analyses of binding data revealed two apparent components. High-affinity sites with an apparent dissociation constant of 5 x 10-9 M were shown to represent cell-specific interactions. The second component, comprising low-affinity/high-capacity sites with an apparent dissociation constant of 0.2 x 10-6 M, was essentially associated with pericellular components. High-affinity ANG binding sites varied with cell density and were found on other endothelial cells from bovine aorta, cornea, and adrenal cortex capillary but not on Chinese hamster lung fibroblasts. Divalent copper, a modulator of angiogenesis, was found to induce a severalfold increase in specific cell-bound radioactivity. Placental ribonuclease inhibitor, a tight-binding inhibitor of both ribonucleolytic and angiogenic activities of ANG, abolished 1251-labeled human recombinant ANG binding only in the absence of copper.

Angiogenin is a potent inducer of blood-vessel formation with ribonucleolytic activity. Angiogenin binds to high affinity endothelial cell receptors and with lower affinity to extracellular matrix components. Here we report the effect of copper and zinc on these interactions. There was a 4.3-fold increase in angiogenin binding to calf pulmonary artery endothelial cells in the presence of Cu2+ in vitro. A 3.8-fold increase was observed with Zn2+, whereas Ni2+, Co2+, or Li+ had no effect. Specific angiogenin binding to the lower affinity matrix sites was increased by 2.7- and 1.9-fold in the presence of Cu2+ and Zn2+ respectively. Metal ion affinity chromatography and atomic absorption spectrometry were used to show the direct interaction of angiogenin with copper and zinc ions. Angiogenin bound 2.4 mol of copper per mole of protein. We suggest that copper, a modulator of angiogenesis in vivo, may be involved in the regulation of the biological activity of angiogenin.

One link in the complex chain of medical economics is the cost of bringing new drugs and biologicals to the market. Advances in recombinant-DNA technology permit production of therapeutically active proteins in effectively unlimited quantities. Nevertheless, each expression system has a characteristic influence on the nature of the product produced and the process required to obtain it. In this case study we compare experiences with recombinant-tissue plasminogen activator (rtPA) produced in Chinese hamster ovary (CHO) cells and in Escherichia coli, with the aim of understanding the roles of some of the parameters that affect process economics. tPA belongs to the group of highly specific serine proteases that convert plasminogen to plasmin, which in turn degrades several protein substrates including fibrin, thus making it an effective thrombolytic agent. The treatment of acute myocardial infarction with such thrombolytic agents can result in early discharge of patients and decreased medical costs. However, there are major differences in the prices of the various available agents. The price of the FDA-licensed tPA product is $2,200 per dose or $22,000 per gram. It is believed that a significant portion of this price relates to manufacturing costs. We examine by way of case study illustration the cost breakdown for the two processes, and highlight important process, design and economic considerations that ultimately define a particular protein product.

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