Induction of angiogenesis by mixtures of two angiogenic proteins, angiogenin and acidic fibroblast growth factor, in the chick chorioallantoic membrane.

The chick chorioallantoic membrane assay was employed to assess the angiogenic response induced by mixtures of human angiogenin with bovine heparin-binding acidic fibroblast growth factor. Statistical evaluation of data accumulated at several molar ratios of the two proteins indicate that the angiogenic activity observed is neither an additive nor a synergistic resultant of the activities of the proteins separately. The possibility exists, however, that at an approximately 1:1 mole ratio an apparent inhibitory effect can be observed. Mechanisms which could underlie such observed effects are discussed.

Angiogenic activity of human tumor plasma membrane components.

Plasma membranes from the human colon adenocarcinoma cell line HT-29 have been isolated and examined for the presence of angiogenic activity. Membrane-associated macromolecules extracted with Triton X-100 were fractionated on immobilized wheat germ agglutinin. The fraction which bound specifically (about 200 ng of protein/mL packed cells) was highly angiogenic when assayed on the chick embryo chorioallantoic membrane. As little as 0.2 ng of this human tumor derived material consistently induced neovascularization. Similarly, 1-2 ng of this material implanted into the rabbit cornea induced new vessel growth (5-8 mm) within 10 days. The plasma membranes of eight other human tumor lines were examined for angiogenic activity. For each, the wheat germ agglutinin bound material induced neovascularization at the low nanogram level. In contrast, the wheat germ agglutinin bound material derived from purified plasma membranes of two normal human diploid fibroblast cell lines failed to induce an angiogenic response on the chick chorioallantoic membrane, even at microgram levels.

Amino acid sequence of human tumor derived angiogenin.

The amino acid sequence and disulfide bond pairing of human tumor derived angiogenin, the first tumor angiogenesis factor to be isolated in pure form from human sources, have been determined by conventional sequencing techniques adapted and applied to nanomole and subnanomole levels of material. Angiogenin, obtained from conditioned media of a human colonic adenocarcinoma cell line, is a single-chain protein consisting of 123 amino acids with the following sequences: less than Glu1-Asp-Asn-Ser-Arg-Tyr-Thr-His- Phe-Leu-Thr-Gln-His-Tyr-Asp15-Ala-Lys-Pro-Gln-Gly-Arg-Asp-Asp- Arg-Tyr-Cys-Glu-Ser-Ile-Met30- Arg-Arg-Arg-Gly-Leu-Thr-Ser-Pro-Cys-Lys-Asp-Ile-Asn-Thr- Phe45-Ile-His-Gly-Asn-Lys-Arg-Ser -Ile-Lys-Ala-Ile-Cys-Glu-Asn-Lys60-Asn-Gly-Asn-Pro-His-Arg-Glu-Asn -Leu-Arg-Ile -Ser-Lys-Ser-Ser75 -Phe-Gln-Val-Thr-Thr-Cys-Lys-Leu-His-Gly-Gly-Ser-Pro-Trp-Pro90-Pro -Cys-Gln-Tyr -Arg-Ala-Thr-Ala -Gly-Phe-Arg-Asn-Val-Val-Val105-Ala-Cys-Glu-Asn-Gly-Leu-Pro-Val- His-Leu-Asp-Gln-Ser-Ile-Phe120-Arg-Arg-Pro123-OH. Three disulfide bonds link the half-cystinyl residues 26-81, 39-92, and 57-107. The sequence is homologous to that of the pancreatic ribonucleases with 35% identity and many of the remaining residues conservatively replaced. Similarities are especially apparent around the major active-site residues His-12, Lys-41, and His-119 of ribonuclease which are conserved as are three of the four disulfide bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of angiogenin, an angiogenic protein from human carcinoma cells.

The first human tumor derived protein with in vivo angiogenic activity to be obtained in pure form has been isolated from serum-free supernatants of an established human adenocarcinoma cell line (HT-29) and named angiogenin. It was purified by cation-exchange and reversed-phase high-performance liquid chromatography; the yield was approximately 0.5 microgram/L of medium. Biological activity of angiogenin was monitored throughout purification by using the chick embryo chorioallantoic membrane assay. Statistical evaluation demonstrates that it displays activity in this system with as little as 35 fmol per egg. Moreover, only 3.5 pmol is required to induce extensive blood vessel growth in the rabbit cornea. The amino acid composition of this basic (isoelectric point greater than 9.5), single-chain protein of molecular weight approximately 14 400 has been determined. The amino terminus is blocked, and the carboxyl-terminal residue is proline.

Tumor-derived angiogenesis factors from rat Walker 256 carcinoma: an experimental investigation and review.

Angiogenesis, the process of developing a hemovascular network, is an essential feature of the growth of solid tumors, and is induced by factors secreted by tumor cells. Assay procedures suitable for the investigation of angiogenesis, and for the screening of angiogenesis factors during purification are reviewed; and a number of reports describing the purification of angiogenesis factors, primarily from the rat Walker 256 carcinoma as starting material, are discussed. Work from the authors' laboratory is also presented. Walker 256 cells grown in large-scale culture were the source of a reproducible and homogeneous source of angiogenic material. Factors secreted by these cells were isolated by a series of chromatographic steps. Ion exchange chromatography on carboxymethyl-Sephadex produced two active fractions, one of which was fractionated into several macromolecular species by lectin affinity and hydrophobic adsorption chromatography. The other gave a high mol.wt, active fraction that was resolved into a low mol.wt, active component and a non-angiogenic but possibly carrier molecule with a mol.wt of 140,000. While none of the angiogenic factors were identified chemically, the results demonstrate the existence of both high and low mol.wt tumor-secreted angiogenic substances, confirming the hypothesis for tumor-induced angiogenesis and predicting potential means to interfere with the process of tumor growth.

Formamide-induced dissociation and inactivation of Escherichia coli alkaline phosphatase. Metal-dependent reassociation and restoration of activity from isolated subunits.

Alkaline phosphatase from Escherichia coli has been reversibly dissociated by treatment with low concentrations of formamide. The monomer retains the capacity to bind metals and to regenerate catalytically active dimer that is identical with the native dimeric enzyme. The rate and extent of dissociation of dimer to monomer depend upon pH, ionic strength, temperature, formamide concentration, and enzyme-bound metal. Under appropriate experimental conditions, reassociation can be greatly slowed, allowing the properties of the monomer to be examined in solution. The formamide-induced apo monomer has a conformation distinct from that of the dimer and zinc- or cobalt-containing monomers. The monomer tightly binds 1 mol of zinc or cobalt in a metal-binding site altered from those of the dimer but is catalytically inactive. pH, ionic strength, and formamide concentration all influence reassociation. Hydrophobic forces are implicated as important in subunit interactions. The effect of metal content on the dissociation--reassociation process underscores the essential role that metals play in maintaining enzyme tertiary structure and reveals a new role in stabilizing the quaternary structure.

Kinetic properties of crystalline enzymes. Carboxypeptidase A.

Spectrochemical probes have demonstrated that the conformations of carboxypeptidase A differ in solution and in the crystalline state. Detailed kinetic studies of carboxypeptidase A crystals and solutions now show that the physical state of the enzyme is also a critical parameter that affects this enzyme's function. Thus, for all substrates examined, crystallization of the enzyme markedly reduces catalytic efficiency, kcat, from 20- to 1000-fold. In addition, substrate inhibition, apparent in solution for some di- and depsipeptides, is abolished with crystals, while longer substrates with normal kinetics in solution may exhibit activation with the crystals. The physical state of the enzyme also affects the mode of action of known modifiers of peptidase activity of the enzyme. In solution, addition of benzoylglycine or cinnamic acid markedly increases the rate of hydrolysis of CbzGly-Phe, but, with the crystalline enzyme, their addition hardly alters the activity. This is in accord with the weakening or absence of inhibitory enzyme-substrate binding modes. Kinetic studies on crystals were carried out over a range of enzyme concentrations, substrate concentrations, and crystal sizes, and in all instances the results are in good agreement with the theory developed by Katchalski for enzymes insolubilized by other means. Importantly, these kinetic parameters are determined under conditions which obviate artifacts due to diffusion limitation of substrates or products. The differences in the kinetic behavior of carboxypeptidase crystals, on the one hand, and of their solutions, on the other hand, bear importantly on efforts to interpret the function of the enzyme in structural terms. Hypothetical modes of substrate-enzyme interaction, generated by superimposing substrate models on the crystal structure of carboxypeptidase to stimulate kinetics in solution, have failed to detect all of these changes which affect inhibitory or activating binding modes.

The physical state dependence of carboxypeptidase Aalpha and Agamma kinetics.

Spectrochemical probes have demonstrated that the conformations of carboxypeptidase A (EC 3.4.12.2) differ in solution and in the crystalline state. Detailed kinetic studies of carboxypeptidase A(alpha) and A(gamma) crystals and solutions now show that the physical state of the enzyme is also a critical parameter that affects the function of the A(alpha) and A(gamma) enzymes in the same manner. The kinetic profiles and the corresponding kinetic constants of substrate hydrolysis are, therefore, important functional indices of the known conformational differences of the enzyme in these two physical states. The complex kinetic behavior of this enzyme, however, precludes meaningful comparisons of activity measurements for crystals and solutions obtained at only one substrate concentration. Underlying differences in varying substrate-inhibiting or -activating binding modes can result in either high or low activity ratios, concealing the true, functional consequences of the change in physical state. Thus, for all substrates examined, crystallization of the enzyme markedly reduces catalytic efficiency, k(cat), from 20- to 1000-fold. Equally as important, the substrate inhibition, apparent in solution for some di- and depsipeptides, is abolished with crystals, while for longer substrates the normal solution kinetics may acquire activation with the crystals. Hypothetical modes of substrate-enzyme interaction, generated by superimposing substrate models on the crystal structure of carboxypeptidase to simulate kinetics in solution, have failed to detect both of these changes, which affect inhibitory or activating binding modes. The only structure of carboxypeptidase yet published and that of its functionally inert complex with the pseudosubstrate, glycyl-L-tyrosine, derive from a unique form of carboxypeptidase A(alpha) crystals. These crystals differ from all others with regard both to their spectral properties and activity toward carbobenzoxy-glycyl-L-phenylalanine, which is 30% of that in solution, though the significance of this value cannot be gauged without knowledge of the relevant kinetic constants. The rapidly accumulating evidence for functional and conformational differences between crystals and solutions and the recent stress on the nonproductive aspects of the carboxypeptidase A(alpha)-glycyl-L-tyrosine complex, based on 30% site occupancy, suggest that the functional implications of its structural features require reevaluation.