Shmaryahu Blumberg

Enhancement of phagocytosis - a newly found activity of substance P residing in its N-terminal tetrapeptide sequence.

Abstract The undecapeptide Substance P stimulates phagocytosis by mouse macrophages and human polymorphonuclear leukocytes. The activity of Substance P resides in its N-terminal tetrapeptide protion. Substance P and its N-terminal tetrapeptide are as active as tuftsin in their phagocytosis-stimulating activity and compete with tuftsin for its binding sites. The phagocytosis-enhancing activity of Substance P may play a role in inflammatory processes of neural origin where the involvement of the peptide has been implicated.

Purification of soybean agglutinin by affinity chromatography On sepharose-N-ϵ-aminocaproyl-β-D-galactopyranosylamine

Affinity chromatography has been successfully used for the purification of proteins with specific binding sites, such as enzymes, antibodies and lectins [l-3]. Soybean agglutinin (SBA), a lectin isolated from soybean oil meal [4,5] has been shown to bind specifically N-acetyl-D-galactosamine and D-galactose [6]. This property has now been utlized by us for the purification of SBA on a column made of a conjugate of Sepharose and N-e-aminocaproyl-/3-D-galactopy ranosylamine (SAG). The method used for the preparation of SAG was essentially the same as that developed very recently by Blumberg et al. [7] for the binding of p-L-fucopyranosylamine to Sepharose. N-e-aminocaproylQ-Dgalactopyranosylamine was prepared by a reaction of /3-D-galactopyranosylamine (1 Q-amino-l -deoxy-Dgalactopyranoside) and N-benzyloxycarbonyl-eaminocaproic acid [8], and removal of the benzyloxycarbonyl group by hydrogenolysis. The product was coupled to Sepharose by the cyanogen bromide method of Ax&r et al. [9] as described by Blumberg et al. [lo]. SBA was adsorbed from a partially purified extract of soybean oil meal to a column made of SAG, and the active material was eluted from the column by a solution of D-galactose. Separation of SBA from minor agglutinating components present in the soybean oil meal [ 111 was achieved by chromatography on DEAE cellulose. From 50 g of soybean oil meal, 80 mg of purified SBA, with a specific ac-

Purification by affinity chromatography of acetylcholinesterase from electric organ tissue of the electric eel subsequent to tryptic treatment

1. 1.|A Sepharose-acetylcholinesterase inhibitor conjugate was prepared by synthesis of the inhibitor [N-(e-aminocaproyl)-p-aminophenyl]trimethylammonium bromide hydrobromide and its covalent linkage to the CNBr-activated resin. 2. 2.|The Sepharose-inhibitor conjugate was employed for purification of acetyl-cholinesterase (acetylcholine hydrolase, EC 3.1.1.7) from electric organ tissue by affinity chromatography. Thus the enzyme was selectively adsorbed on the resin and specifically eluted with the soluble acetylcholinesterase inhibitor, decamethonium bromide. 3. 3.|The Sepharose-inhibitor conjugate adsorbed the different molecular species of acetylcholinesterase (differing in sedimentation coefficient) present in electric organ tissue. Since two of these species aggregate at low ionic strength, the partially purified enzyme also displayed this property. 4. 4.|Highly purified acetylcholinesterase was obtained if affinity chromatography was preceded by controlled tryptic digestion or prolonged autolysis causing conversion of the enzyme to an 11-S form which does not aggregate at low ionic strength. 5. 5.|Purified acetylcholinesterase was obtained in an overall yield of 40%. It was essentially homogeneous on acrylamide-gel electrophoresis, and its sedimentation coefficient (approx. 11 S), specific activity and amino acid composition resembled those previously reported for purified acetylcholinesterase. 6. 6.|Active site titration of the purified enzyme yielded an equivalent weight of 107 000 per active site. Acrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol revealed two major polypeptide components of molecular weights approx. 88 000 and 64 000. 7. 7.|The properties of the purified acetylcholinesterase are compared with those of the purified preparations previously reported, and its relationship to the molecular species present in intact electric organ tissue is discussed.

Biological activity and enzymic degradation of substance P analogs: implications for studies of the substance P receptor.

Abstract Partial sequences of Substance P, either free or blocked at their amino terminal, have been examined for their stability towards inactivation by homogenate or particulate fractions of rat brain and for their relative potencies as smooth muscle contractors. The C-terminal hexapeptide in both the free and blocked forms displays activity comparable to that of the longer C-terminal peptides as well as to that of the native undecapeptide. The blocked peptides, however, are much more stable than their corresponding free peptides. Among the free peptides Substance P is degraded slower than the free hexa- and hepta-peptides, suggesting that the N-terminal tetrapeptide part may play a role in stabilizing the molecule. Blocked hepta- and octapeptide analogs, carrying probe properties, may be useful for studies of the Substance P receptor.

Neurotensin — macrophage interaction: Specific binding and augmentation of phagocytosis

Abstract Neurotensin (NT) was found to bind to thioglycollate-elicited mouse peritoneal macrophages and to modulate their phagocytic capability. A Scatchard analysis of the binding curve of [ 3 H]NT suggests the presence of two subclasses of binding sites having a.−a K D of 0.9 nM (4800 sites per cell) and b.−a K D of 28 nM (33500 sites per cell). NT as well as two of its partial sequences, NT(8–13) and NT(6–13) competed with [ 3 H]NT for its binding whereas NT(1–10) was rather ineffective. [ 3 H]NT was also competitively displaced by tuftsin, substance P (SP) and by SP (1–4). The K I values estimated for all the above competitive inhibitors of [ 3 H]NT binding (except for NT) suggest interaction with the relatively low affinity sites. NT exerts a biphasic effect on the phagocytic response of macrophages. At a concentration range of 10 −14 –10 −9 M NT had a dose dependent augmenting effect on the phagocytic response (up to 2 fold), further increase in concentration (>10 −9 M) of NT resulted in a gradual decrease of the augmented response which disappears at 10 −7 M NT. NT(8–13), NT(6–13) as well as NT(1–10) augment the phagocytic response of macrophages. However the maximal effect with these peptides was attained at about 10 −7 M and stayed at the same level at concentrations up to 10 −5 M. The phagocytosis augmenting dose-response curves of these peptides resembled that of tuftsin and SP, two unrelated peptides. It is suggested that NT-phagocyte interaction may be of relevance in the regulation of the functions of phagocytic cells.

Distinct classes of substance P receptors revealed by a comparison of the activities of substance P and some of its segments

The contracting potency of Substance P and of its C-terminal fragments was studied using four isolated preparations of smooth muscle. The Substance P receptors in the four muscles studied can be differentiated on the basis of their interactions with Substance P and its C-terminal fragments. On the guinea pig ileum, the potency of Substance P is equal to that of the C-terminal octa- and heptapeptide segments and in the rat ileum the potency of Substance P is equal to that of the C-terminal octapeptide and even higher than that of the heptapeptide. In contrast, on the cow pupillary sphincter and guinea pig urinary bladder, Substance P is markedly less potent that the C-terminal octa-, hepta- and hexapeptides. These results suggest the existence of different classes of Substance P receptors and indicate that the N-terminal sequence may be important in regulating Substance P activity.

Inhibition of enkephalin-degrading enzymes from rat brain and of thermolysin by amino acid hydroxamates

Abstract Benzyloxycarbonyl derivatives (Z) of amino acid hydroxamates have been found to inhibit the bacterial metalloendopeptidase thermolysin and enkephalin-degrading enzymes from rat brain. The hydroxamate derivatives of glycine, leucine, phenylalanine and D-phenylalanine inhibit thermolysin with K I values in the range of 3–23 μM. They also inhibit the enkephalin-degrading endopeptidase (enkephalinase) and aminopeptidase with different efficiencies, depending on the structure of the amino acid employed. Thus, Z-Gly-NHOH inhibits the enkephalinase and aminopeptidase with IC 50 values of 1 μM and 300 μM, respectively, whereas Z-D-Phe-NHOH inhibits the corresponding enzymes with IC 50 values of 0.2 μM and 1.5 μM.

Labelling of the active site of β-galactosidase by N-bromoacetyl β-D-galactopyranosylamine

A’-Bromoacetyl P-D-galactopyranosylamine was designed as an affinity label for Zac-permease [l] which has a reactive sulphydryl group at its substrate binding site [2] . The observed irreversible inactivation of the active transport of lac-permease substrates by this reagent is, however, not site-specific since active transport mediated by another permease, a MG-permease, is also inactivated [3] . In the course of our investigations of kc-permease, this reagent was found to inactivate /3-galactosidase in intact E. coli cells [4] . We describe here the inactivation of purified fl-galactosidase by N-bromoacetyl&D-galactopyranosylamine. One mole of reagent is bound to the enzyme per mole of site inactivated. The described labelling procedure should facilitate the study of the peptide sequence in the vicinity of the active site of this enzyme.

The effect of human recombinant erythropoietin on the growth of a human neuroblastoma cell line.

Erythropoietin is a growth factor. Cancer can be described as disturbance of the fine balance of positive and negative growth control mechanisms. The effect of human recombinant erythropoietin (EPO) was studied on the cell growth and differentiation of a human neuroblastoma cell line (h-NMB). Cell growth curves, trypan blue staining and thymidine uptake were used to assess cell proliferation and death. To assess cell differentiation, neutral endopeptidase (cell membrane enzyme marker), creatine kinase (cytosolic enzyme marker), dopamine uptake (dopamine transporter marker) and cell morphology were determined. Specific EPO receptor mRNA, by RT-PCR technique, was demonstrated. The incubation of erythropoietin with the tumor cell line resulted in inhibition of cell proliferation as evidenced in a diminished cell growth. EPO was shown to have induced a differentiation process as seen from the two different enzymatic markers, membranal and cytosolic, and from the cells dopamine uptake studies. However, the morphological changes did not document a full differentiation effect. EPO specific antibodies blocked the effects of EPO on cell proliferation and creatine kinase activity. In this study, EPO did not produce any sign of proliferation in the nervous tumor cell line used.

Enhancement of phagocytosis by neurotensin, a newly found biological activity of the neuropeptide.

Specific binding of neurotensin (NT) to mouse peritoneal thioglycollate-elicited macrophages and macrophages differentiated in vitro from bone marrow cells was demonstrated and characterized. NT binding to these phagocytes modulated their phagocytic capacity in a biphasic manner. At concentrations of 10(-14) to 10(-9) M NT, a dose-dependent augmentation of phagocytosis (up to 2-fold) was observed. Further increases in the concentration of NT resulted in a gradual decrease of the augmented response until the basal phagocytic activity (in the absence of NT) was reached. Three partial sequences of NT, NT (8-13), NT (6-13) and NT (1-10), were also effective in augmenting the phagocytic response of thioglycollate elicited macrophages, but the maximal effect was attained at about 10(-7) M and stayed at that level up to a concentration of 10(-5) M. The activity of the three NT partial sequences was comparable to that of substance P and tuftsin. Scatchard analysis of (3H)NT binding to macrophages suggested the existence of two populations of binding sites, a major population of relatively low affinity binding sites and a small population of high affinity binding sites. NT (8-13), NT (6-13), substance P and tuftsin competed with (3H)NT binding to the low affinity sites with a comparable KI to that of NT. NT (1-10) did not compete for the binding at the low affinity sites. It is suggested that NT binding to the high affinity sites leads to enhancement of phagocytosis, whereas its binding to the low affinity sites leads to inhibition of the augmented response. However, the low affinity sites are the sites of interaction of NT (8-13), NT (6-13), substance P and tuftsin with the phagocytes and their saturation with the peptides leads to augmentation of phagocytosis.