Dieter Gillessen

New coupling reagents in peptide chemistry

2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU) has been applied as coupling reagent to solid phase peptide synthesis. Furthermore, a general synthetic procedure for new derivatives of different N-hydroxy compounds has been developed. They either act as excellent activating reagents causing low racemization during condensation of peptide segments or are useful tools for the formation of active esters suitable for couplings in mixed aqueous / organic media, respectively.

Identification and immunohistochemical mapping of GABAA receptor subtypes containing the δ-subunit in rat brain

Synaptic inhibition in brain is mainly mediated via GABAA receptors which display a striking structural heterogeneity. A novel type of GABAA receptor subunit, the δ‐subunit, has recently been described based on molecular cloning of its cDNA [1]. To identify the prevalence and distrubution of GABAA receptors which contain the δ‐subunit protein in situ, polyclonal site‐directed antisera were developed against three synthetic peptides derived form the rat δ‐subunit cDNA‐sequence. All antisera specifically recognized a 54 kDa protein in GABAA receptor preparation. Nearly 30% of the GABAA receptors contained the δ‐subunit immunoreactivity and displayed high affinity GABA and high affinity benzodiazepine binding sites as shown by immunoprecipitation. Receptors which contain the δ‐subunit were immunohistochemically shown to be restricted to a few brain areas such as the cerebellum, thalamus and dentate gyrus of the hippocampal formation. Thus, those neurons which express GABAA receptors with a δ‐subunit have now been visualized and made accessible for a functional analysis of this GABAA receptors subtype in situ.

Expression, purification, biochemical characterization and inhibition of recombinant Plasmodium falciparum aldolase

The energy metabolism of the blood stage form of the human malaria parasite Plasmodium falciparum is adapted to the host cell. Like erythrocytes, P. falciparum merozoites lack a functional citric acid cycle. Generation of ATP depends therefore fully on the glycolytic pathway. Aldolase is a key enzyme of this pathway and a high degree of sequence diversity between parasite and host makes it a potential drug target. We have expressed the enzyme in its tetrameric form in Escherichia coli and the catalytic constants Vmax and Km of the recombinant enzyme correspond to the constants of parasite-derived aldolase. Rabbit antibodies against the recombinant P. falciparum aldolase inhibit the natural enzyme and no cross-reaction with human aldolase is detectable. Both the recombinant and the natural protein bind to the cytosolic domain of the band 3 membrane protein in vitro. A 19-residue synthetic peptide corresponding to the sequence of the binding domain of band 3 is an inhibitor when included in the binding assay. In addition, this peptide inhibits the catalytic activity of recombinant P. falciparum aldolase when assayed in a buffer system devoid of anions such as chloride or phosphate. The band 3-derived peptides compete with the aldolase substrate fructose-1,6-diphosphate for binding, suggesting that both reagents have a high affinity for the substrate pocket. A similar sequence motif exists in P. falciparum actin II. A 19-residue peptide corresponding to this sequence is also an inhibitor which could suggest that the P. falciparum aldolase can associate with the cytoskeleton of the parasite or of the host.

Ontention of antisera against a hypothalamic decapeptide (luteinizing hormone/follicle stimulating hormone releasing hormone) which stimulates the release of pituitary gonadotropins and development of its radioimmunoassay

Abstract Using the classical approach, a decapeptide was synthesized with the structure of porcine luteinizing hormone/follicle stimulating hormone releasing hormone reported by Matsuo, H., Baba, Y., Nair, R. M. G., Arimura, A. and Schally, A. V. (1971) Biochem. Biophys. Res. Commun. 43, 1393–1399. As already reported, this peptide was capable of inducing in vitro the release of luteinizing hormone and follicle stimulating hormone from rat pituitary glands. A specific antiserum against luteinizing hormone/follicle stimulating hormone releasing hormone has been generated in the guinea pig and this allowed the development of a radioimmunoassay for this peptide. The antisera, at a final dilution of 1 100 to 1 600 depending on the antiserum used, were able to bind 35% of the 131 I-labelled antigen. The sensitivity of this assay method was 50 pg of luteinizing hormone/follicle stimulating hormone releasing hormone. The following substances did not cross-react: oxytocin, lysine-vasopressin, synthetic thyroid stimulating hormone releasing hormone, ovine luteinizing hormone, follicle stimulating hormone and prolactin. Des-Trp 3 luteinizing hormone/follicle stimulating hormone releasing hormone, pyroglutamyl-histidyl-tryptophan and seryl-tyrosyl-glycyl-leucyl-arginyl-prolyl-glycinamide, exhibited flatter curves than luteinizing hormone/follicle stimulating hormone releasing hormone with a cross-reactivity of about 1 1000 . Using this method, luteinizing hormone/follicle stimulating hormone releasing hormone was assayed in extracts of the sheep stalk-median eminence and of the hypothalamus and in jugular vein blood from a normal ram and from normal male rats, from cyclic ewe and from hypophysectomized ram and rats. It was concluded that luteinizing hormone/follicle stimulating hormone releasing hormone is present in hypothalamic extracts and in plasma of sheep and rat.

Identification of the γ2-subunit protein in native GABAA receptors in brain

Abstract GABAA receptors with functional benzodiazepine receptors have been reconstituted by coexpression of α-, β- and γ-subunit cDNAs. In brain, proteins of the α- and β-subunits, but not the γ2-subunit have been identified. Using an antipeptide antiserum, we now demonstrate that the γ2-subunit is a protein of 43 kDa. It is present in at least 50% of GABAA receptors as shown by immunoprecipitation.

α1-microglobulin from normal and pathologigal urines

Summary α 1 -Microglobulin was purified from normal and pathological urines. Significant differences were found in the amino acid compositions of the α 1 -microglobulin isolated from these two sources. In addition electrofocusing of α 1 -microglobulin from normal urine gave rise to two peaks of equal intensity with rather acidic isoelectric points (3.8 and 4.2), whilst α 1 -microglobulin from pathological urine showed two peaks in a 1:5 ratio with less acidic isoelectric points (4.2 and 4.7). Further charge heterogeneity was also observed in the second peaks from both sources. The sugar compositions were also established, as well as the N-terminal sequences of the α 1 -microglobulin of both peaks isolated from normal and pathological urines.

Investigations on the primary structure of human plasminogen. Further evidence for sequence homology.

NH2-Terminal sequences were determined by the automated Edman method in four fragments which were isolated from a mixture of fragments obtained by CNBr cleavage of human plasminogen. One of the fragments whose sequence was determined over the first 31 residues shows sequence homologies with the fragment that forms the linkage between the plasmin chains and also with the non-thrombin part of prothrombin.

Biological activity of the C-terminal octapeptide of cholecystokinin, of three of its analogues and of caerulein in the dog

Dose-response curves of the C-terminal octapeptide (CCK-8) of cholecystokinin, of 3 of its methoxinine analogues, and of caerulein for various variables of exocrine pancreatic secretion have been established in conscious dogs. The following relative potencies were calculated for the protein secretion activity of CCK-8 (100%), [Mox3]-CCK-8 (52%), [Mox6]-CCK-8 (27%), [Mox3,Mox6]-CCK-8 (19%) and caerulein (178%).

Synthesis of [4-leucine]-arginine-vasotocin, a natriuretic analogue of arginine-vasotocin

Es wird die Synthese des 4-Leucin-Analogen des Arginin-vasotocins (=[Leu4, Arg8]-oxytocin) beschrieben.

Inhibition of the reactivation of acid-dissociated lactate dehydrogenase isoenzymes by their aminoterminal CNBr fragments

The catalytic activity of lactate dehydrogenase isoenzymes (LDH) depends on their tetrameric structure. Stabilization of this quaternary structure is achieved by interaction of the N-terminal part of one subunit with the C-terminal region of the other subunit. The N-terminal peptides from pig M-LDH and H-LDH which are responsible for this stabilization were obtained by CNBr-fragmentation and purification on reversed-phase HPLC. The effect of these peptides on the formation of the quaternary structure of LDH-isoenzymes was investigated by monitoring the reconstitution of the catalytic activity after acid-dissociation. Low concentrations of the N-terminal peptides led to an increased, and high concentrations to a decreased yield of reconstituted LDH activity. The effects of these two peptides were isoenzyme specific. The 32 residue peptide derived from M-LDH showed the highest effect when tested with M-LDH as target enzyme but only a poor effect with H-LDH. On the other side the 33 residue peptide generated from H-LDH showed a moderate effect with both isoenzymes. The effects of the N-terminal LDH peptides are antagonized by the coenzymes NAD+ and NADH. The most significant influence was observed with NAD+ in the M-LDH peptide-M-LDH enzyme system. Comparison of the properties of the reactivation antagonists isolated from human origin with the N-terminal CNBr-peptides of LDH revealed identity in all essential properties, suggesting that the former peptides are generated by degradation of LDH.