P. de la Llosa

A new procedure for labelling luteinizing hormone with tritium

Studies of the specific binding of glycoprotein hormones to their receptors require labelled compounds of high specific radioactivity. The most frequent procedure for labelling glycoprotein hormones is iodination, but incorporation of tritium into t.he glycoprotein molecule by modification of the sialic acid residues has also been employed for this purpose [1,2]. We reported recently [3,4] that reductive methylation of ovine luteinizing hormone, using the procedure of Means and Feeney [5], could be achieved without loss of biological activity. We now report a procedure for the tritiation of LH* by reductive methylation of its lysine residues with tritiated NaBH4. The tritiated LH derivative fully retains its biological potency when measured by the ovarian ascorbic acid depletion bioassay of Parlow [6]. Our experiments show this derivative to be of advantage for the study of the binding of this hormone to its receptors ]7,8].

Involvement of lysine residues in the binding of hGH and bGH to somatotropic receptors

The biological activities of human (hGH) and bovine (bGH) growth hormone derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit liver homogenates for somatotropic activity (SA). Control treatment with BH4 − had a very slight effect on the SA, whereas the methylation and ethylation drastically reduced the acitivty of both hormones. Guanidination of these hormones and even acetimidination at a lower rate are accompanied by a considerable loss of biological activity. These results show the involvement of lysine residues in the interaction of hGH and bGH with somatotropic receptors. The structure‐function relationship of these molecules is discussed, suggesting that the lysine or arginine residues in positions 41, 64, 70 and 115 might be particularly implicated.

Involvement of lysine residues in the binding of ovine prolactin and human growth hormone to lactogenic receptors

The lactogenic activity (L.A.) of oPRL and hGH derivatives obtained by chemical modifications of lysine residues was studied by radioreceptor assay. Control treatment with borohydride had a slight effect on the L.A. of hGH but drastically reduced the oPRL activity; this latter was preserved in the presence of iodoacetamide. Methylation, ethylation, guanidination and acetimidination affected the L.A. of both hormones as a function of the degree of modification. The structure‐binding relationships to the lactogenic receptors are discussed, suggesting that the lysine or arginine residues in homologous positions 42, 51, 73, 128, 146 of oPRL and 47, 50, 73, 128, 147 of hGH might be particularly involved.

Isolation and characterization of protein kinase C from human placenta

Protein kinase C (Ca2+ and phospholipid-dependent protein kinase) was detected in human placenta and was partially purified using DEAE cellulose chromatography and Ultrogel filtration. Diolein alone did not act on this enzyme but exerted a strong stimulatory action when associated with phosphatidylserine and Ca2+. Similar results were obtained with the phorbol myristate acetate. The kinetic constants for ATP, histone HI or Mg2+ and the apparent Ka for Ca2+ and phosphatidylserine were determined.

Studies of the binding activity to different gonadal receptors of ovine luteinizing hormone (LH) after chemical modification of lysine residues.

Abstract Several LH derivatives obtained by modification of the lysine residues were studied by the radioligand receptor assay using either corpora lutea homogenates or testis homogenates and tritiated methylated LH. The binding activity found was very similar to the biological activity as determined in the ovarian ascorbic acid depletion test, although some minor differences were observed. The binding activity of the methylated derivatives was no different from that of native LH, whereas the other alkylated derivatives were less active or inactive. As expected, carbamylated LH was inactive and the activity of guanidinated LH was considerably reduced. Some differences were observed in the binding affinities of these derivatives when comparing corpora lutea with testicular homogenates.

Activation of a cyclic nucleotide phosphodiesterase and of a protein kinase by chemically modified calmodulin.

1. Several calmodulin derivatives prepared by chemical modification of lysine residues were tested using bovine heart cyclic nucleotide phosphodiesterase and wheat germ calmodulin-dependent protein kinase. 2. The effect of chemical modification on the activation capacity of calmodulin for the two studied enzymes was different. 3. This was particularly noticeable in the case of alkylated derivatives which exhibited a higher affinity than native calmodulin towards phosphodiesterase but a lower affinity towards protein kinase. 4. The efficiency of these derivatives (maximal activation) was higher than that of native calmodulin in relation with the protein kinase.

Involvement of lysine residues in the binding of ovine chorionic somatomammotropin to lactogenic and somatotropic receptors

The biological activities of several ovine chorionic somatomammotropin (oCS) derivatives obtained by chemical modification of the lysine residues were studied by radioreceptor assays using rabbit mammary homogenates (lactogenic activity, L.A.) and liver homogenates (somatotropic activity, S.A.). Even if the control treatment with BH− 4 markedly decreased the L.A., it was clear that methylation mainly affected the S.A. and that ethylation reduced both activities. Guanidination inactivated almost completely both activities and acetimidination at a very low degree (3 of 14 lysines) led to less than 50% of both activities. These results show the involvement of lysine residues in the interaction of oCS with lactogenic and somatotropic receptors.

The reaction of the histidine residues of luteinizing hormone with ethoxyformyl anhydride

Bovine and porcine luteinizing hormones (B-LH, P-LH) and their subunits were treated by ethoxyformyl anhydride. The acylation of the histidine residues was followed by examination of the absorbance spectrum. All the histidine residues of the luteinizing hormone molecule can be modified at pH5. However 2 His in B-LH and 1 in P-LH appear to be much less reactive at pH 5 than the others and their acylated imidazols more labile at the same pH. At neutral pH, 2 histidines in B-LH (and 1 in P-LH) become unreactive. In the case of the subunits, 1 histidine becomes unreactive in each subunit at neutral pH. These unreactive histidine residues at neutral pH are probably those which appear to be poorly reactive at pH 5. Comparison of the results obtained with B-LH and P-LH suggests that of the 2 histidine residues present in B-LH and absent in P-LH (beta 60, beta 112), only one exhibits a low reactivity. Acylation of 4 His in B-LH do not cause dissociation into subunits of the molecule but supress 95 per cent of the biological activity.

Nitroguanidyl-lutropin, a derivative which inhibits the stimulation of ovarian adenylate cyclase by lutropin

The high reactivity of the e-NH2 group of lysine residues offers the possibility of introducing very different groups in the sidechain of this amino acid. These groups can alter different characteristics: e.g., length of the chain, charge, distance between the charge and the peptide backbone. When the positive charge of the e-NH, groups of lysine in luteinizing hormone is suppressed, the biological activity of this hormone is abolished [1,2]. However, if the positive charge is maintained, various chemical modifications of the e-NH, group can lead to gradual modification of the biological properties of the molecule ranging from the preservation of the total biological activity (e.g., with the methylated derivative) [l] to a combination of agonist and antagonist properties (e.g., with some guanidinated derivatives) [ 31. With the nitroguanidyl group, the positive charge is suppressed and the size of the introduced group rather increased in comparison to the methylated or guanidinated derivatives. This paper describes the physico-chemical and biological properties of the nitroguanidyl derivative of LH, the first well-defined antagonist obtained by chemical modification of the protein moiety in the gonadotropins.

Adenylate cyclase stimulation and luteinizing hormone-receptor interaction in plasma membranes from rat testicular interstitial cells in relation to the chemical structure of the hormone. Role of Mg2+.

To understand more closely the structural requirements of the LH molecule necessary to stimulate adenylate cyclase, we studied the modulation of this enzyme in partially purified plasma membranes prepared from isolated interstitial cells of rat testis submitted to oLH and to some oLH derivatives and natural analogues. The role of Mg2+ was also investigated in relation to the structural modifications of oLH. Some new facts appeared in this study: 1. Methyl oLH, which exhibited the same ability as native oLH to stimulate cAMP accumulation and steroidogenesis in isolated cells, cannot induce the same level of maximal stimulation of adenylate cyclase as native oLH in plasma membranes. This phenomenon is related to the Mg2+ concentration, and the differences between maximal activation induced by methyl oLH and oLH were more apparent at a free Mg2+ concentration of 3.3 mmol/l than at lower concentrations. The maximal activity (in terms of native oLH) of other alkyl derivatives, such as ethyl or isopropyl oLH, on the contrary, was similar in isolated plasma membranes and in intact cells suggesting that the differential behaviour of the membranes specifically concerns the methyl derivative. 2. Guanidyl oLH and guanidyl porcine LH, which were able to induce cAMP accumulation in intact cells, did not exhibit any stimulating activity in plasma membranes. 3. Among the natural analogues, hCG and pLH are distinguished by a lower maximal activity (by comparison with oLH) particularly at high Mg2+ concentration. This work shows that changes in the LH structure have an impact not only on the parameters of the adenylate cyclase complex but also on the transduction of the hormone signal and its modulation by Mg2+.