Novel S-Adenosylmethionine-Dependent Indole-N-Methylation of β-Carbolines in Brain Particulate Fractions

Guinea pig brain S-adenosylmethionine (SAM)-dependent N-methyltransferase activity toward physiologically relevant beta-carboline (BC) substrates was examined with reverse-phase HPLC and radiochemical detection. Representative BCs, norharman and harmine, were enzymatically methylated on the 2[beta]-nitrogen by [3H]CH3-SAM in undialyzed homogenates to yield 2[beta]-methylated BCs and subsequently on the 9[indole]-nitrogen to generate 2,9-dimethylated BC products. This may be the first account of mammalian indole N-methyl transfer. There was no HPLC evidence for 9-methyl BC or (from carbon methylation) 2,6-dimethyl BC products. Capillary gas chromatography-mass spectrometry analysis confirmed the structures of the 2,9-dimethyl and 2-methyl products of norharman. The 2[beta]- and 9[indole]-N-methylation activities were mainly in the nuclear fractions and were negligible in undialyzed cytosol. This differs from the cytosolic SAM-dependent N-methylations reported with other azaheterocyclics, including 1,2,3,4-tetrahydro-BCs. The involvement of a single enzyme was suggested because the two N-methyl transfers with BC substrate had similar subcellular activity patterns, regional brain distributions, and Km and Vmax values. Sequential N-methylation of various BCs that have been observed in vivo may be a unique route to centrally retained N2,N9-dimethylated beta-carbolinium ions. Because they resemble the synthetic parkinsonian toxicant, N-methyl-4-phenylpyridinium, with respect to structure and neurotoxic activity, such "bioactivated" carbolinium ions could be endogenous causative factors in Parkinson's disease.     

Preparation and opioid activities of N-methylated analogs of [D-Ala2,Leu5]enkephalin

Analogs of opioid pentapeptide [D-Ala2,Leu5]enkephalin were prepared using two kinds of N-methylation reactions, namely quaternization and amide-methylation. Quaternization reaction with CH3I-KHCO3 in methanol was applied to the deprotected N-terminal group of the pentapeptide derivatives affording trimethylammonium group-containing analogs. [Me3+Tyr1,D-Ala2,Leu5]enkephalin and its amide were found to show opioid activity on guinea pig ileium assay only slightly lower than the parent unmethylated peptides. Application of amide-methylation reaction using CH3I-Ag2O in DMF to the protected pentapeptide yielded a pentamethyl derivative in which all of the five N atoms were methylated. Deprotection of the derivative gave pentamethyl analogs of [D-Ala2,Leu5]enkephalin, which showed no significant activity on the guinea pig ileum assay and opiate-receptor binding assay.     

Beta-endorphin. Biological activity of synthetic analogs with analgesia inhibiting property in rat vas deferens and guinea pig ileum assays

Three synthetic analogs of human beta-endorphin (beta h-EP) (I, [Gln8, Gly31]-beta h-EP-Gly-Gly-NH2; II, [Arg9,12,24,28,29]-beta h-EP and III, [Cys11,26, Phe27, Gly31]-beta h-EP), which have been shown to possess potent inhibiting activity to beta h-EP-induced analgesia, were assayed in rat vas deferens and guinea pig ileum bioassay systems. In the rat vas deferens assay, relative potencies of these analogs were beta h-EP, 100; I, 30; II, 40; III, 1, whereas in the guinea pig ileum assay: beta h-EP, 100; I, 184; II, 81; III, 163. From previous studies on their analgesia potency in mice and opiate receptor-binding activity in rat brain membranes, their activity in rat vas deferens correlates well with the analgesic potency and the activity from guinea pig ileum assay shows good correlations with that from the opiate receptor-binding assay.     

Species differences in structure-activity relationships of adenosine agonists and xanthine antagonists at brain A1 adenosine receptors

A series of 28 adenosine analogs and 17 xanthines has been assessed as inhibitors of binding of N6-R-[3H]phenylisopropyladenosine binding to A1 adenosine receptors in membranes from rat, calf, and guinea pig brain. Potencies of N6-alkyl- and N6-cycloalkyladenosines are similar in the different species. However, the presence of an aryl or heteroaryl moiety in the N6 substituent results in marked species differences with certain such analogs being about 30-fold more potent at receptors in calf than in guinea pig brain. Potencies at receptors in rat brain are intermediate. Conversely, 2-chloroadenosine and 5'-N-ethylcarboxamidoadenosine are about 10-fold less potent at receptors in calf brain than in guinea pig brain. Potencies of xanthines, such as theophylline, caffeine and 1,3-dipropylxanthine are similar in the different species. However, the presence of an 8-phenyl or 8-cycloalkyl substituent results in marked species differences. For example, a xanthine amine conjugate of 1,3-dipropyl-8-phenylxanthine is 9-fold more potent at receptors in calf than in rat brain and 110-fold more potent in calf than in guinea pig brain. Such differences indicate that brain A1 adenosine receptors are not identical in recognition sites for either agonists or antagonists in different mammalian species.     

Human beta-endorphin: synthesis and biological activity of analogs with substitutions in positions 2, 9, 18, 27 and 31

Four analogs of the opioid peptide human beta-endorphin (Bh-EP) have been synthesized: [D-Lys9, Phe27, Gly31]-beta h-EP, [D-PHe18,Phe27,Gly31]-beta h-EP, [D-Thr2,D-Lys9,Phe27,Gly31]-beta h-EP, and [D-Thr2,D-Phe18,Phe27,Gly31]-beta h-EP. All are practically indistinguishable from beta h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

Enzymes of Choline Synthesis in Spinach (Response of Phospho-Base N-Methyltransferase Activities to Light and Salinity)

In spinach (Spinacia oleracea L.), choline is synthesized by the sequential N-methylation of phosphoethanolamine -> phosphomono- -> phosphodi- -> phosphotrimethylethanolamine (i.e. phosphocholine) followed by hydrolysis to release choline. Differential centrifugation of spinach leaf extracts shows that enzymes catalyzing the three N-methylations are cytosolic. These enzymes were assayed in leaf extracts prepared from plants growing under various light/dark periods. Under a diurnal, 8-h light/16-h dark photoperiod, the activity of the enzyme catalyzing the N-methylation of phosphoethanolamine is highest at the end of the light period and lowest following the dark period. Prolonged dark periods (exceeding 16 h) lead to a further reduction in the activity of this enzyme, although activity is restored when plants are reexposed to light. In contrast, the activity of the enzyme(s) catalyzing the N-methylations of phosphomono- and phosphodimethylethanolamine does not undergo comparable changes in response to light/dark treatments. Salt shock of plants with 200 mM NaCl results in a 2-fold increase in all three N-methylation activities relative to nonsalinized controls but only in plants exposed to light. Thus, light is required for the salt-responsive up-regulation of choline synthesis in spinach.     

High-performance liquid chromatography of tetrahydro-beta-carbolines extracted from plasma and platelets

A fast method for extraction and concentration of tryptamine (TA), 5-hydroxy-TA, and 5-methoxy-TA was developed using reverse-phase C-18 sample preparation columns in combination with an ion-pairing reagent. Using this method, 1,2,3,4-tetrahydro-beta-carboline (THBC), 6-hydroxy-THBC, and 6-methoxy-THBC, the respective reaction products formed after reaction of formaldehyde with the primary amines mentioned above, and beta-carboline (BC, norharman) and 1-methyl-beta-carboline (1-Me-BC, harman) could also be extracted from human and rat platelets and platelet-poor plasma (PPP). A HPLC method combined with fluorometric detection was developed for the quantitative determination of these compounds in the picomole range. The formation of beta-carbolines during the extraction procedure was below the limit of detection of the assay procedure. 6-OH-THBC, THBC, 1-Me-BC, and 5-HT were identified as normal constituents of human platelets, whereas only 5-hydroxytryptamine (5-HT) and 6-OH-THBC could be identified in human PPP. In rat platelets and PPP 5-HT, but no THBCs, could be detected.     

Biochemical and Pharmacological Characterization of Serotonin-O-Carboxymethylglycyl[125I]Iodotyrosinamide5 a New Radioiodinated Probe for 5-HT1B and 5-HT1D Binding Sites

There is a lack of radioactive probes, particularly radioiodinated probes, for the direct labeling of serotonin-1B (5-HT1B) and serotonin-1D (5-HT1D) binding sites. Serotonin-O-carboxymethylglycyltyrosinamide (S-CM-GTNH2) was shown previously to be specific for these two subtypes; we, therefore, linked a 125I to its tyrosine residue. Biochemical and pharmacological properties of S-CM-G[125I]TNH2-binding sites were studied by quantitative autoradiography on rat and guinea pig brain sections. S-CM-G[125I]TNH2 binding is saturable and reversible with a KD value of 1.3 nM in the rat and 6.4 nM in the guinea pig. Binding is heterogeneous, paralleling the anatomical distribution of 5-HT1B sites in the rat and of 5-HT1D sites in the guinea pig. The binding of 0.02 nM S-CM-G[125I]TNH2 was inhibited by low concentrations of 5-HT, S-CM-GTNH2, CGS 12066 B, 5-methoxytryptamine, and tryptamine in both species. Propranolol inhibited the radioligand binding with a greater affinity in the rat than in the guinea pig. Conversely, 8-hydroxy-2-(di-n-propylamino)tetralin inhibited S-CM-G[125I]TNH2 binding with a greater affinity in the guinea pig than in the rat. Other competitors, specific for 5-HT1C, 5-HT2, 5-HT3, and adrenergic receptors, inhibited S-CM-G[125I]TNH2 binding in rat and guinea pig substantia nigra and in other labeled structures known to contain these receptors, but only at high concentrations. S-CM-G[125I]TNH2 is then a useful new probe for the direct study of 5-HT1B and 5-HT1D binding sites.     

The conjugation of tyramine with sulphate by liver and intestine of different animals.

Tyramine was conjugated with sulphate by extracts of monkey intestine and livers of monkey, rat, mouse, guinea pig and man. The activity measured in monkey intestine was almost three times that of monkey liver. Labelled tyramine sulphate synthesized from [14C] tyramine, [3H] tyramine or Na235SO4, on acid hydrolysis, released its radioactive precursor. Liver extracts of monkey, rat, mouse and guinea pig synthesized respectively 145,66,21 and 6 pmol of [14C] tyramine sulphate/min per mg of protein. Except with the monkey, intestine exhibited very low activity. trans-2-Phenylcyclopropylamine, a monoamine oxidase inhibitor, was added as a routine to the enzyme preparation, as its omission resulted in the production of p-hydroxyphenylacetic acid in appreciable amounts. This oxidative deamination of tyramine, however, did not decrease the sulpho-conjugation of tyramine. The low Km (9.1 muM) of sulphotransferase for tyramine is probably responsible.     

Stereoselective reduction of 4-benzoylpyridine in the heart of vertebrates

The stereoselectivity in the reduction of 4-benzoylpyridine (4-BP) was examined in the cytosolic fractions from the heart of 9 vertebrates (pig, rabbit, guinea pig, rat, mouse, chicken, soft-shelled turtle, frog and flounder). 4-BP was stereoselectively reduced to S(-)-alpha-phenyl-4-pyridylmethanol [S(-)-PPOL] in the cytosolic fractions from the heart of pig, rabbit and guinea pig. However, of mammalian heart cytsol tested, only rat heart cytosol had little ability to reduce stereoselectively 4-BP. In an attempt to elucidate this reason, amino acid sequence of rat heart carbonyl reductase (RatHCR) was deduced from the cloned cDNA and compared with that of pig heart carbonyl reductase (PigHCR), which shows a high stereoselectivity in the reduction of 4-BP to S(-)-PPOL. RatHCR showed a high identity with PigHCR in amino acid sequence. Furthermore, recombinant RatHCR was confirmed to reduce stereoselectively 4-BP to S(-)-PPOL with a high optical purity comparable to recombinant PigHCR. It is possible that in the cytosolic fraction from the heart of rat, constitutive reductase other than RatHCR counteracts the stereoselective reduction of 4-BP to S(-)-PPOL, by catalyzing the reduction of 4-BP to the R(+)-enantiomer.     

Comparative aspects of nitrobenzylthioinosine and dipyridamole inhibition of adenosine accumulation in rat and guinea pig synaptoneurosomes

Dipyridamole (DPR) and nitrobenzylthioinosine (NBI) inhibition of adenosine accumulation in synaptoneurosomes derived from rat cerebral cortex, rat cerebellum, guinea pig cerebral cortex and guinea pig cerebellum was investigated. The inhibition of adenosine accumulation by NBI was observed to be distinctly biphasic in both guinea pig and rat synaptoneurosomes. Such biphasic inhibition consisted of a nM potency component to inhibition, accounting for 20–30% of the maximum inhibition, and a μM potency component, accounting for the remaining 70–80% maximum inhibition. Such an inhibitory profile contrasts sharply with that of DPR which appears monophasic, with a mean IC₅₀ of between 10⁻⁷ M and 10⁻⁶ M, in all rat and guinea pig synaptoneurosomes preparations studied.Further differences between the potency of NBI and DPR in inhibiting [³H]adenosine accumulation were also noted. DPR was more potent in inhibiting [³H]adenosine accumulation in guinea pig cerebellar synaptoneurosomes than in cerebral cortex synaptoneurosomes. In rat synaptoneurosomes, the reverse selectivity was observed. DPR was also 2–6 fold (depending on brain region of comparison) more potent in inhibiting adenosine accumulation in guinea pig synaptoneurosomes than in inhibiting such accumulation in rat synaptoneurosomes. In contrast, NBI was approximately equipotent in inhibiting adenosine accumulation in rat and guinea pig synaptoneurosomes. Additional binding studies using [³H]NBI are also reported. The data presented are entirely consistent with the hypotheses that (1) NBI and DPR bind to functionally relevant sites and (2) there are different populations of nucleoside transporters in mammalian brain.     

Distribution of the Two Forms of Monoamine Oxidase Within Monoaminergic Neurons of the Guinea Pig Brain

The relative distribution of type A and type B monoamine oxidase (MAO) inside and outside the monoaminergic synaptosomes in preparations from hypothalamus and striatum of the guinea pig was determined by incubation of synaptosomal preparations of these regions with low concentrations of [14C]5-hydroxytryptamine (5-HT), noradrenaline, and dopamine. The deamination within the monoaminergic synaptosomes was hindered by selective amine uptake inhibitors. In the absence of these inhibitors, both intra- and extraneuronal deamination was measured. The two forms of the enzyme were differentiated with the irreversible and selective MAO-A and MAO-B inhibitors clorgyline and selegiline (l-deprenyl), respectively. [14C]5-HT was deaminated greater than 90% by MAO-A both inside and outside the 5-hydroxytryptaminergic synaptosomes prepared from the guinea pig hypothalamus. The deamination of [14C]noradrenaline within the noradrenergic synaptosomes of the hypothalamic preparation was in the ratio 75:25% for MAO-A:MAO-B; the corresponding ratio outside these synaptosomes was 45:55%. The deamination of [14C]dopamine within dopaminergic synaptosomes in the striatal preparation was 65% type A:35% type B, whereas outside these synaptosomes the ratio was 35:65%. Because the relative amounts and the distribution of the two forms of MAO in the guinea pig brain seem to be similar to those previously detected for the human brain, the MAO in the guinea pig brain may be a good model for the MAO in the human brain.     

Affinities of Histamine H1-Antagonists in Guinea Pig Brain: Similarity of Values Determined from [3H]Mepyramine Binding and from Inhibition of a Functional Response

Affinity constants for five antagonists at histamine H1-receptors in guinea pig brain have been determined from inhibition of the potentiation by histamine of the adenosine-induced accumulation of cyclic AMP in cerebral cortical slices. This action of histamine appeared to be mediated solely through H1-receptors. The affinity constants obtained were similar to those determined on peripheral H1-receptors and from inhibition of high-affinity [3H]mepyramine binding. This provides strong evidence that at least some of the [3H]mepyramine binding sites in guinea pig brain can be identified with functional H1-receptors.     

Effect of mequitazine a non sedative antihistamine on brain H1 receptors

Marked species variations occured in the relative activity of mequitazine (10-[3-quinuclidinylmethyl]-phenothiazine) on the binding of [³H]mepyramine to brain cortical membranes. Mequitazine was as potent as promethazine in the mouse but about 6 times less effective than promethazine in the guinea pig and human. On the other hand in the guinea pig mequitazine was as potent as promethazine on [³H]mepyramine binding in a peripheral organ (lung). Although mequitazine did not displace [³H]mepyramine in vivo in the mouse and guinea pig, its brain concentration (measured by [³H]mequitazine) was largely sufficient and corresponds to 90% of inhibition in vitro. Moreover in the mouse the brain regional distribution of [³H]mequitazine was very different from that of [³H]mepyramine, highest level was obtained in the cerebellum and hypothalamus was the poorest region with mequitazine whereas the reverse was true with mepyramine. All these results could suggest that mequitazine possesses a greater affinity for peripheral H₁ receptors which could explain the absence of sedative side-effects of this potent H₁ antagonist.     

Binding of [35S]GTPgammaS stimulated by (+)-pentazocine sigma receptor agonist, is abundant in the guinea pig spleen

Here we measured sigma receptor agonist, [3H](+)-pentazocine binding and (+)-pentazocine-stimulated [35S]GTPgammaS binding throughout brain regions and peripheral organs of mice and guinea pigs to investigate the distribution of G protein-coupled sigma receptors. There was no significant correlationship between both distributions, in which the [3H](+)-pentazocine binding is highest in the liver of each species, while the [35S]GTP-gammaS binding is highest in the guinea pig spleen. The agonist-stimulated [35S]GTPgammaS binding in the spleen was also confirmed by in situ autoradiography using sections. Thus it is suggested that there are at least two subtypes, metabotropic and nonmetabotropic sigma receptors, and the former ones are abundant in the guinea pig spleen.     

Delayed labelling of brain glutamate after an intra-arterial [13C]glucose bolus: evidence for aerobic metabolism of guinea pig brain glycogen store.

Glycogen in glial cells is the largest store of glucose equivalents in the brain. Here we describe evidence that brain glycogen contributes to aerobic energy metabolism of the guinea pig brain in vivo. Five min after an intra-arterial bolus injection of d-[U-14C]glucose, 28+/-11% of the radioactivity in brain tissue was associated with the glycogen fraction, indicating that a significant proportion of labelled glucose taken up by the brain is converted to glycogen shortly after bolus infusion. Incorporation of 13C-label into lactate generated by brains made ischaemic after d-[1-13C]glucose injection confirms that these glucose equivalents can be mobilised for anaerobic glucose metabolism. Aerobic metabolism was monitored by following the time course of 13C-incorporation into glutamate in guinea pig cortex and cerebellum in vivo. After an intra-arterial bolus injection of d-[1-13C]glucose, glutamate labelling reached a maximum 40-60 min after injection, suggesting that a slowly metabolised pool of labelled glucose equivalents was present. As the concentration of 13C-labelled glucose in blood was shown to decrease below detectable levels within 5 min of bolus injection, this late phase of glutamate labelling must occur with mobilisation of a brain storage pool of labelled glucose equivalents. We interpret this as evidence that glucose equivalents in glycogen may contribute to energy metabolism in the aerobic guinea pig brain.     

Evidence for the presence of dihydro (reduced) somatostatin-14 in the guinea pig brain

Analysis of somatostatin-like immunoreactivity (SLI) in guinea pig brain by HPLC and radioimmunoassay revealed an unexpected peak of SLI eluting at a retention time slightly later than standard somatostatin-14. The following evidence argues that this peak represents dihydro (H2) somatostatin-14. (1) The peak had the same retention time as standard [H2]somatostatin. (2) The possibility of a reduction artefact due to tissue processing was excluded by adding exogenous somatostatin-14 or 125I-labeled N-Tyr-somatostatin-14 to tissue and observing that no corresponding reduced peptides were generated. (3) Mild oxidation of brain extracts with H2O2 decreased, whereas mild reduction with dithiothreitol increased, the proposed peak of [H2]somatostatin. (4) Reaction of tissue extracts with iodoacetamide decreased the size of the proposed [H2]somatostatin peak but resulted in generation of a new peak co-eluting with standard carboxymethylated somatostatin-14. The proportion of the [H2]somatostatin peak in five brain regions, the hypothalamus, amygdala, cerebral cortex, brainstem and cerebellum, ranged from 6 to 20% of total SLI. The probability of somatostatin-14 existing endogenously in reduced or oxidized forms may have implications for its biological function in the guinea pig.     

The mammalian beta 2-adrenergic receptor: purification and characterization

The beta 2-adrenergic receptors from hamster, guinea pig, and rat lungs have been solubilized with digitonin and purified by sequential Sepharose-alprenolol affinity and high-performance steric-exclusion liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of iodinated purified receptor preparations reveal a peptide with an apparent Mr of 64 000 in all three systems that coincides with the peptide labeled by the specific beta-adrenergic photoaffinity probe (p-azido-m-[125I]iodobenzyl)carazolol. A single polypeptide was observed in all three systems, suggesting that lower molecular weight peptides identified previously by affinity labeling or purification in mammalian systems may represent proteolyzed forms of the receptor. Purification of the beta-adrenergic receptor has also been assessed by silver staining, iodinated lectin binding, and measurement of the specific activity (approximately 15 000 pmol of [3H]dihydroalprenolol bound/mg of protein). Overall yields approximate 10% of the initial crude particulate binding, with 1-3 pmol of purified receptor obtained/g of tissue. The purified receptor preparations bind agonist and antagonist ligands with the expected beta 2-adrenergic specificity and stereoselectivity. Peptide mapping and lectin binding studies of the hamster, guinea pig, and rat lung beta 2-adrenergic receptors reveal significant similarities suggestive of evolutionary homology.     

Comparison of the nuclear and cytosolic forms of the Ah receptor from Hepa 1c1c7 cells: charge heterogeneity and ATP binding properties.

2-[125I]iodo-7,8-dibromo-p-dioxin ([125I]Br2DpD) and 2-[125I]iodo-3-azido-7,8-dibromo-p-dioxin ([125I]N3Br2-DpD) are both capable of binding to the Ah receptor (AhR) with a high degree of specificity in cultured Hepa 1c1c7 cells. After incubation with either [125I]N3Br2DpD or [125I]Br2DpD Hepa 1c1c7 cytosolic and high salt nuclear extracts were analyzed by sucrose density gradient analysis with the following results: (i) With both radioligands an approximately 9 S form of the AhR was observed in cytosolic extracts. (ii) Nuclear extracts labeled with [125I]N3Br2DpD revealed both approximately 6 S and approximately 9 S forms of the AhR. (iii) In contrast, analysis of nuclear extracts labeled with [125I]Br2DpD revealed only an approximately 6 S form of the AhR. The approximately 9 S [125I]N3Br2DpD-labeled AhR was preferentially extracted with 100 mM KCl from a nuclear fraction and mixed with monoclonal antibody 8D3, an anti-90-kDa heat shock protein antibody. Monoclonal antibody 8D3 was able to bind to the approximately 9 S nuclear form of the AhR and caused the receptor to sediment as a heavier complex on sucrose density gradients. This would indicate that the AhR can reside in the nucleus bound to 90-kDa heat shock protein. The [125I]N3Br2DpD-labeled approximately 6 S peak fractions were collected and subjected to denaturing two-dimensional gel electrophoresis. A comparison of [125I]N3Br2DpD-labeled cytosolic (9 S) AhR preparations with the nuclear (6 S) AhR by 2-D gel electrophoresis was performed. The cytosolic form of the AhR was present in the apparent pI range of 5.2-5.7; the nuclear form focused between 5.5 and 6.2. The [125I]N3Br2DpD-labeled nuclear extracts were incubated with ATP-agarose and 43% of the photoaffinity-labeled AhR bound to the affinity gel. In contrast, approximately threefold lower binding of [125I]N3Br2DpD-labeled receptor was obtained when GTP-, AMP-, or ADP-agarose was used. Only 2% of the [125I]N3Br2DpD-labeled cytosolic AhR was able to bind to ATP-agarose. These results suggest that after the AhR translocates into the nucleus the following biochemical changes occur: (i) The sedimentation value for the AhR changes from an approximately 9 S to an approximately 6 S species. (ii) The AhR attains the ability to bind with specificity to ATP. (iii) The AhR undergoes a shift to a more basic pI.     

Non-equivalent role of inter- and intramolecular hydrogen bonds in the insulin dimer interface

Apart from its role in insulin receptor (IR) activation, the C terminus of the B-chain of insulin is also responsible for the formation of insulin dimers. The dimerization of insulin plays an important role in the endogenous delivery of the hormone and in the administration of insulin to patients. Here, we investigated insulin analogues with selective N-methylations of peptide bond amides at positions B24, B25, or B26 to delineate their structural and functional contribution to the dimer interface. All N-methylated analogues showed impaired binding affinities to IR, which suggests a direct IR-interacting role for the respective amide hydrogens. The dimerization capabilities of analogues were investigated by isothermal microcalorimetry. Selective N-methylations of B24, B25, or B26 amides resulted in reduced dimerization abilities compared with native insulin (K(d) = 8.8 μM). Interestingly, although the N-methylation in [NMeTyrB26]-insulin or [NMePheB24]-insulin resulted in K(d) values of 142 and 587 μM, respectively, the [NMePheB25]-insulin did not form dimers even at high concentrations. This effect may be attributed to the loss of intramolecular hydrogen bonding between NHB25 and COA19, which connects the B-chain β-strand to the core of the molecule. The release of the B-chain β-strand from this hydrogen bond lock may result in its higher mobility, thereby shifting solution equilibrium toward the monomeric state of the hormone. The study was complemented by analyses of two novel analogue crystal structures. All examined analogues crystallized only in the most stable R(6) form of insulin oligomers (even if the dimer interface was totally disrupted), confirming the role of R(6)-specific intra/intermolecular interactions for hexamer stability.