Protein Concentration-dependent Manner Research Articles

Biochemical Analysis of the DNA Unwinding and Strand Annealing Activities Catalyzed by Human RECQ1*[boxs

RecQ helicases play an important role in preserving genomic integrity, and their cellular roles in DNA repair, recombination, and replication have been of considerable interest. Of the five human RecQ helicases identified, three are associated with genetic disorders characterized by an elevated incidence of cancer or premature aging: Werner syndrome, Bloom syndrome, and Rothmund-Thomson syndrome. Although the biochemical properties and protein interactions of the WRN and BLM helicases defective in Werner syndrome and Bloom syndrome, respectively, have been extensively investigated, less information is available concerning the functions of the other human RecQ helicases. We have focused our attention on human RECQ1, a DNA helicase whose cellular functions remain largely uncharacterized. In this work, we have characterized the DNA substrate specificity and optimal cofactor requirements for efficient RECQ1-catalyzed DNA unwinding and determined that RECQ1 has certain properties that are distinct from those of other RecQ helicases. RECQ1 stably bound to a variety of DNA structures, enabling it to unwind a diverse set of DNA substrates. In addition to its DNA binding and helicase activities, RECQ1 catalyzed efficient strand annealing between complementary single-stranded DNA molecules. The ability of RECQ1 to promote strand annealing was modulated by ATP binding, which induced a conformational change in the protein. The enzymatic properties of the RECQ1 helicase and strand annealing activities are discussed in the context of proposed cellular DNA metabolic pathways that are important in the maintenance of genomic stability.

Cooperative Interaction of Human XPA Stabilizes and Enhances Specific Binding of XPA to DNA Damage

Human xeroderma pigmentosum group A (XPA) is an essential protein for nucleotide excision repair (NER). We have previously reported that XPA forms a homodimer in the absence of DNA. However, what oligomeric forms of XPA are involved in DNA damage recognition and how the interaction occurs in terms of biochemical understanding remain unclear. Using the homogeneous XPA protein purified from baculovirus-infected Sf21 insect cells and the methods of gel mobility shift assays, gel filtration chromatography, and UV-cross-linking, we demonstrated that both monomeric and dimeric XPA bound to the DNA adduct of N-acetyl-2-aminofluorene (AAF), while showing little affinity for nondamaged DNA. The binding occurred in a sequential and protein concentration-dependent manner. At relatively low-protein concentrations, XPA formed a complex with DNA adduct as a monomer, while at the higher concentrations, an XPA dimer was involved in the specific binding. Results from fluorescence spectroscopic and competitive binding analyses indicated that the specific binding of XPA to the adduct was significantly facilitated and stabilized by the presence of the second XPA in a positive cooperative manner. This cooperative binding exhibited a Hill coefficient of 1.9 and the step binding constants of K(1) = 1.4 x 10(6) M(-)(1) and K(2) = 1.8 x 10(7) M(-)(1). When interaction of XPA and RPA with DNA was studied, even though binding of RPA-XPA complex to adducted DNA was observed, the presence of RPA had little effect on the overall binding efficiency. Our results suggest that the dominant form for XPA to efficiently bind to DNA damage is the XPA dimer. We hypothesized that the concentration-dependent formation of different types of XPA-damaged DNA complex may play a role in cellular regulation of XPA activity.

Identification of the partitioning site within the repABC-type replicon of the composite Paracoccus versutus plasmid pTAV1.

The replicator region of composite plasmid pTAV1 of Paracoccus versutus (included in mini-replicon pTAV320) belongs to the family of repABC replicons commonly found in plasmids harbored by Agrobacterium and Rhizobium spp. The repABC replicons encode three genes clustered in an operon, which are involved in partitioning (repA and repB) and replication (repC). In order to localize the partitioning site of pTAV320, the two identified incompatibility determinants of this mini-replicon (inc1, located in the intergenic sequence between repB and repC; and inc2, situated downstream of the repC gene) were PCR amplified and used together with purified RepB fusion protein (homologous to the type B partitioning proteins binding to the partitioning sites) in an electrophoretic mobility shift assay. The protein bound only inc2, forming two complexes in a protein concentration-dependent manner. The inc2 region contains two long (14-bp) repeated sequences (R1 and R2). Disruption of these sequences completely eliminates RepB binding ability. R1 and R2 have sequence similarities with analogous repeats of another repABC replicon of plasmid pPAN1 of Paracoccus pantotrophus DSM 82.5 and with centromeric sequences of the Bacillus subtilis chromosome. Excess RepB protein resulted in destabilization of the inc2-containing plasmid in Escherichia coli. On the other hand, the inc2 region could stabilize another unstable replicon in P. versutus when RepA and RepB were delivered in trans, proving that this region has centromere-like activity. Thus, it was demonstrated that repA, repB, and inc2 constitute a functional system for active partitioning of pTAV320.

Isolation of calcifiable vesicles from aortas of rabbits fed with high cholesterol diets

Advanced arterial wall calcification in atherosclerosis imposes a serious rupturing effect on the aorta. However, the mechanism of dystrophic calcification linked to hyperlipidemia, that causes atherosclerosis remains unknown. Emerging morphological and biochemical evidence reveals that calcifiable vesicles may have a role in plaque calcification. To determine whether a high cholesterol diet can induce arterial calcification and produce or activate calcifiable vesicles in aortas, a rabbit model was used. After 2 months of daily high lipid feeding (supplemented with 2% cholesterol and 6% peanut oil), typical atherosclerotic lesions developed. However, the mineral, if present in aortas, was insufficient to be detected by Fourier transform-infrared spectroscopy (FT-IR) or alizarin red staining, indicative of a non-calcifying stage of atherosclerosis. Small segments of thoracic aortas were digested in a crude collagenase solution to release calcifiable vesicles. Vesicles were also isolated from normal aortas as control to consider the possibility that membrane vesicles may be produced by crude collagenase digestion, which could cause the degradation of some cells. Calcifiable vesicles were precipitated at 300 000× g after subcellular particles were removed by centrifugation at 30 000× g. Calcifiability of isolated vesicles was then tested using calcifying media containing physiological levels of Ca 2+ and Pi and 1 mM ATP. Electron microscopic observations showed that the isolated vesicles were heterogeneous in size and shape and capable of depositing electron dense particles. Fourier transform infrared spectroscopic analysis of the deposited particles revealed the presence of an amorphous mineral phase. The spectroscopic mineral to matrix ratios, related to the amount of mineralization, indicated that vesicles from cholesterol-fed rabbits produced more minerals than control vesicles obtained from the normal aortas. Alizarin red staining for mineral further demonstrated substantially higher calcifiability of the experimental vesicles. A 3–5 h exposure of the vesicles to calcifying media caused significant deposition of 45Ca and 32Pi in a vesicle protein-concentration dependent manner. Similar to previously reported observations with human atherosclerotic aorta vesicles, rabbit vesicles were enriched in ATP-hydrolyzing enzymes including Mg 2+- or Ca 2+-ATPase and NTP pyrophosphohydrolase that are implicated in normal and pathological calcification. Altogether, these observations suggest that accumulation of the released calcifiable vesicles, as a result of high cholesterol diets, may have a role in dystrophic calcification in hyperlipidemia-related atherosclerosis.

Isolation of calcifiable vesicles from human atherosclerotic aortas

Advanced mineralization can cause brittleness of aortic walls with decreased elasticity thereby causing the wall to rupture. Although the precise mechanisms of dystrophic calcification remain unknown, morphological evidence reveals the presence of mineral-associated vesicles in the lesions and defective bioprosthetic valves. In an attempt to demonstrate the calcifiability of the vesicles, small segments of human atherosclerotic aortas with calcified lesions were removed at autopsy and then digested in a crude collagenase solution to release vesicles. A differential centrifugation was then used to isolate calcifiable vesicles, which was precipitated at 300 000× g for 20 min. An exposure of the vesicles to a calcifying medium containing physiologic levels of Ca 2+, Pi, and 1 mM ATP caused Ca deposition in a vesicle protein-concentration dependent manner. The calcifiability of the vesicles was further demonstrated by electron microscopy. Fourier transform spectroscopic analysis of the deposited mineral revealed the presence of a hydroxyapatite phase, closely resembling the native form of mineral in atherosclerotic plaques. In addition, calcifiable vesicles were enriched in ATP-hydrolyzing enzymes including Mg 2+- or Ca 2+-ATPase and NTP pyrophosphohydrolase that may be involved in normal and pathological calcification. Triton X-100 at 0.01% abolished 80% of both ATPase activity and ATP-initiated calcification. A comparison of vesicles isolated from non-atherosclerotic and atherosclerotic aortas indicated that atherosclerotic vesicles tended to have higher calcifiability. These observations suggest that the calcifiable vesicles play a part in dystrophic calcification of aortas in atherosclerosis.

Characterization of neuropeptide Y receptor subtypes in the normal human brain, including the hypothalamus

The aim of the present study was to investigate the existence and distribution of neuropeptide Y receptor subtypes in various regions of the normal human brain using the peptide YY derivative receptor probes, [ 125I][Leu 31,Pro 34]polypeptide YY/Y 1 and [ 125I]polypeptide YY 3–36/Y 2, in addition to the non-selective ligand [ 125I]polypeptide YY. Membrane binding assays performed with post mortem frontal cortex homogenates revealed that [ 125I]polypeptide YY and [ 125I]polypeptide YY 3–36 bound in a time- and protein concentration-dependent manner. Very low amounts of specific [ 125I][Leu 31,Pro 34]polypeptide YY binding could be detected even in the presence of high amounts of protein, contrasting with results obtained with [ 125I]polypeptide YY and [ 125I]polypeptide YY 3–36, a preferential Y 2 receptor probe. Analysis of saturation isotherms revealed that [ 125I]polypeptide YY 3–36 bound to a single class of high-affinity sites (0.5–2 nM). Significantly higher binding capacities were evident for [ 125I]polypeptide YY 3–36 as compared to [ 125I][Leu 31,Pro 34]polypeptide YY, suggesting that the human frontal cortex, in contrast to the rat, is mostly enriched with Y 2 receptors. Ligand selectivity profile confirmed the hypothesis that polypeptide YY 3–36, neuropeptide Y and polypeptide YY but not the [Leu 31,Pro 34] derivatives are potent competitors of [ 125I]polypeptide YY and [ 125I]polypeptide YY 3–36 binding sites. Autoradiographic studies demonstrated further that cortical areas, as well as most other regions of the human brain, are particularly enriched with Y 2/[ 125I]polypeptide YY 3–36 sites, while only low to very low amounts of Y 1 binding were detected except in the dentate gyrus of the hippocampal formation. In the human hypothalamus, a preponderance of Y 2 binding sites was also noted. Taken together, these results clearly establish that the distribution of the Y 1 and Y 2 receptor subtypes in human is different from the rodent brain, the Y 2 subtype being most abundant in the human brain.

A Structural Phase of Heat-Denatured Lysozyme with Novel Antimicrobial Action

The structure and antimicrobial function of hen egg white lysozyme was investigated by means of thermal denaturation at 80 °C (pH 7.2), which leads to irreversible denaturation. With an increase in the heating time (up to 30 min) of lysozyme, the soluble fraction showed progressive decrease in its enzyme activity that coincided with the formation of a slower migrating band on the acid PAGE. Fluorescence spectra revealed that, as the extent of denaturation increases, the surface hydrophobicity and the exposure of tryptophan residues were greatly promoted. In parallel to these conformational changes of lysozyme there has been consistent increase in its antimicrobial activities against Gram-negative bacteria, with no detrimental effect on its inherent action to Gram-positive bacteria. Interestingly, lysozyme heated for 20 min, devoid of enzyme activity (HDLz), killed Escherichia coli K12 in a dose-dependent manner, while its bactericidal activity to Staphylococcus aureus was almost similar to that of the native lysozyme. The binding capacity of HDLz to membrane fractions of E. coli K12 was greatly promoted, particularly to the inner membrane, as determined by ELISA. The HDLz permeabilized liposomal membranes made from E. coli phospholipids, as demonstrated by calcein efflux, in a protein concentration-dependent manner. Good correlations between the degree of heat inactivation of lysozyme (or dimerization), increased hydrophobicity, and enhanced bactericidal activity against Gram-negative E. coli K12 were observed. The results of this study, first of all, suggest that susceptibility of Gram-negative or even Gram-positive bacteria to lysozyme is independent of enzymatic activity. It is likely that denatured lysozyme, e.g., the dimeric form, has an intrinsic structural motif which is generally lethal to the bacteria through membrane perturbation. Keywords: Lysozyme; denaturation; conformational changes; antimicrobial action; membrane interaction; liposome

A phospholipase D-mediated pathway for generating diacylglycerol in nuclei from Madin-Darby canine kidney cells.

Many receptors, in response to their specific ligands, trigger activation of phospholipase D (PLD), resulting in the production of phosphatidic acid which, in turn, is acted upon by a specific phosphatase, phosphatidate phosphohydrolase, to produce diacylglycerol. We report here that isolated nuclei from Madin-Darby canine kidneys (MDCK)-D1 cells exhibit a PLD activity that is enhanced by the presence of ATP. PLD activity was measured in the presence of ethanol, by quantitating the production of phosphatidylethanol. Non-phosphorylating ATP analogs were unable to substitute for ATP in activating PLD, indicating that ATP acts as a phosphoryl group donor in a kinase-mediated phosphorylation reaction. The protein kinase C inhibitors chelerythrine and calphostin completely suppressed the ATP-induced nuclear PLD, implicating protein kinase C as the kinase involved in ATP-dependent PLD activity in nuclei from MDCK-D1 cells. In the absence of ethanol, phosphatidic acid was detected in ATP-treated nuclei. Accumulation of phosphatidic acid preceded or closely paralleled that of diacylglycerol, suggesting a precursor-product relationship. Consistent with those results, we detected phosphatidate phosphohydrolase activity in MDCK-D1 cell nuclei. Measurements of phosphatidic acid and diacylglycerol levels at increasing amounts of ethanol demonstrated that PLD and phosphatidate phosphohydrolase are responsible for generating the majority of the diacylglycerol accumulating in MDCK-D1 cell nuclei. The ability of nuclei to generate diacylglycerol from the concerted action of those two enzymes provides a means to regulate nuclear lipid synthesis as well as protein kinase C activity.

Binding of the nicotinic acetylcholine receptor to SH2 domains of Fyn and Fyk protein tyrosine kinases.

The nicotinic acetylcholine receptor (AChR) is phosphorylated on tyrosine both in vitro and in vivo. To identify the protein tyrosine kinase that phosphorylates the receptor, we have previously cloned and characterized two protein tyrosine kinases, Fyn and Fyk, that are highly expressed in Torpedo electric organ, a tissue enriched in the AChR. Both kinases were shown by coimmunoprecipitation to be specifically associated with the AChR. In this study, we examined the molecular basis for the interaction of Fyn and Fyk with the AChR using fusion proteins containing the SH2 domains of the two kinases as affinity reagents. The AChR bound specifically and in a protein concentration-dependent manner to the SH2 domain fusion proteins of Fyn and Fyk. Quantitation of the association revealed that the binding of the AChR to Fyn and Fyk SH2 domain fusion proteins was to a single class of saturable high affinity sites. In addition, the association of the AChR with the SH2 domain fusion proteins was dependent on tyrosine phosphorylation of the AChR and was mediated by the delta subunit of the receptor. Furthermore, upon dissociation of the AChR into subunits, the delta subunit coimmunoprecipitated with both Fyn and Fyk. These data suggest that the association of the AChR with Fyn and Fyk is mediated by an interaction of the tyrosine-phosphorylated delta subunit of the receptor with the SH2 domains of the protein tyrosine kinases.

Catalytic Properties of a Newly Discovered Acyltransferase That Synthesizes N-Acylphosphatidylethanolamine in Cottonseed (Gossypium hirsutum L.) Microsomes

We recently demonstrated that cotyledons of cotton (Gossypium hirsutum L.) seedlings synthesize N-acylphosphatidylethanolamine (NAPE), an unusual acylated derivative of phosphatidylethanolamine (PE), during postgerminative growth (K.D. Chapman and T.S. Moore [1993] Arch Biochem Biophys 301: 21-33). Here, we report the discovery of an acyltransferase enzyme, fatty acid: diacylphosphatidylethanolamine N-acyltransferase (designated NAPE synthase), that synthesizes NAPE from PE and free fatty acids (FFA) in cottonseed microsomes. [14C]NAPE was synthesized from [14C]palmitic acid and endogenous PE in a time-, pH-, temperature-, and protein concentration-dependent manner. [14C]Palmitic acid was incorporated exclusively into the N-acyl position of NAPE. [14C]palmitoyl coenzyme A (CoA) and [14C]-dipalmitoyl phosphatidylcholine (PC) were poor acyl donors for the synthesis of NAPE (i.e. 200- and 3000-fold lower incorporation efficiency than palmitic acid, respectively). Synthesis of NAPE from palmitoyl-CoA and dipalmitoyl-PC was observed only after the release of FFA in microsomes. We observed a temperature optimum of 45[deg]C and a pH optimum of 8.0 for the synthesis of [14C]NAPE from [14C]palmitic acid (or from [14C]PE). NAPE synthase activity showed no apparent divalent cation requirement. Notably, activity was stimulated by HPO42-, HCO3-, SO42-, and NADPH, whereas activity was inhibited by Ca2+, Mn2+, Cd2+, ATP, ADP, flavin adenine disnucleotide, and flavin mononucleotide. Other nucleotide triphosphates (GTP and CTP) and pyridine dinucleotides (NAD, NADH, and NADP) did not appreciably affect NAPE synthase activity. Initial velocity measurements of NAPE synthase activity at increasing concentrations of palmitic acid showed non-Michaelis-Menten, biphasic kinetics. A high-affinity site (S0.5 = 7.2 [mu]M, Vmax = 18.8 nmol h-1 mg-1 of protein) and a low-affinity site (S0.5 = 32.0 [mu]M, Vmax = 44.9 nmol h-1 mg-1 of protein) were identified. Both sites exhibited positive cooperativity. Adding myristic, stearic, or oleic acids at equimolar amounts reduced the incorporation of [14C]palmitic acid into NAPE at low concentrations (10 [mu]M, high-affinity site) but not at high concentrations (50 [mu]M, low-affinity site), indicating that the two putative sites can be distinguished by their fatty acid preferences.

Effect of protein precipitating agents on the recovery of plasma free amino acids

The effect of the protein precipitants acetone, acetonitrile, perchloric acid and trichloroacetic acid on free amino acid concentrations in supernatants from ovine plasma and bovine serum albumin solutions was determined. The organic precipitants decreased (P < 0.05) free amino acid concentrations in a protein concentration dependent manner while the acid precipitants had no effect. Key words: Amino acids, protein precipitating agents

Regulation of Rat Hypothalamic PreproGrowth Hormone-Releasing Factor Messenger Ribonucleic Acid by Dietary Protein

To further evaluate nutrient regulation of GRF synthesis, we measured hypothalamic preproGRF messenger (m) RNA in food-deprived rats refed diets varying in nutrient composition by nuclease protection analysis. Adult male Sprague-Dawley rats were allowed free access to food (Fed), food deprived for 72 h (72-h FD), or 72 h FD then refed for 72 h with either a normal (NF) diet or isocaloric diets containing no protein (PF), carbohydrate (CF), or fat (FF). Seventy-two-hour FD rats displayed the expected 80% reduction in hypothalamic preproGRF mRNA. Upon refeeding, levels were normalized in rats refed NF, CF, or FF diets. In contrast, preproGRF mRNA in rats refed a PF diet was similar to that in 72-h FD rats. Rats refed a PF diet failed to gain weight and consumed less food than animals refed NF, CF, or FF diets. However, the lack of the GRF response to the PF diet was due to protein deprivation rather than caloric restriction, since hypothalamic preproGRF mRNA returned to 66% of Fed values in rats refed an equivalent amount (grams per day) of a NF diet. In 72-h FD rats refed isocaloric diets containing 4%, 8%, or 12% protein, preproGRF mRNA was restored to Fed values in a protein concentration-dependent manner being completely restored by the 12% diet. A lack of dietary protein was sufficient to regulate hypothalamic preproGRF mRNA since feeding rats a PF diet without prior food deprivation resulted in 70% reduction in preproGRF mRNA, whereas CF and FF diets were without effect. These data indicate that decreased hypothalamic preproGRF mRNA expression in 72-h FD rats occurs as a result of dietary protein deprivation.

Localization of N‐Methyl‐d‐Aspartate Receptors in the Rat Striatum: Effects of Specific Lesions on the [3H]3‐(2‐Carboxypiperazin‐4‐yl)propyl‐1‐Phosphonic Acid Binding

The binding of [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid ([3H]CPP), a rigid analogue of 2-amino-7-phosphonoheptanoic acid (AP7) and reported to be a selective N-methyl-D-aspartate (NMDA) antagonist, was studied in rat striatal membranes using a centrifugation procedure to separate bound and free radioligand. [3H]CPP bound with high affinity (KD = 272 nM) in a saturable, reversible, and protein concentration-dependent manner. Specific binding was suggested to involve a single class of noninteracting binding sites. The most potent [3H]CPP binding inhibitors tested were CPP, L-glutamate, 2-amino-5-phosphonovalerate, and AP7. NMDA, L-aspartate, and alpha-aminoadipate were also shown to be efficient in inhibiting the binding, whereas quisqualate, D,L-2-amino-4-phosphonobutyrate, kainate, L-glutamate diethylester, and alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid were found to be essentially inactive. These data are therefore consistent with the view that [3H]CPP selectively binds to NMDA receptors in the rat striatum. Lesions of intrastriatal neurons using local injections of kainic acid revealed a marked decrease in [3H]CPP binding, suggesting an almost exclusively postsynaptic location of binding sites in the striatum. Conversely, bilateral lesion of corticostriatal glutamatergic fibers resulted in an increased number of [3H]CPP striatal binding sites, providing evidence for a putative supersensitivity response to this striatal deafferentation. Interestingly, lesion of the nigrostriatal dopaminergic neurons using intranigral 6-hydroxydopamine injections resulted, 2-3 weeks later, in a similar increase in the number of [3H]CPP striatal binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)

Histamine acting on H2 receptors stimulates phospholipid methylation in synaptic membranes of rat brain.

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.

O-61 - Histamine acting on H2-receptors stimulates phospholipid methylation in synaptic membranes of rat brain

Histamine stimulated [3H]methyl group incorporation into phospholipids in crude synaptic membranes of rat whole brain (without cerebellum) in modified Krebs-Ringer solution containing the methyl donor S-adenosyl-[methyl-3H]methionine. The transient increase of [3H]methyl incorporation into lipids peaked within 45 s after addition of histamine (5 or 10 microM) and decreased the basal level in 60 s. Histamine-stimulated [3H]methyl incorporation was increased linearly in a protein concentration-dependent manner. The stimulation was temperature and histamine concentration dependent. TLC analysis of a chloroform/methanol extract indicated that radioactive phospholipids (phosphatidylcholine, phosphatidyl-N,N-dimethylethanolamine, and phosphatidyl-N-monomethylethanolamine) accounted for 60-65% of the total radioactivity recovered. The synaptosomal fraction had the highest specific activity of all the subfractions of crude synaptic membranes (P2). Histamine-induced [3H]methyl incorporation was inhibited by addition of cimetidine (0.01-10 microM) or famotidine (0.01-1.0 microM) in a concentration-dependent manner but not by mepyramine (0.1-10 microM) or diphenhydramine (0.1-10 microM). The stimulation of [3H]methyl incorporation was also observed by addition of impromidine (0.01-10 microM) or dimaprit (1.0 microM-1.0 mM) in a concentration-dependent manner but not by 2-pyridylethylamine (1.0 microM-1.0 mM). These results indicate that phospholipid methylation is induced by histamine acting on H2 receptors in rat brain synaptosomes.

Insulin and glucagon binding and degradation by kidney cell membranes.

The binding and degradation of insulin and glucagon to kidney cell membranes was examined. Time- and temperature-dependent specific binding of [125I]iodo-insulin to kidney cell membranes was demonstrated. The membranes also degraded insulin in a time-, temperature-, and protein concentration-dependent manner. The apparent Km of the degradation was 2.7 x 10(-7) M. Glucagon degradation by the kidney membranes was extremely active. Per milligram of protein the kidney membrane was over 20 times as active as the liver membrane. Even at 4 C, significant glucagon degradation occurred. Because of this very active degradation, glucagon binding could not be accurately assessed. The kidney glucagon-degrading activity was inhibited by glutahione and EDTA but unaffected by N-ethylmaleimide, ACTH, or insulin, all potent inhibitors of liver glucagon degradation. The apparent Km for glucagon degradation by the kidney, however, was essentially identical with that for the liver, 2.4 x 10(-6) M.