Radioactive labeling of alpha1-antitrypsin, trypsin, and chymotrypsin by reductive methylation: properties of the labeled derivatives.

Human plasma α₁-antitrypsin (α₁-AT), bovine trypsin, and α-chymotrypsin were labeled with either ¹⁴C or ³H by reductive methylation. The labeled inhibitor retained the capacity to inactivate and to form 1:1 molar complexes with either the unlabeled or labeled trypsin and α-chymotrypsin. After intravenous injection of reductively methylated α₁-AT into rats, the labeled glycoprotein showed a circulating half-life of 12 h. When the N-acetylneuraminic acid residues were removed from the labeled α₁-AT by neuraminidase in vitro, injection into rats of this product resulted in a rapid (half-life of about 5 min) and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver of over 75% of the injected dose. The reductively methylated radioactively labeled trypsin and chymotrypsin experienced no loss of enzymatic activities. They showed the ability to form complexes in vivo with the two major plasma inhibitors, namely, α₁-AT and α₂-macroglobulin. High-voltage paper electrophoretic separation of acid hydrolysates of the labeled proteins revealed that ?-N-monomethyllysine and ?N,N-dimethyllysine are the only residues found to be radioactive.     

The role of sialic acid and galactose residues in determining the survival of human plasma alpha-antitrypsin in the blood circulation.

Human plasma α₁-antitrypsin (α₁-AT) is a glycoprotein known to contain terminal sialic acids (N-acetylneuraminic acids) in the carbohydrate units. These residues were converted to a radioactive seven-carbon analog (NANA-7) by sequential periodate oxidiation and tritiated borohydride reduction. Modified α₁-AT prepartions, namely, (a) periodate oxidized α₁-AT, (b) asialo α₁-AT (neuraminidase-treated α₁-AT), (c) (NANA 7)-α₁-AT (periodate-oxidized, -tritiated, borohydride-reduced α₁-AT), (d) (NANA-7)-α₁ AT (partially desialylated by neuraminidase), and (e) partially desialylated (NANA 7)-α₁-AT oxidized with galactose oxidase, all retained the following properties attributable to native α₁-AT: trypsin-inhibitory and chymotrypsin-inhibitory activities, immunological reactivity to antibody against native α₁-AT, and the ability to bind to concanavalin A-Sepharose 4-B columns. After intravenous injection of intact (NANA-7)-α₁-AT into rats, the labeled material had a circulating half-life of 18 h. When (NANA-7)-α₁-AT was partially desialylated (four residues of NANA-7 out of a total of six were removed, thus exposing an equivalent number of galactose residues at the terminal positions) by neuraminidase, injection into rats of this material resulted in a rapid and almost complete disappearance of the label from the circulation in 30 min. There was a concomitant accumulation of radioactivity in the liver. The rate of this rapid transfer depended on the presence of intact galactose residues as the terminal, nonreducing sugar in the carbohydrate units. Galactose oxidase treatment of the partially desialylated (NANA-7)-α₂-AT, which presumably oxidized the primary alcohol of galactose at C-6 to an aldehyde group, caused a reversion of its survival time in the circulation to that of the intact (NANA-7)-α₁-AT.     

Chemical modifications of lysyl and arginyl residues of human plasma α1-antitrypsin

Reactions of human plasma α₁-antitrypsin (α₁-AT) with reagents known to modify the lysyl residues [citraconic anhydride, acetic anhydride, 2,4,6-trinitrobenzenesulfonic acid (TNBS)] and arginyl residues [1,2-cyclohexanedione (CHD) and phenylglyoxal (PGO)] in proteins have been studied. Native and modified human plasma α₁-AT preparations were tested for their inhibitory activities against trypsin and α-chymotrypsin. TNBS was utilized to modify and quantitate free amino groups (?-NH₂ groups of lysine residues) in human plasma α₁-AT. The number of lysine residues determined by the TNBS spectrophotometric procedure agreed well with that found by amino acid analyses. Both the trypsin-inhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed by modification with TNBS. CHD was employed to modify the arginyl residues of α₁-AT. Neither the trypsin-inhibitory nor the chymotrypsin-inhibitory activity of α₁-AT was affected by modification of its arginyl residues. Amino acid analyses of the CHD-treated α₁AT revealed that only the arginine residues were modified. PGO was also utilized for the modification of the arginyl residues in α₁-AT. Both the trypsininhibitory and chymotrypsin-inhibitory activities of α₁-AT were destroyed after modification. However, amino acid analyses showed that not only the arginyl, but also the lysyl residues of the PGO-treated inhibitor were modified. The side reaction of PGO with the lysyl residues could explain the loss of inhibitory activities. Reaction of a α₁-AT with citraconic anhydride resulted in an extensive modification of the amino groups accompanied by a 100% loss in inhibitory activity against both trypsin and α-chymotrypsin. Comparable results were observed when acetic anhydride was utilized as the acylating reagent. With the exception of the citraconylated α₁AT, all of the other chemically modified α₁-AT derivatives studied presently retained their immunological reactivities against antisera to native α₁-AT. Regeneration of about 60% of the PGO-blocked arginyl residues in α₁-AT did not lead to any recovery of the proteinase inhibitory activities. Full recovery of trypsin-inhibitory and immunological activities were achieved when about 50% of the citraconylated amino groups were deblocked. The CHD-treated α₁-AT still retained the capacity to form complexes with both trypsin and chymotrypsin. On the other hand, the other chemically modified α₁-AT derivatives have completely lost the ability to form complexes with the enzymes. Recovery of the ability to form complexes with the enzymes was, however, recovered when about 50% of the citraconylyl groups was removed from the α₁-AT molecule. Based on these modification studies, it is concluded that α₁-AT is a lysyl inhibitor type (i.e., the reactive site is Lys-X bond) and that the interaction of α₁-AT with trypsin or chymotrypsin very likely involves or requires the same site as in the case of the soybean trypsin inhibitor (Kunitz).     

o-phthalaldehyde for the fluorometric assay of nonprotein amino compounds.

Procedures for the preparation of UDP-N-[1-¹⁴C]acetyl-d-glucosamine and UDP-N-[1-¹⁴C]acetyl-d-galactosamine with very high specific activities are deseribed. The overall yield based on the amount of [1-¹⁴C]acetate used is greater than 80%. The N-acetyl-d-glucosamine-α-1-phosphate used in this synthesis is prepared by phosphorylation of tetraacetyl-d-N-acetylglucosamine with crystalline phosphoric acid. N-acetyl-d-glucosamine-α-1-phosphate is then deacetylated in anhydrous hydrazine with hydrazine sulfate as a catalyst. d-glucosamine-α-1-phosphate is N-acetylated with [¹⁴C]acetate using N-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline as the coupling agent. The acetylated product is coverted to the UDP derivative with yeast UDP-N-acetyl-d-glucosamine pyrophosphorylase. UDP-N-[1-¹⁴C]acetylgalactosamine is prepared by acetylation of UDP-galactosamine using [1-¹⁴C]acetate and N-ethoxy-carbonyl-2-ethoxy-1,2-dihydroquinoline. UDP-galactosamine is prepared enzymatically using galactokinase and galactose-1-phosphate uridyltransferase. The labeled products, isolated and characterized by ion-exchange and paper chromatography, were active as substrates in glycosyl transferase systems.     

Synthesis of carbon-11-labeled casimiroin analogues as new potential PET agents for imaging of quinone reductase 2 and aromatase expression in breast cancer

Carbon-11-labeled casimiroin analogues were first designed and synthesized as new potential PET agents for imaging of quinone reductase (QR) 2 and aromatase expression in breast cancer. [¹¹C]casimiroin (6-[¹¹C]methoxy-9-methyl-[1,3]dioxolo[4,5-h]quinolin-8(9H)-one, [¹¹C]11) and its carbon-11-labeled analogues 5,6,8-trimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]17), 8-methoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21a), 6,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21b), and 5,8-dimethoxy-1-[¹¹C]methyl-4-methylquinolin-2(1H)-one ([¹¹C]21c), were prepared from their corresponding precursors with [¹¹C]methyl triflate ([¹¹C]CH₃OTf) under basic conditions (NaH) through either O- or N-[¹¹C]methylation and isolated by semi-preparative HPLC method in 40-50% radiochemical yields decay corrected to end of bombardment (EOB), based on [¹¹C]CO₂, and 111-185 GBq/μmol specific activity at the end of synthesis (EOS).     

Cell association and degradation of α2-macroglobulin-trypsin complexes in hepatocytes and adipocytes

¹²⁵I-labelled α₂-macroglobulin-typrin complex (¹²⁵I-labelled α₂-macroglobulin·trypsin) was associated to isolated rat adipocytes and hepatocytes with a half-time of about 60 min at 37°C. The association of 0.5 μg/ml ¹²⁵I-labelled α₂-macroglobulin·trypsin was inhibited by unlabelled α₂-macroglobulin·trypsin with a half-inhibition constant of about 8 μg/ml (11 nM). ¹²⁵I-Labelled α₂-macrioglubulin became cell-associated to a smaller extent (10–40% of that of α₂-macroglobulin·trypsin) and the half-inhibition constant was about 35 μg/ml in adipocytes. The cell associated of ¹²⁵I-labelled α-macroglobulin·trypsin was markedly inhibited by dansylcadaverin, bacitracin, omission of Ca²⁺ from the medium or pretreatment of the cell with trypsin. After incubation for 180 min more than 60% of the cell-associated ¹²⁵-Ilabelled α₂-macroglobulin·trypsin was not removed by treatment of the cells with trypsin-EDTA and represented probably internalized marterial. ¹²⁵I-Labelled α₂-macroglobulin·trypsin was degraded to trichloroacetic acid-soluble fragments by suspensions of both cell types but only to a negligible extent by incubation media preincubated with these cells. The rate of degradation of 0.5 μg/ml ¹²⁵I-labelled α₂-macroglobulin was approx. 40% of that of ¹²⁵I-labelled α₂-macroglobulin·trypsin. Degradation of ¹²⁵I-labelled α₂-macroglobulin·trypsin was abolished by a high concentration (0.5 mg/ml) and α₂-macroglobulin·trypsin. It is concluded that α₂-macroglobulin·trypsin by a specific and saturable mechanism is bound to, internalized and degraded by isolated rat adipocytes and hepatocytes.     

Characterization of particulate D-apiosyl-and D-xylosyltransferase from Lemna minor.

A particulate enzyme preparation capable of catalyzing the transfer of d-[U-¹⁴C]apiose and d-[U-¹⁴C]xylose from uridine 5′-(α-d-[U-¹⁴C]apio-d-furanosyl pyrophosphate) (UDP[U-¹⁴C]Api) and uridine 5′-(α-d-[U-¹⁴C]xylopyranosyl pyrophosphate) (UDP[U-¹⁴C]Xyl) to endogenous acceptor molecules was isolated from Lemna minor. The two enzymes were named UDP-d-apiose:acceptor d-apiosyltransferase and UDP-d-xylose:acceptor d-xylosyltransferase and were associated with particulate material sedimenting between 480 and 34,800g. The rate of d-[U-¹⁴C]apiose or d-[U-¹⁴C]xylose incorporation was proportional to the quantity of enzyme preparation used and was constant with time to 1.5 min. Both enzymes showed a pH optimum of 5.7 in citrate-phosphate buffer. The d-apiosyltransferase has a K_m for UDP[U-¹⁴C]Api of 4.9 μm. Bovine serum albumin and sucrose stimulated the rate of incorporation of both pentoses. Both enzymes rapidly lost activity; with our best conditions, approximately 50% of each enzyme activity was lost in 6 min at 25 °C or in 3 h at 4 °C. Incorporation of d-[U-¹⁴C]apiose was obtained in the absence of added uridine 5′-(α-d-galactopyranosyluronic acid pyrophosphate) (UDPGalUA); however, the addition of UDPGalUA not only almost doubled the rate of incorporation, but also increased the total incorporation of d-[U-^l4C]apiose and extended the proportional range of incorporation at 25 °C from 1.5 to 2 min.     

Uptake of 3-O-methyl-D-[U-14C]glucose into brain of rainbow trout: possible effects of melatonin

The influx of glucose into the brain and plasma glucose disappearance were estimated in rainbow trout (Oncorhynchus mykiss) intravenously injected (1 ml · kg⁻¹ body weight) with a single dose (15 μCi · kg⁻¹ body weight) of 3-O-methyl-D-[U-¹⁴C]glucose ([U-¹⁴C]-3-OMG) at different times (2–160 min), and after intravenous injection at 15 min of increased doses (10–60 μCi · kg⁻¹ body weight) of [U-¹⁴C]-3-OMG. Brain and plasma radiotracer concentrations were measured, and several kinetic parameters were calculated. The apparent brain glucose influx showed a maximum after 15–20 min of injection then decreased to a plateau after 80 min. Brain distribution space of 3-OMG increased from 2 min to 20 min reaching equilibrium from that time onwards at a value of 0.14 ml · g⁻¹. The unidirectional clearance of glucose from blood to brain (k₁) and the fractional clearance of glucose from brain to blood (k₂) were estimated to be 0.093 ml · min⁻¹ · g⁻¹, and 0.867 min⁻¹, respectively. A linear increase was observed in brain and plasma radiotracer concentrations when increased doses of [U-¹⁴C]-3-OMG were used. All these findings support a facilitative transport of glucose through the blood-brain barrier of rainbow trout with characteristics similar to those observed in mammals. The injection of different doses of melatonin (0.25–1.0 mg · kg⁻¹) significantly increased brain glucose influx suggesting a possible role for melatonin in the regulation of glucose transport into the brain. Accepted: 26 January 2000     

High-performance liquid chromatographic determination of [C]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide in mouse plasma and tissue and in human plasma

The high-performance liquid chromatographic determination of 1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3-isoquinoline carboxamide ([¹¹C]PK 11195) is described. The method was successfully applied for plasma and tissue analysis after i.v. injection of [¹¹C]PK 11195 in mice and for plasma analysis after administration of [¹¹C]PK 11195 to humans. Separation is effected on a RP-C₁₈ column, using a mixture of acetonitrile–water–triethylamine (65:35:0.5, v/v). Quantitative measurements of radioactivity are performed on a one-channel γ-ray spectrometer equipped with a 2×2 in. NaI(Tl) detector. For humans rapid metabolisation of [¹¹C]PK 11195 was observed. At 5, 20 and 35 min post injection 5%, 22% and 32%, respectively, of the plasma activity consisted of at least two more polar metabolites. Despite the extensive metabolisation rate in mice (up to 42% at 10 min post injection of [¹¹C]PK 11195), no ¹¹C-labelled metabolites could be detected in the extracts of brain and heart.     

Synthesis and biodistribution of [11C]fludiazepam for imaging benzodiazepine receptors

As a tracer for in vivo studies on benzodiazepine receptors, 7-chloro-1,3-dihydro-5-(2-fluorophenyl)-1-[¹¹C]methyl-2H-1, 4-benzodiazepin-2-one, [¹¹C]fludiazepam, was synthesized by the methylation of norderivative with [¹¹C]CH₃I, and purified by high-performance liquid chromatography. Within 60 min [¹¹C]fludiazepam was obtained for injection in high radiochemical yields and in high radiochemical purity with a specific activity of up to 230mCi/μmol.After i.v. injection of [¹¹C]fludiazepam in rats the radioactivity was rapidly incorporated into many tissues and the blood clearance of the radioactivity was very rapid. The brain uptake was high and decreased gradually. The adrenal uptake was the highest and decreased with high loading doses. The effect of the loading dose on the uptake was also found in the heart and lungs. By autoradiography using [¹¹C]fludiazepam, a higher accumulation was visualized in the cortex and thalamus than in other regions.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

Denitrosation of carcinostatic nitrosoureas by purified NADPH cytochrome P-450 reductase and rat liver microsomes to yield nitric oxide under anaerobic conditions

The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [¹⁴C]CCNU (1-(2-[¹⁴C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [¹⁴C]CCU (1-(2-[¹⁴C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [¹⁴C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [¹⁴C]CCNU was shown to undergo denitrosation to [¹⁴C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.     

Evidence for the enzymatic transfer of N-acetylglucosamine from UDP-N-acetylglucosamine into dolichol derivatives and glycoproteins by calf brain membranes

Incubating white matter membranes with UDP-N-acetyl-[¹⁴C]glucosamine in the presence of Mg²⁺ and AMP resulted in the labeling of two major glycolipids, a minor glycolipid and several membrane-associated glycoproteins. The addition of AMP protected the labeled sugar nucleotide from degradation by a membrane-bound sugar nucleotide pyrophosphatase activity. While no labeled oligosaccharide lipid was recovered in a CHCl₃CH₃OHH₂O (10:10:3) extract after incubating with only UDP-N-acetyl-[¹⁴C] glucosamine, Mg²⁺, and AMP, the inclusion of unlabeled GDP-mannose led to the formation of an N-acetyl-[¹⁴C]glucosamine-labeled oligosaccharide lipid that was soluble in CHCl₃CH₃OHH₂O (10:10:3). The [GlcNAc-¹⁴C]oligosaccharide unit was released by treatment with 0.1 N HCl in 80% tetrahydrofuran at 50 °C for 30 min and appears to have the same molecular size as the lipid-linked [mannose-¹⁴C] oligosaccharide, formed enzymatically by white matter membranes as judged by their elution behavior on Bio-Gel P-6. The incorporation of N-acetyl-[¹⁴C]glucosamine into glycolipid was stimulated by exogenous dolichol monophosphate, but inhibited by UMP or tunicamycin, a glucosamine-containing antibiotic. Although UMP and tunicamycin drastically inhibited the labeling of glycolipid, these compounds had very little effect on the labeling of glycoproteins. The major glycolipids have the chemical and Chromatographic characteristics of N-acetylglucosaminylpyrophosphoryldolichol and N,N′-diacetylchitobiosylpyrophosphoryldolichol. When the labeled glycoproteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, four labeled polypeptides were observed, having apparent molecular weights of 145,000, 105,000, 54,000, and 35,000. Virtually all of the N-acetyl-[¹⁴C]glucosamine was released when the labeled glycopeptides, produced by pronase digestion, were incubated with an exo-β-N-acetylglucosaminidase, indicating that all of the N-acetyl-[¹⁴C]glucosamine incorporated under these conditions is attached to white matter membrane glycoproteins at nonreducing termini.     

Enzymatic synthesis of 2-O-α-d-mannopyranosyl-methyl-α-d-mannopyranoside by a cell-free particulate system of mycobacterium smegmatis

A cell-free particulate enzyme preparation of Mycobacterium smegmatis ATCC 607 catalyzed the transfer of labeled mannose from GDP[¹⁴C]mannose to methyl-α-mannopyranoside (an exogenously added acceptor) to form a product that was characterized to be $\text{2-O-α-}\text{d}\text{-[}{}^{14}\text{C}\text{]}\text{mannopyranosyl-methyl}\text{-α-}\text{D-mannopyranoside}$. This tranmannosylase activity was specific for both the sugar nucleotide donor and methyl monosaccharide acceptor. The reaction was stimulated by the addition of various metal ions and had a pH optimum of 6.0. The apparent K_m of this transmannosylase reaction for methyl-α-d-mannopyranoside was 35 mM.The possible relationship between this “artificial” mannosyl-transfer system and the “natural” system which leads to the formation of the oligomannosides and glycoproteins is discussed.     

Biosynthesis of piperlongumine

The incorporation of l-[U-¹⁴C]lysine and l-[U-¹⁴C]phenylalanine into piperlongumine has been demonstrated in Piper longum. The subsequent stepwise degradation to methyl-(3,4,5-trimethoxyphenyl)-propanoate and δ-aminovaleric acid revealed that the C₆-C₃ moiety of the alkamide arises from phenylalanine; the heterocyclic ring is biosynthesised from lysine. It has also been shown that dl-[2-¹⁴C]tyrosine and [2-¹⁴C]sodium acetate are poor precursors of piperlongumine.     

A high-yield route to synthesize the P-glycoprotein radioligand [11C]N-desmethyl-loperamide and its parent radioligand [11C]loperamide

N-Desmethyl-loperamide and loperamide were synthesized from α,α-diphenyl-γ-butyrolactone and 4-(4-chlorophenyl)-4-hydroxypiperidine in five and four steps with 8% and 16% overall yield, respectively. The amide precursor was synthesized from 4-bromo-2,2-diphenylbutyronitrile and 4-(4-chlorophenyl)-4-hydroxypiperidine in 2 steps with 21–57% overall yield. [¹¹C]N-Desmethyl-loperamide and [¹¹C]loperamide were prepared from their corresponding amide precursor and N-desmethyl-loperamide with [¹¹C]CH₃OTf through N-[¹¹C]methylation and isolated by HPLC combined with solid-phase extraction (SPE) in 20–30% and 10–15% radiochemical yields, respectively, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB), with 370–740 GBq/μmol specific activity at EOB.     

Targeting of auxin carriers to the plasma membrane: differential effects of brefeldin A on the traffic of auxin uptake and efflux carriers

Monensin and brefeldin A (BFA), inhibitors of Golgi-mediated protein secretion, rapidly perturb the transport catalytic activity of specific plasma membrane-associated efflux carriers for indole-3-acetic acid (IAA) and inhibit polar transport of IAA. To determine if these responses result solely from perturbation of the efflux carrier or whether specific auxin uptake carrier function is also affected, the influence of BFA on the cellular transport of a range of auxins with contrasting affinities for specific auxin uptake and efflux carriers was investigated in zucchini (Cucurbita pepo L.) hypocotyl tissue. In-flight addition of BFA (3 · 10⁻⁵ mol · dm⁻³) caused a rapid (lag < 10 min) and substantial (fourfold) increase in the rate of [1-¹⁴C]IAA net uptake by zucchini hypocotyl tissue. In the presence of the specific auxin efflux carrier inhibitor N-1-naphthylphthalamic acid (NPA; 3 · 10⁻⁶ mol · dm⁻³), BFA slightly reduced the rate of [1-¹⁴C]IAA net uptake. Stimulation of [1-¹⁴C]IAA net uptake by BFA was concentration-dependent. In the absence of BFA, net uptake of [1-¹⁴C]IAA exhibited the characteristic biphasic response to increasing concentrations of competing cold IAA but in the presence of BFA, [1-¹⁴C]IAA uptake decreased smoothly with increase in concentration of competing unlabelled IAA, indicating a loss of auxin efflux carrier activity but retention of functional uptake carriers. The half-time for mediated efflux of [1-¹⁴C]IAA from preloaded zucchini tissue was substantially increased by BFA (t_1/2 = 51 min, controls; 107 min, BFA-treated). Treatment with BFA and/or NPA did not significantly affect the net uptake by, or efflux from, zucchini tissue of [1-¹⁴C]2,4-dichlorophenoxyacetic acid ([1-¹⁴C]2,4-D), a substrate for the auxin uptake carrier but not the auxin efflux carrier. Uptake of [1-¹⁴C]2,4-D declined smoothly with increasing concentrations of competing unlabelled IAA whether or not BFA was included in the uptake medium, confirming the failure of BFA to perturb auxin uptake carrier function. Transport of 1-[4-³H]naphthaleneacetic acid (1-NAA) exhibited little response to BFA or NPA, confirming that it is only a weakly transported substrate for the efflux carrier in zucchini cells. Received: 12 November 1997 / Accepted: 27 January 1998     

The plasma clearance of human α2-macroglobulin-trypsin complex in the rat is mainly accounted for by uptake into hepatocytes

Rats were given intravenous injections of ¹²⁵I-labelled human α₂-macroglobulin·trypsin. The half-time of disappearance of radioactivity from arterial blood was 2 min. External counting showed that radioactivity in the liver was maximal by 10 min and then decreased slowly. 87% of the injected dose was recovered in the liver by 10 min. Light- and electron microscopic autoradiography carried out on samples of liver fixed with glutaraldehyde 3 min or 30 min after the injection showed that 85–90% of the grains were over the hepatocytes and 4–9% were over the Kupffer cells. Thus, uptake into hepatocytes, and not into Kupffer cells as believed previously, appears to account for the major part of the uptake of α₂-macroglobulin·trypsin by the liver and thereby for its rapid removal from the blood.     

The biliary excretion of circulating asialoglycoproteins in the rat.

Following intravenous injection into the rat a small proportion (0.5 – 3.0%) of asialo α₁-acid glycoprotein, asialo fetuin, asialo CEA¹ and native CEA are excreted in an apparently unchanged form in the bile. The maximum excretion rate occurs one hour after injection in all cases. The possibility of a novel pathway for glycoprotein uptake by the liver is discussed.     

Studies on the production and assessment of experimental histidinemia in the rat

Intraperitoneal administration to rats of D- or DL-α-hydrazunoimidazolylpropionic acid was found to produce a substantial inactivation of hepatic histidine ammonia-lysase (EC 4.3.1.3) in vivo. Proportional to this loss in enzyme activity was an impairment of the ability of treated rats to oxidize l-[ring-2-¹⁴C] histidine to ¹⁴CO₂. Rats in which hepatic histadine ammonia-lyase activity was either depressed by dl-hydrazunoimidazolylproprionic acid injection or elevated by feeding a high protein diet displayed proportionately altered rates of ³H₂O release into plasma water following l-[3-H]histidine administration. Plasma l-histidine clearance following loading with this amino acid was similarly affected by these treatments. Administration of dl-α-hydrazinoimisazolyl-proprionic acid to rats was also found to inactivate non-specifically pyridoxal 5-phosphate enzymes in vivo; pyridoxine injection was found to reverse the dl-α-hydrazinoimidazolylproprionic acid-induced inactivation of hepatic aspartate aminotransferase (EC 2.6.1.1) in vivo, but not that of hepatic histidine ammonia-lyase. These findings demonstrate that histidine ammonia-lyase is the rate-limiting factor in l-histidine degradation in the rat. The potential usefulness of dl-hydrazinoimidazolylproprionic acid in the production of an animal model for histidinemia (hereditary histidine ammonia-lyase deficiency) is discussed.