Transfer of N, N'-diacetylchitobiose from dolichyl diphosphate into a gray matter membrane glycoprotein

Gray matter and white matter membranes catalyze the transfer of label from UDP-N-acetyl-[${}^{14}\text{C}$] glucosamine into N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, N,N′-diacetyl [${}^{14}\text{C}$]chitobiosyl-pyrophosphoryl-dolichol, and N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein. Gel filtration of the Pronase digests of gray matter N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein reveals two N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide fractions. One fraction (A) contains approximately eight glycose units. All of the radioactivity is at nonreducing termini and can be released by treatment with an exo-β-N-acetylglucosaminidase. A smaller N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (B) is recovered in the elution volume expected for an asparaginyl disaccharide. Structural studies show that the labeled saccharide unit in glycopeptide B is N,N′-diacetyl[${}^{14}\text{C}$]chitobiose. The linkage between the ${}^{14}\text{C}$-labeled disaccharide and the polypeptide has the properties of an N-glycosidic attachment to asparagine. Only the larger N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycopeptide (A) is found in Pronase digests of white matter membrane N-acetyl[${}^{14}\text{C}$]glucosamine-labeled glycoprotein after incubation with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine. When gray matter membranes are incubated with UDP-N-acetyl[${}^{14}\text{C}$]glucosamine in the presence of tunicamycin or UMP, the labeling of glycolipid and the asparaginyl disaccharide is inhibited. UMP and tunicamycin have no effect on the transfer of N-acetyl[${}^{14}\text{C}$]glucosamine to external acceptor sites of the larger glycopeptide (A). The transfer of N,N′-diacetyl[${}^{14}\text{C}$]-chitobiose from carrier lipid to protein is observed when extensively washed membranes containing endogenous, prelabeled ${}^{14}\text{C}$-labeled glycolipids are incubated in the presence or absence of unlabeled GDP-mannose. UMP treatment of the prelabeled membranes selectively discharged over 80% of the label from N-acetyl[${}^{14}\text{C}$]glucosaminyl-pyrophosphoryl-dolichol, but had no effect on the transfer of the ${}^{14}\text{C}$-labeled disaccharide to protein. All of these results are concordant with transfer of N,N′-diacetylchitobiose from dolichyl diphosphate to gray matter glycoprotein. The major membrane glycoprotein labeled by the lipid-mediated [${}^{14}\text{C}$]disaccharide transfer reaction has an apparent molecular weight of 24,000. Tunicamycin prevents the enzymatic labeling of the gray matter glycoprotein having an apparent molecular weight of 24,000.     

Esterification of chlorophyllide by geranylgeranyl pyrophosphate in a cell-free system from maize shoots.

A cell-free system is described which incorporates [${}^{14}\text{C}$]-geranylgeranyl pyrophosphate, but not free [${}^{14}\text{C}$]-geranylgeraniol, into chlorophyll a_{geranylgeraniol}. The esterifying enzyme is found in the 75,000 g pellet of a homogenate from maize shoots whereas most of the phosphatase activity remains in the supernatant. The enzyme is different from chlorophyllase which has been discussed in the literature as the possible esterifying enzyme.     

Non-sterol metabolism of mevalonate in vitro: artifacts and realities.

Commercial [5-¹⁴C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-¹⁴C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated $\text{R}\text{-5-phospho-[5-}{}^{14}\text{C]mevalonate}$ by ion-exchange chromatography. The artifactual ${}^{14}\text{CO}{}_2$ results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the ${}^{14}\text{CO}{}_2$ and [${}^{14}\text{C}$]cholesterol produced by rat renal cortex slices incubated with purified [5-¹⁴C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that $\text{R}\text{[5-}{}^{14}\text{C]mevalonate}$ is genuinely oxidized to ${}^{14}\text{CO}{}_2\text{in}\text{vitro}$, and that purification of substrate before its use is necessary. Production of ${}^{14}\text{CO}{}_2$ and various [${}^{14}\text{C}$]lipids from purified [5-¹⁴C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described.     

Studies on the metabolism of guanidine compounds in mammals

[¹⁴C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-¹⁴C]arginine or l-[guanidino-¹⁴C]canavanine. [${}^{14}\text{C}$]Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-¹⁴C]canavanine. These ${}^{14}\text{C}$ metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [¹⁴C]hydroxyguanidine to [¹⁴C]guanidine, and by repeated recrystallization of isolated urinary [${}^{14}\text{C}$]guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat.     

Tyrosine metabolism for cuticle tanning in the tobacco hornworm, Manduca sexta (L.) and other Lepidoptera: identification of beta-D-glucopyranosyl-O-L-tyrosine and other metabolites

When [${}^{14}\text{C}$]tyrosine and [${}^{14}\text{C}$]glucose were fed or injected into feeding fifth-instar larvae of the tobacco hornworm, Manduca sexta (L.), they were incorporated into a conjugate identified in hemolymph and carcass extracts as β-d-glucopyranosyl-O-l-tyrosine. In wandering larvae and pupae, the conjugate was hydrolyzed, and tyrosine was hydroxylated and decarboxylated to dihydroxyphenylalanine and 2-(dihydroxyphenyl)ethylamine. None of these metabolites were formed in fourth-instar larvae or in adults. [${}^{14}\text{C}$]Phenylalanine was hydroxylated to tyrosine in all stages of insect development. β-d-Glucopyranosyl-O-l-tyrosine was also detected in 18 other species of Lepidoptera but not in species from other insect orders. This conjugate appears to be the major tyrosine storage metabolite for production of tanning diphenol substrates in Lepidoptera.     

Assembly of the vesicular stomatitis virus envelope: incorporation of viral polypeptides into the host plasma membrane

Incorporation of viral polypeptides into the host plasma membrane is an essential step in the formation of the lipoprotein envelope of vesicular stomatitis virus. A quantitative study of this process was carried out using a double-isotope labeling procedure. Infected cells were incubated for two hours with ¹⁴C-labeled amino acids, pulse-labeled with [³H]leucine and incubated for various times with an excess of non-radioactive leucine. The ${}^3\text{H}{}^{14}\text{C}$ ratio was determined for each viral polypeptide in isolated plasma membranes and in the whole cell by polyacrylamide gel electrophoresis. It was found that [³H]leucine-labeled viral polypeptides could be detected in the plasma membranes immediately following a 30-second pulse but that the ${}^3\text{H}{}^{14}\text{C}$ ratios of polypeptides in the plasma membrane did not reach the ${}^3\text{H}{}^{14}\text{C}$ ratios in the whole cells until the end of a two-minute chase period. The addition of puromycin to the cultures at the end of the pulse period did not affect subsequent incorporation of [³H]leucine-labeled polypeptides into the plasma membrane. The incorporation of various amino acid analogs into the viral polypeptides did not affect the efficiency with which they were incorporated into the plasma membranes. It is proposed that viral polypeptides are selected for incorporation into the plasma membrane from a small interior pool of completed molecules.     

Invivo conversion of 3-ketoceramide to ceramide in rat liver

A doubly labeled 3-ketoceramide, [1-¹⁴C] lignoceroyl [1-³H₂] 3-ketosphingosine ($\text{3H}{}^{14}\text{C}$ ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal $\text{3H}\text{>}{}^{14}\text{C}$ ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver.     

Tissue-specificity of N-nitrosodibutylamine metabolism in Sprague-Dawley rats

The distribution and metabolism of $\text{N-[}{}^{14}\text{C}\text{]}$nitrosodibutylamine were studied in Sprague-Dawley rats. The results indicated that in addition to the liver, metabolism of the substance occurred in the nasal mucosa, the lung and the oesophagus. Metyrapone and diethyldithiocarbamate reduced the production of ¹⁴CO₂ from $\text{N-[}{}^{14}\text{C}\text{]}$nitrosodibutylamine by all these tissues. There was no indication of metabolic capacity in the urinary bladder or the kidney. The results fit with the assumption that tumours of the urinary tract are induced by metabolites reaching these tissues via the urine. Besides the liver, the oesophagus and the lung are target tissues for the carcinogenicity of N-nitrosodibutylamine in Sprague-Dawley rats and in these tissues the local formation of reactive metabolites may play a role in the pathogenesis of N-nitrosodibutylamine-induced lesions.     

Interaction of kanamycin and related antibiotics with the large subunit of ribosomes and the inhibition of translocation.

The binding of [${}^3\text{H}$]kanamycin to $\text{E. coli}$ ribosomes and ribosomal subunits was studied by equilibrium dialysis and Millipore filter methods. The 70S ribosome bound ca. two molecules up to the antibiotic concentration of 10 uM, and more at higher concentrations. Each ribosomal subunit was observed to possess one major binding site, and the affinity of the small ribosomal subunit was greater than that of the large subunit. The binding of [${}^3\text{H}$]kanamycin to ribosomes and ribosomal subunits was reversed by neomycin or gentamicin, but not by streptomycin and chloramphenicol. Kanamycin, neomycin and gentamicin interfered with the binding of [${}^{14}\text{C}$] tuberactinomycin O. Translocation of N-Ac-Phe-tRNA was markedly inhibited by kanamycin, neomycin or gentamicin, but not by streptomycin.     

Magnetic resonance study of 13C-enriched glycophorin A reconstituted into phospholipid vesicles

$\text{[S-[}{}^{13}\text{C}\text{]}\text{methylmethionine-8 and -81]glycophorin}$ A was reconstituted into l-α-phosphatidyl choline vesicles. Results indicate that the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethyionine}\text{-81}$ residue in the phospholipid bilayer has limited mobility and is not susceptible to dealkylation, whereas the opposite effects are indicated for the $\text{S-[}{}^{13}\text{C}\text{]}\text{methylmethionine}\text{-8}$ residue.     

An overall radioassay for the first three reactions of de novo pyrimidine biosynthesis

A sensitive radioassay is described for the overall biosynthetic activity of the multienzymatic protein which catalyzes the first three reactions of de novo pyrimidine biosynthesis in mammals. The ability of the multienzymatic protein to synthesize dihydroorotate can be assayed using $\text{[}{}^{14}\text{C]HCO}{}_3{}^{\mathrm{?}}$, l-[¹⁴C]aspartate, or [${}^{14}\text{C}$] carbamyl phosphate as substrate. The synthesis of the final product, l-dihydroorotate, may be coupled to synthesis of orotidine 5′-monophosphate to overcome the unfavorable equilibrium existing between l-dihydroorotate and its precursor, N-carbamyl-l-aspartate, in the physiological pH range (Christopherson, R. I., and Jones, M. E., 1979, J. Biol. Chem.254, 12506–12512). l-Aspartate and all pyrimidine intermediates from carbamyl phosphate to orotidine 5′-monophosphate can be clearly separated by ion-exchange chromatography in a single dimension on polyethyleneimine-cellulose chromatograms and carbamyl phosphate and its degradation product cyanate may be quantitated directly along with the other intermediates.     

The stimulating effect of 3′,5′-(cyclic)adenosine monophosphate and lipolytic hormones on 3-O-methylglucose transport and 45Ca2+ release in adipocytes and skeletal muscle of the rat

(1) In order to assess the possible role of 3′,5′-(cyclic)adenosine monophosphate (cAMP) in the control of glucose transport, the effect of the nucleotide or agents known to increase its intracellular concentration on sugar transport or ⁴⁵Ca²⁺ washout were characterized in epididymal fat pads, free fat cells and soleus muscles of the rat. (2) When added to the incubation medium, cAMP (0.1–2.0 mM) stimulated $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ washout from fat pads. This effect was abolished by cytochalasin B, and additive to that induced by submaximal (10–25 μU/ml), but not by supramaximal (10 mU/ml) concentrations of insulin. (3) cAMP (2 mM) stimulated the conversion of [U-¹⁴C]glucose into CO₂ and triacylglycerols. This effect was additive to that of insulin (100 μU/ml). (4) ACTH, glucagon, adrenaline, noradrenaline and salbutamol, which are all known to increase the cAMP content of adipose tissue, stimulated the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺ from preloaded fat pads. The fractional losses of the two isotopes were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.73}$). (5) In free fat cells, adrenaline (10^?6 M) and salbutamol (10^?5 M) stimulated the uptake of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$, and salbutamol (10^?5 M) did not interfere with the stimulating effect of insulin (25 μU/ml) on sugar uptake. (6) In rat soleus muscles, adrenaline and salbutamol produced a dose-dependent stimulation of the washout of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca²⁺. The effect of adrenaline on sugar efflux was abolished by propranolol. (7) It is concluded that the activation of the glucose transport system by insulin is unlikely to be mediated by a drop in the cellular concentration of cAMP. An increase in cAMP brought about by β-adrenoceptor agonists or lipolytic hormones may induce a mobilization of calcium ions from cellular pools into the cytoplasm, which in turn leads to the activation of the glucose transport system demonstrated in the present as well as in several earlier studies.     

The relationship between the transport of glucose and cations across cell membranes in isolated tissues XI. The effect of vanadate on 45Ca-efflux and sugar transport in adipose tissue and skeletal muscle

(1) The effects of vanadate of hexose transport, ⁴⁵Ca-exchange and (Na⁺, K⁺)-contents have been characterized in isolated adipose tissue and skeletal muscles of the rat. (2) In whole epididymal fat pads, vanadate (0.5–5.0 mM) markedly stimulated the uptake of 2-deoxyl[¹⁴C]glucose as well as the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$. (3) Within the same concentration range, vanadate induced an early increase in ⁴⁵Ca-washout from preloaded fat pads. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.001}$, $\text{r = 0.98}$). (4) In extensor digitorum longus and soleus muscles, vanadate (0.5–5.0 mM) stimulated the efflux of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methylglucose}$ and this effect was preceded by a rise in the washout of ⁴⁵Ca. The maximum increases in the fractional losses of $\text{3-O-[}{}^{14}\text{C}\text{]}\text{methyglucose}$ and ⁴⁵Ca were significantly correlated ($\text{P < 0.005}$, $\text{r = 0.98}$). (5) In extensor digitorum longus and soleus muscles, vanadate increased K⁺-contents and decreased Na⁺ contents. (6) The stimulation of ⁴⁵Ca-washout presumably reflects an increase in the cytoplasmic Ca²⁺ level, brought about by an inhibitory effect of vanadate on the Ca²⁺-sensitive ATPase of the sarcoplasmic or the endoplasmic reticulum. As demonstrated for most other insulin-like agents (Sørensen, S.S., Christensen, F. and Clausen, T. (1980) Biochim. Biophys. Acta 602, 433–445), the stimulating effect of vanadate on glucose transport appears to be associated with or mediated by a rise in the cytoplasmic Ca²⁺ level.     

Demonstration of a very short post-mitotic G1 period in proliferating PHA-stimulated lymphocytes

Evidence indicating a very short duration of the post-mitotic G1 phase of PHA-stimulated lymphocytes was obtained in experiments where proliferating lymphocytes were pulsed for 1 h with ³H-TdR and then, following different periods of incubation in ‘cold’ medium, again pulsed for 1 h with ¹⁴C-TdR. Cells labelled with ³H-and (³H + ¹⁴C)-TdR were counted in double layer autoradiograms. Cells labelled with ³H-TdR should gradually leave the S phase when incubated in ‘cold’ medium and would consequently not incorporate ¹⁴C-TdR. Thus, the quotient

$\text{\%(}{}^3\text{H}\text{+}{}^{14}\text{C}\text{)}\text{cells}\text{\%}{}^3\text{H}\text{+ \%(}{}^3\text{H}\text{+}{}^{14}\text{C}\text{)}\text{cells}$

should decrease with time in ‘cold’ medium and should be zero after an incubation time corresponding to the length of the S phase. If, however, the sum of the G2 + M + G1 phases is shorter than the length of the S phase, the zero value will not be obtained. This was actually the case, and the quotients obtained agreed well with those that could be calculated on basis of the G1 period being 0 h, whereas they did not agree with those calculated on basis of G1 being 4–6 h.     

Magnetic resonance study of 13C reductively methylated glycophorin and glycophorin glycopeptides

¹³C nuclear magnetic resonance (n.m.r.) spectral data for ¹³C reductively methylated N-terminal tryptic glycopeptides and for ¹³C reductively methylated N-terminal glyco-octapeptides derived from homozygous glycophorins A^M and A^N are presented. Their ¹³C chemical shift data are compared with the previously published ¹³C n.m.r. data for ¹³C reductively methylated homozygous glycophorins A^M and A^N in order to investigate the means of display of the MN blood determinants by these species. The pH dependence of the ¹³C resonances of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glyco-octapeptide A^N and of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of glyco-octapepti A^M indicated that only a slight structural perturbation occurs at the N-terminus when a large portion of the glycoprotein molecule is removed. However, one structural ‘state’ of ¹³C reductively methylated glycophorin A^M is lost when the glyco-octapeptide A^M is produced. The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ leucine of glycooctapeptide A^N titrated with a $\text{p}\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of 7.7 (Hill coefficient $\text{～ 1}$). The ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine, on the other hand, exhibited an unusual pH dependence, indicating the existence of some possible steric constraints or hydrogen bonding in this molecule. In comparison to the data obtained for ¹³C-labelled glycooctapeptide A^M molecule, the pH dependence of the chemical shift of the ¹³C resonance of $\text{N}{}^{\mathrm{\alpha }}\text{,N-[}{}^{13}\text{C}\text{]}\text{dimethyl}$ serine of tripeptide tri-L-serine is also presented. Circular dichroism (c.d.) spectra indicated that the reductive methylation technique does not cause a large perturbation of the glycophorin A molecule.     

Accumulation of 2-deoxyglucose against its concentration gradient in rat adipocytes

Rat adipocytes were incubated at 37°C with 2-deoxy-d-[1-¹⁴C]glucose ([¹⁴C]2dGlc) at various concentrations and the intracellular concentrations of [¹⁴C]2dGlc and deoxy[¹⁴C]glucose phosphate ($\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$) were measured. Using 7 μM extracellular [¹⁴C]2dGlc, the intracellular [¹⁴C]2dGlc concentration approached the extracellular by 5 min in insulin-stimulated cells and by 60 min it exceeded the extracellular concentration by 50-fold. A maximum accumulation ratio of 3.5 was reached by 7 min using 1 mM and a ratio of 1.6 was reached by 1 to 3 min using 10 mM extracellular 2dGlc. The time at which the concentration of intracellular 2dGlc exceeded the extracellular was inversely related to the accumulation of $\text{2}\text{dGlc}\text{P}$. The rate of accumulation of total radioactivity ([¹⁴C]2dGlc plus $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$ decreased after 20 min using 7 μM extracellular [¹⁴C]2dGlc. This change occurred later at 22°C or in the absence of insulin and sooner at higher concentrations of 2dGlc. Experiments where uptake was stopped by dilution indicated that radioactivity appearing in the medium was [¹⁴C]2dGlc, but radioactivity disappearing from the cells was largerly $\text{[}{}^{14}\text{C}\text{]2}\text{dGlc}\text{P}$. Addition of 10 mM unlabelled 2dGlc or glucose to cells preincubated with 7 μM [¹⁴C]2dGlc resulted in a more rapid loss of accumulated label from the cells, while addition of 10 mM $\text{3-O-}\text{methylglucose}$, a non-metabolizeable sugar analogue with about the same affinity for the transport system as 2dGlc, was without effect. The results show that 2dGlc is accumulated against its concentration gradient. It is suggested that the mechanism involves first, dephosphorylation of $\text{2}\text{dGlc}\text{P}$ and second, the presence of a diffusion barrier between the site of dephosphorylation and the transport site.     

Occurrence of selenocysteine in the selenium-dependent formate dehydrogenase of Methanococcus vannielii.

The selenium-dependent formate dehydrogenase of Methanococcus vannielii was isolated from bacteria grown in the presence of [${}^{75}\text{Se}$]selenite. Purification under strictly anaerobic conditions resulted in the simultaneous enrichment of formate dehydrogenase activity, ${}^{75}\text{Se}$, and a brown chromophore that absorbs maximally at 380 nm. Acid hydrolysis of the enzyme after reduction with borohydride and alkylation with iodoacetamide, released a radioactive selenoamino acid derivative that was identified as [${}^{75}\text{Se}$]carboxymethyl-selenocysteine. This is the third selenoenzyme shown to contain selenocysteine.     

Purification of cytochromes P-448 from β-naphthoflavone-treated rainbow trout

Rainbow trout were treated with β-naphthoflavone and the hepatic microsomal cytochrome P-450 solubilized with 3-[(3-cholaminopropyl)dimethylammonio]-1-propanesulfonate. Chromatography on tryptamine-Sepharose 4B gave a single cytochrome P-450 peak which was further resolved into three components by elution from DEAE-Sepharose. The two main peaks were then chromatographed on hydroxyapatite and a total of four fractions obtained. Two of these fractions had similar properties and significantly metabolized $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ in a reconstituted system containing rat cytochrome P-450 reductase. This activity was inhibited by α-naphthoflavone but not by metyrapone or SKF-525A. Purified cytochromes P-448 from 3-methylcholanthrene-treated rat had similar spectral properties and activity towards $\text{[}{}^{14}\text{C}\text{]}\text{benzo}\text{[a]}\text{pyrene}$ suggesting similarities between these forms.