Agarose-DTNB column for binding sulfhydryl-containing proteins and peptides.

The counting rate of [1-¹⁴C]trichloroacetic acid (TCA) was not stable in a standard toluene/Triton X-100 liquid scintillation solution because this compound becomes partially degraded to ¹⁴CO₂ and CHCl₃. Both toluene and Triton were contributing factors in causing this degradation. The NCS solubilizer added to the toluene/Triton scintillation solution trapped ¹⁴CO₂ and stabilized counting rates of [1-¹⁴C]TCA. When [2-¹⁴C]TCA was used, ¹⁴CHCl₃ remained in the scintillation solution resulting in stable counting rates without the addition of NCS.     

Biodegradation of Trichloroacetic Acid in Norway Spruce/Soil System

Trichloroacetic acid (TCA) belongs to secondary atmospheric pollutants affecting the forest health. Distribution of [1,2-¹⁴C]TCA-residues and TCA biodegradation were investigated in 4-year-old nursery-grown trees of Norway spruce [Picea abies (L.) Karst.] in the whole plant/soil system. Radioactivity was monitored in needles, wood, roots and soil as well as in the air. During two weeks of exposure TCA was continuously degraded, especially in the soil. Estimates of radioactivity balance showed loss of radioactivity into the atmosphere in the form of ¹⁴CO₂; unincorporated [1,2-¹⁴C]TCA, chloroform, carbon monoxide and methane were not detected at all. TCA degradation to CO₂ was indicated also in the spruce needles. Moreover, it was found that soil litter contained [1,2-¹⁴C]TCA unavailable to microorganisms.     

The synthesis of diacetylated histone H4-(1–37) for studies on the mechanism of histone deacetylation

The heptatriacontapeptide [Lys([¹⁴C]Ac)¹², Lys([³H]Ac)¹⁶]histone H4-(1–37) was synthesized by the automated solid phase method. This dual-labeled peptide was designed for studies on the mechanism of histone deacetylation. The synthesis was monitored by an automated picrate method; and the product, purified by affinity chromatography and ion exchange chromatography, was homogeneous by polyacrylamide gel electrophoresis and gave excellent amino acid and radiolabel ratios. A purified calf thymus histone deacetylase released acetyl groups from this synthetic peptide at essentially the same rate as from a diacetylated 1–37 peptide derived from native calf thymus histone H4. The relative rate of release of [¹⁴C]acetyl from Lys¹² and of [³H]acetyl from Lys¹⁶ was 1.03 ± 0.03 throughout the time course of the enzymatic assay. Based on these results, possible mechanisms of histone deacetylation are proposed.     

Rapid Methylation of Cell Proteins and Lipids in Trypanosoma brucei

ABSTRACT. The fate of the [methyl-¹⁴C] group of S-adenosylmethionine (AdoMet) in bloodstream forms of Trypanosoma brucei brucei, was studied. Trypanosomes were incubated with either [methyl-¹⁴C]methionine, [U-¹⁴C]methionine, S-[methyl-¹⁴C]AdoMet or [³⁵S]methionine and incorporation into the total TCA precipitable fractions was followed. Incorporation of label into protein through methylation was estimated by comparing molar incorporation of [methyl-¹⁴C] and [U-¹⁴C]methionine to [³⁵S]methionine. After 4-h incubation with [U-¹⁴C]methionine, [methyl-¹⁴C]methionine or [³⁵S]methionine, cells incorporated label at mean rates of 2,880 pmol, 1,305 pmol and 296 pmol per mg total cellular protein, respectively. Cells incubated with [U-¹⁴C] or [methyl-¹⁴C]methionine in the presence of cycloheximide (50 μg/ml) for four hours incorporated label eight- and twofold more rapidly, respectively, than cells incubated with [³⁵S]methionine and cycloheximide. [Methyl-¹⁴C] and [U-¹⁴C]methionine incorporation were > 85% decreased by co-incubation with unlabeled AdoMet (1 mM). The level of protein methylation remaining after 4-h treatment with cycloheximide was also inhibited with unlabeled AdoMet. The acid precipitable label from [U-¹⁴C]methionine incorporation was not appreciably hydrolyzed by DNAse or RNAse treatment but was 95% solubilized by proteinase K. [U-¹⁴C]methionine incorporated into the TCA precipitable fraction was susceptible to alkaline borate treatment, indicating that much of this label (55%) was incorporated as carboxymethyl groups. The rate of total lipid methylation was found to be 1.5 times that of protein methylation by incubating cells with [U-¹⁴C]methionine for six hours and differential extraction of the TCA lysate. These studies show T. b. brucei maintains rapid lipid and protein methylation, confirming previous studies demonstrating rapid conversion of methionine to AdoMet and subsequent production of post-methylation products of AdoMet in African trypanosomes.     

Measurement of intracellular collagen degradation

Intracellular degradation of newly synthesized collagen is quantitated by incubating fibroblasts with [¹⁴C]proline and determining the percentage of total [¹⁴C]hydroxyproline that is present in a low molecular weight fraction. Several problems make this difficult. (1) Commercial [¹⁴C]proline is often contaminated with [¹⁴C]hydroxyproline and must be purified before use. (2) Salt and [¹⁴C]proline interfere with the determination of [¹⁴C]hydroxyproline in the low molecular weight fraction and must be removed by preparative ion-exchange chromatography. (3) Epimerization of trans- to cis-hydroxyproline during acid hydrolysis is variable and must be taken into account. (4) Loss of [¹⁴C]hydroxyproline during processing varies; [³H]hydroxyproline can be used as an internal measure of recovery, even though tritium may be lost during hydrolysis. An analytic cation-exchange resin is used for the final quantitation of [¹⁴C]hydroxyproline in the low and high molecular weight fractions. With these methods, degradation of newly synthesized collagen can be determined with a precision of ± 3%.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

Determination of the specific radioactivity of 14C-labeled glutamic acid and glutamine

A new, simple, and accurate method for the sequential determination of the specific radioactivity of [1-¹⁴C]glutamic acid and [1-¹⁴C]glutamine is described. Using this method, radioactivity in H¹⁴CO₃^?, in [¹⁴C]glutamic acid, and in [¹⁴C]-glutamine can be readily determined on a single sample of blood plasma. Radioactivity is released as ¹⁴CO₂ in a stepwise fashion, trapped in the center wells, and counted in a liquid scintillation counter. The applicability of the method is discussed.     

Histones from exponential and stationary L-cells. Evidence for metabolic heterogeneity of histone fractions retained after isolation of nuclei

Histones of exponential and G₁-arrested mouse L-cells were double-labeled with either [³H]lysine and [³²P]phosphate or with [¹⁴C]arginine and [³H]acetate in order to investigate cell cycle-dependent changes in rates of synthesis and metabolic modifications as well as the effect of the method of nuclear isolation on retention of the labeled fractions. Results indicate that newly synthesized lysine-rich histones incorporate ³²P at the highest rate. However, phosphorylation can also be detected in G₁-arrested cells and in the case of F_2b, F_1⁰, and F_1¹, with no detectable new synthesis.On the other hand, acetylation proceeded at similar rates in both exponential and stationary cells with the exception of F_{2a 2} and F₃ which showed higher acetylation levels in the latter.In relation to the method used for nuclear isolation, we observed that, even in cases where no difference in the relative amount of a given histone could be detected, the species recovered were not identical. We conclude that none of the methods commonly employed retains all of the histones. In general, the acetylated species appear to be best preserved at a higher divalent cation concentration while the newly synthesized ones are better conserved at lower ionic strengths.     

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.     

Measurement of mouse brain glucose utilization in vivo using [U-14C]glucose

The rate of [2-¹⁴C]glucose uptake has been used as an indication of the status of energy consumption by the rat brain, but the cost of this radiolabel can be prohibitive and the surgical manipulation involved in published methods is extensive. A method for measuring glucose utilization in vivo in mouse brain with [U-¹⁴C]glucose is described in this article. Glucose consumption in whole mouse brain obtained with [U-¹⁴C]glucose or [2-¹⁴C]glucose was 0.650±0.022 and 0.716±0.36 nmol/mg/min, respectively. In all instances the rate obtained with the uniformly labeled isotope was somewhat lower than that found with [2-¹⁴C]glucose. The rate of glucose utilization measured with either isotope was significantly depressed in sodium pentobarbital anesthetized mice. The method described here is advantageous because [U-¹⁴C]glucose is substantially less expensive than [2-¹⁴C]glucose and surgical intervention is avoided.     

Nonenzymatic acetylation of histones with acetyl phosphate and acetyl adenylate.

Nonenzymatic acetylation of calf-thymus lysine- and arginine-rich histones was demonstrated to occur when these proteins were incubated with [14C]acetyl phosphate and [14C]acetyl adenylate. The levels of acetylation depend on both pH and on reagent concentration. When acetyl [33P]phosphate and acetyl [3H]adenylate were used as reagents, we found neither histone phosphorylation nor adenylylation. Most of the radioactivity of 14C-labeled acetylated histones was recovered as Ne-acetyllysine. Furthermore, only a small amount of O-bound radioactivity was released by the 14C-labeled acetylated arginine-rich histone during treatment with hydroxylamine. Experiments on the acetylation of histones, in the presence of increasing salt concentration, gave different results for the two acetylating agents.     

Simple procedures for determination of [14C] hydroxyproline.

Two improved and simplified procedures are presented for determination of [¹⁴C]hydroxyproline. Simplification is achieved by boiling samples without previous toluene extractions and, after boiling, passing the toluene extracts through a silicic acid column.Procedure I avoids incomplete toluene extraction of [¹⁴C]proline derivatives and uses instead a specific adsorption to a silicic acid column.Procedure IIA introduces inter alia the silicic acid column to reduce the interference of incompletely extracted [¹⁴C]proline.Procedure IIB simplifies procedure IIA by replacing extraction of [¹⁴C]proline derivatives with a silicic acid column.The new procedures are simple, handy, specific and reproducible methods for determination of [¹⁴C]hydroxyproline and are preferable to any other method known today. Procedure IIB is specially recommended for routine use.     

Effect of water stress on proline catabolism in tobacco leaves

Proline-[¹⁴C] infiltrated into leaf disks of tobacco (Nicotiana tabacum cv BY-4) in the dark was converted to glutamic acid and then metabolized through the TCA cycle. A smaller amount of proline-[¹⁴C] was metabolized when the leaf disks were wilted than when turgid. During a 6 hr period following rehydration, disks converted a larger amount of proline-[¹⁴C] to oxidized products than when wilted, although the proline content of rehydrated disks had not declined. These results indicate that proline oxidation is inhibited by water stress.     

Enzymatic methods for the determination of L-serine concentration and L-[14C]serine specific radioactivity in blood plasma

Methods are desribed for the use of l-serine dehydratase purified from Clostridium acidiurici for the determination of l-serine concentration and l[¹⁴C]serine specific radioactivity in sheep plasma. A spectrophotometric assay using this enzyme accurately measured the concentration of l-serine in standard solutions and in a commercially available mixture of amino acids and related compounds. This assay was shown to be suitable for measurement of plasma l-serine concentrations in excess of 30 μm. The reverse isotope dilution method was used for plasma l-[¹⁴C]serine specific radioactivity measurements. Carrier l-serine was added to plasma and separated from neutral and anionic compounds using ion-exchange chromatography. The l-serine was then converted to pyruvate with l-serine dehydratase and this was purified as the phenylhydrazone derivative. After recrystallization, drying and weighing, the derivative was assayed for radioactivity. The accuracy of this method was verified by adding l-[U-¹⁴C]serine to plasma and comparing the experimentally determined l-[¹⁴C]serine specific radioactivity with the calculated value. The method yielded a value which was 98.6 ± 0.8% (5) of this calculated value.     

Specific changes in the biosynthesis and acetylation of nucleosomal histones in the early stages of embryogenesis of sea urchin.

Histones of the sea urchin Strongylocentrotus droebachiensis were labelled in vivo with [¹⁴C]protein hydrolysate and [¹⁴C]sodium acetate at the developmental stages of blastula, gastrula and pluteus. They were further resolved by electrophoresis in 15% polyacrylamide gels in the presence of 0.9 M acetic acid, 8 M urea as well as in 20% polyacrylamide gels containing the same concentrations of acetic acid and urea, but in addition, 4.3 mM Triton X-100. On comparison of electrophoretic patterns with autoradiograms it was shown that during the early stages of differentiation the synthesis of several new subfractions of some of the histones takes place, namely of two of H1, of two of H2b and of nine of H2a. At the same time the electrophoretic pattern of the arginine-rich histones remained constant, H3 consisting of three and H4 of four subfractions. The basic reason for this heterogeneity seems to be the specific post-synthetic acetylation of the histones. At the blastula stage all the newly synthesized H4 molecules are equally acetylated, while the histone H3 molecules are acetylated to varying extents. After gastrulation both H3 and H4 are also subject to a different level of acetylation.     

Incorporation of labeled small molecules into rubratoxin

A sterile glucose-mineral salts broth was inoculated with conidia of Penicillium rubrum P-13 and P-3290. Radiolabeled compounds were added to some cultures, these being incubated quiescently at 28° C for 14 days. Other stationary cultures were grown for 21 days, received labeled compounds, and were then grown for 5 more days. The remaining cultures were inoculated with 72-h-old mycelial pellets, received labeled materials and were incubated with shaking for 60 h. Rubratoxin was resolved by thin-layer chromatography. Labeled [1¹⁴C]acetate, [1,5¹⁴C]citrate, [2¹⁴C]malonate, [1¹⁴C]glucose, [U¹⁴C]glucose or [1¹⁴C]hexanoate were incorporated into rubratoxins A and B by P. rubrum 3290 and into rubratoxin B by P. rubrum 13. Incorporation of [1¹⁴C]acetate and [2¹⁴C]malonate increased when exogenous unlabeled acetate, malonate, pyruvate, or phosphoenol-pyruvate was added. Acetate incorporation was influenced by cultural conditions, attaining maximum amounts in quiescent cultures which received labeled acetate after 21 days of incubation. Acetate incorporation in shake cultures was enhanced by reduced nicotinamide adenine dinucleotide phosphate (NADPH) and by unlabeled exogenous citrate.Abbreviations GMS glucose-mineral salts - RCM replacement culture medium - TCA tricarboxylic acid - PEP phosphoenolpyruvate - RIC relative isotopic content - PI percent incorporation     

Involvement of a lipid-linked intermediate in the transfer of galactose from UDP [14C]galactose to exogenous protein in castor bean endosperm homogenates

A crude organelle preparation from germinating castor bean endosperm catalysed the incorporation of galactose from UDP[¹⁴C]galactose into chloroform/methanol (2:1)-soluble glactolipids. At least two galactolipids were formed. Most of the [¹⁴C]galactose was present in a galactolipid synthesized by the microsomal membranes, the remainder was present in a second galactolipid synthesized by other cellular membranes, possibly Golgi-derived. The addition of asialo-agalacto-fetuin reduced incorporation of [¹⁴C]galactose into the microsomal galactolipid with a concomitant increase in microsomal [¹⁴C]galactoprotein. Asialo-agalacto-fetuin did not affect galactolipid or galactoprotein synthesis by nonmicrosomal fractions. The results suggest that the endoplasmic reticulum is a major site of protein galactosylation in castor bean endosperm cells, and that galactose transfer from UDP-galactose to protein occurs via a lipid-linked intermediate.Abbreviations ER endoplasmic reticulum - ASGF asialoagalacto-fetuin - IDPase inosine diphosphatase - TCA trichloroacetic acid     

The incorporation of glucose into alpha-1,4-glucan proteins of bovine retina membranes.

—Incubation of bovine retina membranes with UDP-[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous α-1, 4-glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m -sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein-I (GP-I), insoluble in TCA, and glucan protein-II (GP-II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP-I and GP-II were precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA). A third fraction, glucan protein-III (GP-III) was found when incubations were carried out with UDP-[¹⁴C]glucose at millimolar instead of micromolar concentrations. GP-III was soluble in TCA and in TCA-PTA and precipitable by ethanol from the TCA soluble fraction. GP-II was excluded from a Sephadex G-200 column and showed a greater size than GP-I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G-25 column and were of a greater size than the labelled residues released with salivary α-amylase. Only radioactive maltose was found after a-amylase treatment. When membranes containing labelled GP-I and GP-II were incubated with unlabelled UDP-glucose at millimolar concentrations, GP-I was converted into GP-II and GP-III was formed.     

N-[1-C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: Acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.     

N-[1-13C]acetylimidazole as a reagent for tyrosyl modification in protein NMR studies: acetylation of cytochrome c

The reaction of N-[1-¹³C] acetylimidazole with cytochrome c and guanidinated cytochrome c was evaluated as a means of introducing NMR-detectable groups as conformation-dependent probes. Resonances from both N-[1-¹³C]acetyl lysyl and O-[1-¹³C]acetyl tyrosyl groups were observed when ferricytochrome c was acetylated. However, only O-[1-¹³C]acetyl tyrosyl resonances were seen with acetylated guanidinated ferricytochrome c. Chemical shifts of the four O-[1-¹³C]acetyl tyrosyl groups were conformation dependent and ranged from 172 to 176 ppm. A convenient method for the preparation of N-[1-¹³C]acetylimidazole is described.