Protein sequencing: thermal conversion of thiazolinone derivatives of amino acids to thiohydantoins.

A tlc method is presented for the separation on PEI-cellulose of adenine derivatives from the acid-soluble fraction of brain tissue. The procedure permits the quantitative estimation of radioactivity distributed among adenine nucleotides and the determination of ATP specific activity following labeling with [¹⁴C] adenosine. Two rapid one-dimensional separations produce excellent resolution of AMP, ADP, ATP, adenine, and adenosine from each other and from the corresponding guanosine derivatives. A two-dimensional procedure gives excellent separation of ATP from all other deoxy- and ribonucleotides. The recovery of the separated components by elution is better than 94% and requires few technical manipulations. The technique possesses the advantages of simplicity and sensitivity and is applicable to small amounts of biological material.     

Custom fluorescent-nucleotide synthesis as an alternative method for nucleic acid labeling

The variety of potentially useful dyes or haptenes available for fluorescent nucleic acid hybridization assays is far greater than what can be obtained from commercial sources. Since this diversity could be useful in many laboratory applications, we have developed a simple and inexpensive procedure for preparing nonpurified labeled nucleotides, for use in common nucleic acid labeling reactions, such as PCR and nick translation. The modified nucleotides were synthesized by coupling allylamine-dUTP to the succinimidyl-ester derivatives of the fluorescent dyes or haptenes such as biotin or digoxigenin, which require fluorescently labeled proteins for detection. This method allows custom preparation of most common fluorescent nucleotides and rapid testing of new ones, while reducing the cost of procedures such as multiplex fluorescent in situ hybridization (M-FISH) by 100-200 fold.     

Evaluation of velvet bean meal as an alternative protein ingredient for poultry feed

The effect of certain simple and cost-effective processing methods on the nutritional and anti-nutritional properties of seed materials of an under-utilized food legume, Mucuna pruriens (L.) DC. var. utilis (Wall. ex Wight) Baker ex Burck (velvet bean, VB), collected from Valanadu, Kerala, India was analyzed in experiment 1. The raw VB seeds were found to contain appreciable levels of crude protein (263.2 g/kg dry matter (DM)); ether extract (79.6 g/kg DM); crude fiber (95.8 g/kg DM) and ash content (38.4 g/kg DM). Among the different treatments used, soaking in sodium bicarbonate solution + autoclaving was more effective in reducing maximum levels of various anti-nutritional compounds of VB seeds. Furthermore, in experiment 2, the effect of inclusion of different levels of velvet bean meal (VBM; subjected to soaking in sodium bicarbonate solution + autoclaving) as an alternative protein source in poultry feed on the growth performance of commercial-type broiler birds was investigated. The results indicate that the inclusion of VBM up to the 40% level exhibited better growth performance of the broiler birds such as feed intake, body weight gain, feed conversion ratio and protein efficiency ratio in both the starter and finisher phases.     

Peracetic acid as an alternative wastewater disinfectant to chlorine dioxide

AIMS: The aim of this study was to compare the efficiency of peracetic acid with that of chlorine dioxide in the disinfection of wastewater from a sewage treatment plant (serving about 650 000 inhabitants) that has been using peracetic acid as a disinfectant since 1998. METHODS AND RESULTS: A total of 23 samplings were made, each consisting of three samples: from secondary effluent, effluent disinfected with 2 mg l(-1) of peracetic acid and effluent disinfected with 2.2 mg l(-1) of chlorine dioxide (contact time 20 min). For each sample, measurements were made of the heterotrophic plate count at 36 degrees C, total and faecal coliforms, Escherichia coli, enterococci, pH, suspended solids and chemical oxygen demand (COD). During the first phase of the experiment the peracetic acid was seen to be less efficient than chlorine dioxide. To improve the disinfectant action a system of mechanical agitation was added which led to a greater efficiency in the inactivation of bacteria of faecal origin. CONCLUSIONS: Both products were found to be influenced by the level of microbial contamination, the amount of suspended solids and COD but not by the pH of the effluent before disinfection. The immediate mixing of the wastewater and disinfectant caused a greater reduction in enterococci. SIGNIFICANCE AND IMPACT OF THE STUDY: Since peracetic acid was seen to produce a high abatement of micro-organisms, it can be considered as a valid alternative to chlorine dioxide in the disinfection of wastewaters.     

Metabolism of amino acid amides in Pseudomonas putida ATCC 12633

The metabolism of the natural amino acid l-valine, the unnatural amino acids d-valine, and d-, l-phenyglycine (d-, l-PG), and the unnatural amino acid amides d-, l-phenylglycine amide (d-, l-PG-NH₂) and l-valine amide (l-Val-NH₂) was studied in Pseudomonas putida ATCC 12633. The organism possessed constitutive l-amidase activities towards l-PG-NH₂ and l-Val-NH₂, both following the same pattern of expression, suggesting the involvement of similarly regulated enzymes, or a common enzyme. Quite surprisingly, growth in mineral media with l-PG-NH₂ resulted in variable, long lag phases of growth and strongly reduced l-amidase activities. Conversion of d-PG-NH₂ into d-PG and l-PG also occurred and could be attributed to the presence of an inducible d-amidase and the racemization of the amino acid amide in combination with l-amidase activity, respectively. The further degradation of l-PG and d-PG involved constitutive l-PG aminotransferase and inducible d-PG dehydrogenase activities, respectively, both with a high degree of enantioselectivity. Amino acid racemase activity for d- and l-PG was not detected. Correspondence to: L. Dijkhuizen     

Effective concentrations of amino acid side chains in an unfolded protein.

Preferential interactions between chain segments are studied in unfolded cytochrome c. The method takes advantage of heme ligation in the unfolded protein, a feature unique to proteins with covalently attached heme. The approach allows estimation of the effective concentration of one polypeptide chain segment relative to another, and is successful in detecting differences for peptide chain segments separated by different numbers of residues in the linear sequence. The method uses proton NMR spectroscopy to monitor displacement of the histidine heme ligands by imidazole as guanidine hydrochloride unfolded cytochrome c is titrated with deuterated imidazole. When the imidazole concentration exceeds the effective (local) concentration of histidine ligands, the protein ligands are displaced by deuterated imidazole. On displacement, the histidine ring proton resonances move from the paramagnetic region of the spectrum to the diamagnetic region. Titrations have been carried out for members of the mitochondrial cytochrome c family that contain different numbers of histidine residues. These include cytochromes c from tuna (2), yeast iso-2 (3), and yeast iso-1-MS (4). At high imidazole concentration, the number of proton resonances that appear in the histidine ring C2H region of the NMR spectrum is one less than the number of histidine residues in the protein. So one histidine, probably His-18, remains as a heme ligand. The effective local concentrations of histidines-26, -33, and -39 relative to the heme (position 14-17) are estimated to be (3-16) X 10(-3) M.(ABSTRACT TRUNCATED AT 250 WORDS)     

New enzymatic method of chiral amino acid synthesis by dynamic kinetic resolution of amino acid amides: use of stereoselective amino acid amidases in the presence of alpha-amino-epsilon-caprolactam racemase

D- and L-amino acids were produced from L- and D-amino acid amides by D-aminopeptidase from Ochrobactrum anthropi C1-38 and L-amino acid amidase from Pseudomonas azotoformans IAM 1603, respectively, in the presence of alpha-amino-epsilon-caprolactam racemase from Achromobacter obae as the catalyst by dynamic kinetic resolution of amino acid amides.     

N-terminal amino acid sequencing of EDP208 conjugative pili.

EDP208 conjugative pili contain a single polypeptide subunit of 11,500 daltons with a blocked N-terminus. This N-terminal blocking moiety was identified as an N-acetyl group by 1H nuclear magnetic resonance analysis of an N-terminal tripeptide isolated from pronase digests of EDP208 pilin. Limited acid hydrolysis of the tripeptide allowed its sequence to be determined as acetyl-NH-Thr-Asp-Leu. Trypsin digestion of EDP208 pilin resulted in the quantitative release of a fragment containing 12 residues from the N-terminus of the protein. The sequence of this dodecapeptide was determined to be acetyl-NH-Thr-Asp-Leu-Leu-Ala-Gly-Gly-Lys-Asp-Val-Asp-Lys.     

Accumulation of amino acids by lysosomes incubated with amino acid methyl esters.

Rat liver lysosomal preparations incubated with 10(-5) M L-[4,5-3H]leucine methyl ester hydrolyzed the methyl ester and accumulated radioactivity within a particulate compartment. The acculated radioactivity was identified as free leucine by thin layer chromatography. Free leucine was not itself taken up by the lysosomal preparations. The capacity to accumulate leucine was identified as a specific property of lysosomes and was thought to result from the trapping of the free amino acid within the lysosome following the hydrolysis of the methyl ester. Lysosomes also accumulated phenylalanine, serine, and alanine when incubated with the corresponding methyl esters. Leucine accumulation was inhibited by submillimolar concentrations of chloroquine, by the protease inhibitor L-1-tosylamido-2-phenylethyl chloromethyl ketone, and by lowering the pH below 7.0. Efflux of leucine from the lysosomes was highly temperature dependent (activation energy 33 kcal/mol). No evidence was found to suggest that leucine efflux was a carrier-mediated process. The results provide a new methodology for the study of amino acid movements across lysosomal membranes.     

A new method to model membrane protein structure based on silent amino acid substitutions.

The importance of accurately modeling membrane proteins cannot be overstated, in lieu of the difficulties in solving their structures experimentally. Often, however, modeling procedures (e.g., global searching molecular dynamics) generate several possible candidates rather then pointing to a single model. Herein we present a new approach to select among candidate models based on the general hypothesis that silent amino acid substitutions, present in variants identified from evolutionary conservation data or mutagenesis analysis, do not affect the stability of a native structure but may destabilize the non-native structures also found. The proof of this hypothesis has been tested on the alpha-helical transmembrane domains of two homodimers, human glycophorin A and human CD3-zeta, a component of the T-cell receptor. For both proteins, only one structure was identified using all the variants. For glycophorin A, this structure is virtually identical to the structure determined experimentally by NMR. We present a model for the transmembrane domain of CD3-zeta that is consistent with predictions based on mutagenesis, homology modeling, and the presence of a disulfide bond. Our experiments suggest that this method allows the prediction of transmembrane domain structure based only on widely available evolutionary conservation data.     

Coupling sap flow velocity and amino acid concentrations as an alternative method to (15)N labeling for quantifying nitrogen remobilization by walnut trees

The temporal dynamics of N remobilization was studied in walnut (Juglans nigra x regia) trees growing in sand culture. Trees were fed with labeled N ((15)N) during 1999 and unlabeled N in 2000. Total N and (15)N contents in different tree compartments were measured during 80 d after bud burst and were used to estimate N remobilization for spring growth. The seasonal (and occasionally diurnal) dynamics of the concentration and (15)N enrichment of the major amino acids in xylem sap were determined concurrently. Sap flow velocity was also measured for sample trees. A new approach coupling amino acid concentrations to sap flow velocity for quantifying N remobilization was tested. A decrease of the labeled N contents of medium roots, tap roots, and trunk was observed concurrently to the increase in the labeled N content of new shoots. Remobilized N represented from previous year storage 54% of N recovered in new shoots. Arginine, citruline, gamma-amino butyric acid, glutamic acid, and aspartic acid always represented around 80% of total amino acid and amide N in xylem sap and exhibited specific seasonal trends and significant diurnal trends. N translocation was mainly insured by arginine during the first 15 d after bud burst, and then by glutamic acid and citruline. The pattern of N remobilization estimated by the new approach was consistent with that measured by the classical labeling technique. Implications for quantifying N remobilization for large, field-growing trees are discussed.     

14C-methylamine-glutaraldehyde conjugation as an alternative to iodination for protein labeling

Radiolabeling of native proteins conventionally has required iodination using 125Iodine (125I). Although radioiodination can result in high specific activity, there are several drawbacks in the use of 125I (e.g., radiological hazards and short half-life). 14C-Methylamine-glutaraldehyde conjugation to proteins offers an alternative for radiolabeling of proteins that is safer and longer-lived alpha-2-Macroglobulin was radiolabeled by conjugation to a 14C-methylamine-glutaraldehyde conjugate. Analysis of the labeling procedure was performed using scintillation counting, gel filtration chromatography, and protein assays. The radiolabeled alpha-2-macroglobulin was activated using established protocols and tested for functional integrity using competitive binding assays in the presence of recombinant receptor associated protein, an alternative ligand for the alpha-2-macroglobulin cellular receptor. The function of alpha-2-macroglobulin was unaffected by the labeling procedure. Comparison of 14C-methylamine-labeling and iodination by Scatchard analysis yielded nonlinear plots that suggested the presence of two sets of receptors with different binding affinities but that do not show cooperativity. This technique offers an alternative to radioiodination for the sensitive labeling of proteins.     

An ultramicro method of amino acid analysis: application to studies of protein metabolism in cultured cells.

Analysis of the quantity and specific radioactivity of amino acids derived from intra-cellular pools, aminoacyl-transfer RNA, and protein hydrolysates of cultured cells has been achieved using a radiolabeled amino group ligand, dansyl chloride. Speeific activities of ¹⁴C- or ³H-labeled amino acids are calculated after reaction with appropriately labeled dansyl chloride of known specific activity. The quantity of amino acid is determined as a function of its diluting influence on a radioactive standard. The specific activity of as little as 2 pmol of amino acid can be measured using [¹⁴C]dansyl chloride the less sensitive of the two isotopic species available. Thus, cells from a single 60-mm culture dish provide sufficient material for analysis of both intracellular and transfer RNA amino acid pools, and one can easily analyze the amino acids in hydrolysates made from individual bands in polyacrylamide gels. The method offers significant improvement in speed, sensitivity, and economy over conventional methods of amino acid analysis and, because of its double-label design, gives accurate results with a minimum of technical expertise and no major equipment other than a scintillation counter.