Total Amino Acid Analysis Research Articles

Proteins in Oats; their Synthesis and Changes during Germination: A Review

Oats (Avena sativa L.) are distinct among cereals due to their considerably higher protein concentration. At the same time oats possess a protein quality of high nutritional value and a special protein composition. Most cereals like wheat, barley, and rye have a high percentage of prolamins, the alcohol-soluble fraction, which usually contains most of the storage proteins, but oats are an exception. Their major storage proteins belong to the salt-water soluble globulin fraction, whereas oats prolamins are a minor component. During oats groat development, most obvious is the fairly linear increase in the globulin fraction. Oats globulins share structural features with the 11 S globulins of legumes and other dicots. Amino acid composition of oats is superior to that of other cereals due to the higher amount of limiting amino acids like lysine and threonine. During germination, total amino acid analysis revealed an increase in essential amino acids like lysine and tryptophan, which leads to an increased nutritional value of germinated oats. Oats protein products including globulin have been chemically modified by various methods to improve their functional properties.

다시마 열수추출물의 성분 및 항산화활성 측정

Contents of amino acids, vitamins, and minerals as well as its antioxidant activitiy of Laminaria japonica water extract have been analyzed for preparation of functional foods and cosmetic products. From the analysis of total amino acids, eighteen kinds of amino acids were found in the water extract of Laminaria japonica. Among total amino acids, the order of contents was glutamic acid (2.07 mg/g), alanine (0.51 mg/g), aspartic acid (0.44 mg/g), glycine (0.34 mg/g), and valine (0.34 mg/g). In case of free amino acids, glutamic acid (0.95 mg/g), prolin (0.54 mg/g), aspartic acid (0.44 mg/g), leucine (0.07 mg/g), and phenylalanine (0.07 mg/g) were dominant compositions. Vitamin E was only detected in water extract of Laminaria japonica. The mineral contents were as follows: K 752.60 mg, Na 259.20 mg, Ca 80.20 mg, P 29.50 mg, and Fe 8.32 mg based on 100 g Laminaria japonica water extract. The nitrite scavenging activity of the extract were gradually increased with the extracts contents to 86.2% at concentration of 100 mg/mL and DPPH radical scavenging activity of the extracts were 86.4% at concentration of 50 mg/mL.

The Isolation and Characterization of Glycosylated Phosphoproteins from Herring Fish Bones

Past studies of bone extracellular matrix phosphoproteins such as osteopontin and bone sialoprotein have yielded important biological information regarding their role in calcification and the regulation of cellular activity. Most of these studies have been limited to proteins extracted from mammalian and avian vertebrates and nonvertebrates. The present work describes the isolation and purification of two major highly glycosylated and phosphorylated extracellular matrix proteins of 70 and 22 kDa from herring fish bones. The 70-kDa phosphoprotein has some characteristics of osteopontin with respect to amino acid composition and susceptibility to thrombin cleavage. Unlike osteopontin, however, it was found to contain high levels of sialic acid similar to bone sialoprotein. The 22-kDa protein has very different properties such as very high content of phosphoserine (∼270 Ser(P) residues/1000 amino acid residues), Ala, and Asx residues. The N-terminal amino acid sequence analysis of both the 70-kDa (NPIMA(M)ETTS(M)DSKVNPLL) and the 22-kDa (NQDMAMEASSDPEAA) fish phosphoproteins indicate that these unique amino acid sequences are unlike any published in protein databases. An enzyme-linked immunosorbent assay revealed that the 70-kDa phosphoprotein was present principally in bone and in calcified scales, whereas the 22-kDa phosphoprotein was detected only in bone. Immunohistological analysis revealed diffusely positive immunostaining for both the 70- and 22-kDa phosphoproteins throughout the matrix of the bone. Overall, this work adds additional support to the concept that the mechanism of biological calcification has common evolutionary and fundamental bases throughout vertebrate species.

Microwave-assisted high-throughput acid hydrolysis in silicon carbide microtiter platforms—A rapid and low volume sample preparation technique for total amino acid analysis in proteins and peptides

An efficient microwave-assisted high-throughput protein hydrolysis protocol was developed utilizing strongly microwave absorbing silicon carbide-based microtiter platforms. The plates are equipped with 20 bore holes having the proper dimensions for holding standard screw-capped HPLC/GC vials. Due to the possibility of heating up to four heating platforms simultaneously (80 vials), parallel microwave-assisted acid hydrolyses can be performed under carefully controlled conditions significantly reducing the overall time required for protein hydrolysis and the subsequent evaporation step required for larger volumes of acid. An extensive optimization of the hydrolysis conditions has demonstrated that 5 min irradiation at 160 °C with 6 N HCl leads to comparable results in terms of total and individual amino acid recovery as the traditional method requiring 24 h heating at 110 °C. Complete hydrolysis of several proteins and synthetic peptides was performed using 25 μg of sample material and 100 μL of 6 N HCl in a dedicated low-volume HPLC/GC vial. Since the hydrolysis and subsequent analysis can be performed from the same vial, errors caused by sample transfer can be minimized. Control experiments have demonstrated that the observed rate enhancements are the result of a purely thermal/kinetic effect as a consequence of the considerable higher reaction temperatures.

The association between biogenic and inorganic minerals and the amino acid composition of settling particles

To test the hypothesis that calcium carbonate (rather than opal) carries most organic carbon to the deep sea, total hydrolysable amino acid (THAA) analysis was applied to deep‐sea (3000 m) sediment trap material from the northeast Atlantic (PAP Site), a variable but intrinsically carbonate‐dominated system. THAAs were analyzed in conjunction with total organic carbon, biogenic silica, calcium carbonate, and inferred lithogenic fluxes. The THAA‐based degradation state of organic carbon could not be systematically explained by changes in the flux of different mineral phases, which could account for only 16% of the observed variability. In addition amino acid parameters indicative of source organisms indicate that diatom cell walls are an important residual component of organic carbon reaching the deep ocean, a finding supported by comparison with data from previous studies of diverse oceanic environments. Finally, during 2001, very high organic carbon fluxes were associated with elevated lithogenic fluxes and low organic matter degradation relative to surrounding years. In accordance with other recent experimental and observational studies, the data indicate that under specific export scenarios, lithogenic fluxes can act as highly significant mediators of organic carbon transfer to the deep ocean.

A strategic crossflow filtration methodology for the initial purification of promegapoietin from inclusion bodies

A novel crossflow filtration methodology is demonstrated for the initial purification of the therapeutic protein, promegapoietin-1a (PMP), produced as inclusion bodies (IBs) in a recombinant Escherichia coli bioprocess. Two strategic separation steps were performed by utilizing a filtration unit with a 1000 kDa polyethersulphone membrane. The first step, aiming for separation of soluble contaminants, resulted in a 50% reduction of the host cell proteins, quantified by total amino acid analysis and a 70% reduction of all DNA, quantified by fluorometry, when washing the particulate material with a 10 mM EDTA in 50 mM phosphate buffer, pH 8. The second step, aiming for separation of particulate contaminants from solubilized IBs, resulted in a 97–99.5% reduction of endotoxin, used as a marker for cell debris, and was quantified by the kinetic turbidimetric LAL endotoxin assay. The overall PMP yield was 58% and 33% respectively for the two solubilizations investigated, guanidine hydrochloride and arginine, as measured by RP-HPLC. The scope was also to investigate the physical characteristics of the intermediate product/s with regard to the choice of IB solvent. Preliminary results from circular dichroism spectroscopy measurements indicate that the protein secondary structure was restored when arginine was used in the second step.

A Trypanosoma cruzi Phosphatidylinositol 3-Kinase (TcVps34) Is Involved in Osmoregulation and Receptor-mediated Endocytosis

Trypanosoma cruzi, the etiological agent of Chagas disease, has the ability to respond to a variety of environmental changes during its life cycle both in the insect vector and in the vertebrate host. Because regulation of transcription initiation seems to be nonfunctional in this parasite, it is important to investigate other regulatory mechanisms of adaptation. Regulatory mechanisms at the level of signal transduction pathways involving phosphoinositides are good candidates for this purpose. Here we report the identification of the first phosphatidylinositol 3-kinase (PI3K) in T. cruzi, with similarity with its yeast counterpart, Vps34p. TcVps34 specifically phosphorylates phosphatidylinositol to produce phosphatidylinositol 3-phosphate, thus confirming that it belongs to class III PI3K family. Overexpression of TcVps34 resulted in morphological and functional alterations related to vesicular trafficking. Although inhibition of TcVps34 with specific PI3K inhibitors, such as wortmannin and LY294,000, resulted in reduced regulatory volume decrease after hyposmotic stress, cells overexpressing this enzyme were resistant to these inhibitors. Furthermore, these cells were able to recover their original volume faster than wild type cells when they were submitted to severe hyposmotic stress. In addition, in TcVps34-overexpressing cells, the activities of vacuolar-H+-ATPase and vacuolar H+-pyrophosphatase were altered, suggesting defects in the acidification of intracellular compartments. Furthermore, receptor-mediated endocytosis was partially blocked although fluid phase endocytosis was not affected, confirming a function for TcVps34 in membrane trafficking. Taken together, these results strongly support that TcVps34 plays a prominent role in vital processes for T. cruzi survival such as osmoregulation, acidification, and vesicular trafficking.

The influence of extrusion on loss and racemization of amino acids

The influence of the operation conditions (temperature and residence time) of a thermic treatment on the total amount (free and protein-bound) of amino acid enantiomers of dry fullfat soya was investigated. Total amino acid content was determined using conventional ion-exchange amino acid analysis of total hydrolysates and chiral amino acid analysis was performed by HPLC after precolumn derivatization with o-phthaldialdehyde and 1-thio-beta-D-glucose tetraacetate. Contrary to corn that was investigated previously, notable racemization was detected even at lower temperatures. At 140 degrees C the ratio of the D-enantiomer was 0.87% for glutamic acid, 2.81% for serine, and 1.92% for phenylalanine; at 220 degrees C the ratios of the D-enantiomer of the above amino acids were 1.43, 4.61, and 4.68%, respectively. The concentration of several L-amino acids decreased. At 220 degrees C there was 10% less L-glutamic acid, 17% less L-serine, 5% less L-phenylalanine, 6.6% less L-aspartic, acid and 21% less L-lysine than in the control; their loss can be assigned to different degrees of L - D conversion. While nearly complete transformation of L-phenylalanine can be attributed to racemization, the main cause of the loss of L-lysine is not racemization. The treatments in the same order of magnitude resulted in the formation of more D-amino acids and greater extent of racemization of amino acids in fullfat soya than that of maize.

Quantitative analysis of residual protein contamination on reprocessed surgical instruments

'Ready-for-use' instruments from surgical instrument trays were examined after routine cleaning and sterilization in a blinded study. These reprocessed instruments originated from five National Health Service hospital trust sterile service departments in England and Wales. Determination of residual protein and peptide contamination was carried out by acid stripping of the instrument surfaces, hydrolysis of the constituent amino acids and quantitative total amino acid analysis. One hundred and twenty instruments were analysed, and the median levels of residual protein contamination per instrument for the individual trays were 267, 260, 163, 456 and 756 microg. Scanning electron microscopy and energy dispersive X-ray spectroscopic analyses of the instruments showed that tissue deposits were localized on surfaces, but there was no significant correlation between overall protein soiling and instrument complexity. The highest levels of residual contamination were found on instruments used for tonsillectomy and adenoid surgery.

Characterization of a calcium-soluble protein fraction from yellow mustard (Sinapis alba) seed meal with potential application as an additive to calcium-rich drinks.

A calcium-soluble protein isolate (CSPI) was prepared from the supernatant obtained after addition of 0.75 M calcium chloride to a pH 5.0 aqueous extract of yellow mustard (Sinapis alba) seed meal. Total amino acid analysis showed that the CSPI has significantly higher (p < 0.05) contents of glutamic acid + glutamine, cysteine, and proline when compared to the precipitated, calcium-insoluble proteins. Peptide mass fingerprinting of tryptic peptides of the major polypeptides by mass spectrometry indicated that the CSPI is composed mainly of cruciferin proteins with a contribution from napins (the major allergenic proteins of S. alba). The S. alba CSPI had significantly higher (p < 0.05) protein solubility and emulsion formation ability in the presence of 0.75 M calcium chloride when compared to similar isolates prepared from Brassica juncea (brown mustard) and soybean seed meals. We suggest that the S. alba CSPI could be used to prepare calcium-fortified high protein liquid products. However, the presence of allergenic proteins in this extract may limit its widespread food use.

DISTRIBUTION OF INTRACELLULAR NITROGEN IN MARINE MICROALGAE: BASIS FOR THE CALCULATION OF SPECIFIC NITROGEN‐TO‐PROTEIN CONVERSION FACTORS

The utilization of nitrogen‐to‐protein conversion factors (N‐Prot factors) is a widely accepted and practical way to determine total protein content. The accuracy of protein determination depends on the establishment of specific N‐Prot factors, since the conventional factor of 6.25 may be unsuitable for all species. This study was designed to determine the concentrations of the main nitrogenous compounds and to establish N‐Prot factors specific for the following marine microalgae: Chlorella minutissima, Dunaliella tertiolecta, Hillea sp., Isochrysis galbana, Nannochloropsis oculata, Phaeodactylum tricornutum, Prorocentrum minimum, Skeletonema costatum, Synechococcus subsalsus, and Tetraselmis gracilis. Cultures were maintained under a 12‐h photoperiod (300 μmol photons·m−2·s−1) at temperatures of 20.0°± 1.0° C (dark) to 23.0°± 2.0° C (light) in Walne’s culture medium without additional external carbon sources. The distribution of intracellular nitrogen was studied by determining total nitrogen (TN, by CHN [carbon, hydrogen, and nitrogen] analysis), protein N (PN, by analysis of total amino acids), and nonprotein N (NPN, determined by analysis of DNA, RNA, chlorophylls (chl) a,b, and c, and intracellular inorganic nitrogen—NO3−, NO2−, and NH3+ NH4+) in logarithmic and stationary growth phases of cultures. Variations occurred in both accumulation and distribution of PN and NPN among the species, as well as in each species during the different growth phases. Inorganic nitrogen compounds were observed to be the most important NPN source (from 6.4 ± 0.1% to 41.8 ± 4.2% of total N) in all species (except D. tertiolecta), followed by nucleic acids (from 0.8 ± 0.1% to 26.1 ± 2.4% of TN) and chlorophylls (from 0.2 ± 0.0% to 3.1 ± 0.3% of TN). Total amino acid residues ranged from 63.1 ± 4.6% up to 88.1 ± 11.2% of TN, which is in agreement with the presence of high NPN concentrations. N‐Prot factors are proposed for each growth phase in the studied species, based on the ratio of amino acid residues to TN, establishing specific N‐prot factors ranging from 3.60 ± 0.27 to 4.99 ± 0.64. The mean N‐Prot factor for all species/growth phases was 4.58 ± 0.11. The present study shows that the use of the traditional factor 6.25 is not suitable for these marine microalgae, and possibly for other species, because it overestimates their actual protein content.

Formation of Carbonyls during Attack on Insulin by Submolar Amounts of Hypochlorite

Bovine insulin was reacted at pH 4.0 with submolar amounts of hypochlorite. At least one molecule of insulin was modified per two molecules of hypochlorite added, as estimated by HPLC of native and modified insulin. About 5% of the hypochlorite-modified insulin reacted with dinitrophenylhydrazine (DNPH), a reagent which specifically labels carbonyl groups. The major DNPH-labeled product was isolated from the native insulin on reverse-phase HPLC, using trifluoroacetic acid/water/acetonitrile gradients. The UV spectrum of the major peak on the HPLC diode-array detector was representative of DNPH adducts, with λmax= 365 nm. Several methods, including total amino acid analysis, tryptic digestion, and collision-induced dissociation–electrospray MS, indicate that the major carbonyl in the DNPH-labeled product was on the amino-terminal phenylalanine of the insulin B-chain. Amino acid analysis indicated that tyrosine was also degraded by hypochlorite, but we could not detect a carbonyl group formed at tyrosine. These findings suggest that the terminal amino groups of proteins are highly vulnerable to carbonyl formation during hypochlorite attack. The use of relatively low amounts of active oxygen species (such as hypochlorite), followed by chromatographic isolation of the protein labeled with a carbonyl-specific reagent, can be a useful approach to the study of reactive sites on proteins.

The Amino Acids Precursory to Proteins Are Primary Human Food: Proline, Glutamine, and Arginine Found Free in the Juices of Common Vegetables and Herbs

Selection of vegetable, salad, and fruit plant crops for the free amino acids is now a potential direction for agriculture. It is both general and specific, with a broad horizon that focuses on improving our nutrition and, as a result, our health. We present analyses for the free amino acids in the juices of the pods of a dozen bush beans, six varieties of cucumbers, the petals of two sunflowers, the leaves of three radicchios, shingiku and fenugreek, two chicories, an endive, licorice root, and garbanzo bean miso. Analysis of total and free amino acids in commercial organic garbanzo bean miso shows that about 60% of the total protein is fermented into free amino acids. Analyses of free amino acids in the fresh juices of a dozen onions are also presented. Glutamine, arginine, threonine, and asparagine were found to be most abundant. The innermost bulb leaves are 9 times higher in arginine than the outermost. A green and a white broccoli were analyzed an inch at a time for free amino acids. The stems and tops were found to be very similar in content and distribution. The most abundant free amino acids found were glutamine, glutamic acid, serine, and alanine. Ratios of glutamine to glutamic acid differed. Unusually large amounts of proline were found in licorice root and in several other legumes. A summary of the highest proline concentrations in fresh juices is provided for 33 sources. Summaries for free glutamine and arginine in a variety of vegetables are also provided. The roles of proline in cellular biochemistry, collagen biosynthesis, and body flexibility are discussed in the context that all the amino acids used in protein synthesis will be found to act at several principal levels of body and organ physiology beyond that currently recognized and understood. The essential nutritional roles of arginine and glutamine in a variety of physiological processes at the cell, organ, and whole body levels are also discussed. Their presence in the juices of common vegetables is an unheralded aspect of the importance of vegetables in our diets.

Lysozyme fragmentation induced by gamma-radiolysis

Irradiation of lysozyme in frozen states in the absence of oxygen induces specific fragmentation at defined sites along the backbone chain. This paper localizes radiofragmentation sites by two methods. First, N-terminal sequencing of radiolysis fragments after separation by SDS-polyacrylamide gel electrophoresis and estimation of their molecular masses. Secondly, after purification of radiolysis fragments by reverse phase-HPLC and determination of their molecular mass by electro-spray-ionization mass-spectrometric analysis, combined to N-terminal sequencing and total amino acid analysis. Evidence for the breakage of the peptide bond itself (CO-NH) is given, with radio-fragmentation sites mostly found at the surface of irradiated lysozyme in solvent exposed loops and turns.

Dityrosine: Preparation, Isolation, and Analysis

This article describes chromatographic and spectroscopic techniques that have multiple applications in the preparation, isolation, and analysis of dityrosine. A three-step chromatographic procedure facilitates the preparation of 120 mg or more (yield >26% of theoretical maximum) of dityrosine from the enzyme-catalyzed oxidation of tyrosine. DEAE–cellulose chromatography performed in a boric acid–sodium borate buffer removes most of the contaminating pigments. Two-dimensional pH-dependent chromatography on BioGel P-2 separates dityrosine from tyrosine, residual pigments, salts, etc. Elemental analysis indicates that the purified product is ∼92% dityrosine by weight. Fast atom bombardment mass spectrometry and two types of reverse-phase high-performance liquid chromatography (HPLC), monitored in fluorescence and absorbance measurements, verify the purity of the dityrosine. The distinctive pH-dependent chromatography of dityrosine on BioGel P-2, with reversible adsorption to the matrix occurring at pH values less than 3, is useful for the isolation of varying quantities of dityrosine and for analysis per se. Affinity chromatography on immobilized phenyl boronate (Matrex Gel PBA-60) is an alternate method for the isolation and determination of dityrosine, which undergoes specific interactions with the boronate moiety and possible hydrophobic association with the phenyl group. Two new reverse-phase HPLC techniques expedite the analysis of picomole quantities of dityrosine. One employs isocratic elution (92% H2O, 8% acetonitrile, and 0.1% trifluoroacetic acid) of an ODS II Spherisorb column, with both fluorometric and spectrophotometric detection. The other procedure may be performed in conjunction with total amino acid analysis. A rapid gradient program, developed with a Phenomenex Ultracarb 20 column, clearly separates dabsylated dityrosine and tyrosine from other dabsylated amino acids. It is especially useful when dityrosine is a trace component.

Protein Contents, Amino Acid Compositions and Nitrogen-to-Protein Conversion Factors for Cassava Roots

Root protein contents of 15 cassava varieties (Manihot esculenta Crantz) ranged from 5 to 19 g kg -1 dry matter. Intervarietal differences in amino acid profiles of cassava roots were evident. Differences in the levels of aspartic acid, glutamic acid and arginine were most notable. The nitrogen-to-protein conversion factors (k AA ) based on nitrogen recovered from total amino acid analyses including ammonia ranged from 4.75 to 5.87, showing that the traditional conversion factor of 6.25 was not valid for cassava root proteins. Conversion factors (k p ) for 15 cassava varieties based on Kjeldahl nitrogen ranged from 2.49 to 3.67. Therefore an average k p value of 3.24 ± 0.31 may provide a better estimate of the protein content in cassava roots.

High Performance Liquid Chromatographic Determination of Free Amino Acids in Shrimp

Abstract A reverse phase high performance liquid chromatographic method for the analysis of total free amino acids in shrimp, utilizing pre column fluorescence derivatization is described. The primary amino acids were treated with o-phthalaldehyde (OPA). The reaction products were separated on a Microsorb Short-ones 3-μm reversed-phase column with gradient elution development. 15-OPA amino acids were separated in 21 min. Secondary amino acids were reacted with 4-chloro-7-nitrobenzofurazan (NBD). The separation was carried out on a Lichrosorb RP-C18, 5μm column. Wild and cultured Mexican shrimp species (Penaeus vannamei) were analyzed. Free amino acid contents of glycine, alanine and proline were higher than those of other amino acids. Total Free amino acid content was significantly higher in cultured than wild shrimp. The OPA-retention time could be measured within ±0.1 relative standard deviation and the relative peak areas, based on the internal standard calculation methodology, were within ±3% or less....

Efficient purification and rigorous characterisation of a recombinant gp120 for HIV vaccine studies

A recombinant HIV-1 gp120 (rgp120) was expressed in a permanent Chinese Hamster Ovary (CHO) cell line (L761h) that constitutively secretes the product of clone p4 derived from the env gene of HIV-1 isolate GB8. The rgp120 was isolated from cell culture supernatants by a simple, rapid, non-denaturing and efficient purification procedure based on a novel combination of lectin affinity and FPLC ion-exchange chromatography. The purity of the isolated glycoprotein was rigorously confirmed by SDS-PAGE, capillary electrophoresis, laser desorption mass spectrometry, total amino acid analysis and N-terminal amino acid sequencing. The retention of biological activity by the purified rgp120 was assessed by determining the dissociation constant of rgp120 binding to sCD4. After formulation of this highly purified and biologically active rgp120 with “conventional” adjuvants, including types already used in clinical trials of candidate gp120-based HIV vaccines, antibody responses in immunised rabbits were analysed using panels of overlapping synthetic peptides. The consequences of using currently available adjuvants to deliver highly specialised and perhaps conformation-dependent molecules, like HIV gp120, are presented and discussed in the context of HIV vaccine development.

Genetic and amino-acid analysis of two maize threonine-overproducing, lysine-insensitive aspartate kinase mutants

The aspartate-derived amino-acid pathway leads to the production of the essential amino-acids lysine, methionine, threonine and isoleucine. Aspartate kinase (AK) is the first enzyme in this pathway and exists in isoforms that are feedback inhibited by lysine and threonine. Two maize (Zea mays L.) threonine-overproducing, lysine-insensitive AK mutants (Ask1-LT19 and Ask2-LT20) were previously isolated. The present study was conducted to determine the map location of Ask2 and to examine the amino-acid profiles of the Ask mutants. The threonine-overproducing trait conferred by Ask2-LT20 was mapped to the long arm of chromosome 2. Both mutants exhibited increased free threonine concentrations (nmol/mg dry weight) over wild-type. The percent free threonine increased from approximately 2% in wild-type kernels to 37-54% of the total free amino-acid pool in homozygous mutant kernels. Free methionine concentrations also increased significantly in homozygous mutants. Free lysine concentrations were increased but to a much lesser extent than threonine or methionine. In contrast to previous studies, free aspartate concentrations were observed to decrease, indicating a possible limiting factor in threonine synthesis. Total (free plus protein-bound) amino-acid analyses demonstrated a consistent, significant increase in threonine, methionine and lysine concentrations in the homozygous mutants. Significant increases in protein-bound (total minus free) threonine, methionine and lysine were observed in the Ask mutants, indicating adequate protein sinks to incorporate the increased free amino-acid concentrations. Total amino-acid contents (nmol/kernel) were approximately the same for mutant and wild-type kernels. In five inbred lines both Ask mutations conferred the threonine-overproducing phenotype, indicating high expressivity in different genetic backgrounds. These analyses are discussed in the context of the regulation of the aspartate-derived amino-acid pathway.

Biocytin-specific 110-kDa biotinidase from human serum

Biotinidase purification from human serum was performed under new protocol. With HPLC biotinidase assay instead of colorimetric method and using non-ionic surfactant, 110-kDa biotinidase was discovered and co-purified in addition to the previously identified 76-kDa biotinidase. This newly identified enzyme accounted for 5% of the total biotinidase activity. Protein core of 110 kDa was estimated as 72 kDa by use of N-glycanase and SDS-PAGE analysis, while that of the 76-kDa enzyme was estimated as 59 kDa. Total amino acid analysis indicated 30% higher absolute amounts of amino acids in 110-kDa enzyme. The following differences were observed from kinetic study: the 110-kDa enzyme showed a 10-fold lower K m value and a 9-fold higher kcat/K m value for biocytin than those of 76-kDa enzyme. Thus, 110-kDa enzyme is more likely to be the physiological biocytin hydrolase (biocytinase), since the biocytin concentration in human serum is extremely low. The pathogenesis of an inborn error of the metabolism such as a variant form of biotinidase deficiency, which presented an atypical clinical course, might be related to these isoenzymes in terms of their different roles in the body.