Regulation of membrane protein degradation by starvation-response pathways

The multivesicular body (MVB) pathway delivers membrane proteins to the lumen of the vacuole/lysosome for degradation. The resulting amino acids are transported to the cytoplasm for reuse in protein synthesis. Our study shows that this amino acid recycling system plays an essential role in the adaptation of cells to starvation conditions. Cells respond to amino acid starvation by upregulating both endocytosis and the MVB pathway, thereby providing amino acids through increased protein turnover. Our data suggest that increased Rsp5-dependent ubiquitination of membrane proteins and a drop in Ist1 levels, a negative regulator of endosomal sorting complex required for transport (ESCRT) activity, cause this response. Furthermore, we found that target of rapamycin complex 1 (TORC1) and a second, unknown nutrient-sensing system are responsible for the starvation-induced protein turnover. Together, the data indicate that protein synthesis and turnover are linked by a common regulatory system that ensures adaptation and survival under nutrient-stress conditions.     

Some features of the regulation of the free amino acids in adult Calliphora erythrocephala

The amounts of nearly every amino acid in Calliphora remain unchanged inspite of stress. Thus the free amino acid pool is regulated. The amounts of free amino acid in the haemolymph account for only a small part of the total free amino acid in a fly and therefore most free amino acid is located and regulated intracellularly. A comparison of the rates of conversion and turnover of glycine-C¹⁴ in flies fed protein and sugar and in flies fed sugar alone indicates that in the absence of a dietary source of amino acid, not only is the rate of total turnover appreciably lower but the rates of conversion to other amino acids also change. With a dietary source of amino acid, the rates of incorporation into protein account for only a part of the total turnover of each amino acid. With a sufficiency of amino acid, substantial amounts are probably used as an energy source.     

Kinetics of amino acid incorporation into serum proteins

1. The effect of varying body temperature on the rate of amino acid incorporation into serum protein does not give support to the idea that the rate of this process is adjusted in vivo to restore those protein molecules destroyed by thermal denaturation. The experimentally observed Q₁₀ was about 3.9. 2. When amino acids are injected into the blood of animals in a steady state of serum protein turnover, a period of time elapses before these amino acids can be found in the serum proteins. This has been called transit time. At a given temperature (31°) it is the same in rabbits, turtles, and Limulus (1 hour). In rabbits and turtles it has a Q₁₀ of 3.2. It appears to be specifically related to the process of synthesis (or release) of serum proteins. 3. It was not possible to affect the transit time or the incorporation rate by the administration of amino acid analogues.     

Some paradoxical effects of inhibitors of protein synthesis on protein turnover in cultured human cells

Summary Low concentrations ofcycloheximide, sufficient to block net protein synthesis in growing normal and cancer cells, had no effect on protein turnover, i.e. either the incorporation of labeled amino acids from media lacking other amino acids essential for growth, or the loss to the medium of amino acids from prelabeled cells. At the concentrations that blocked growth, the rate of amino acid incorporation from complete medium was reduced to the “quo;turnover level,” i.e. the rate of incorporation seen in amino acid-deficient media. Protein turnover was inhibited only at higher concentrations of the inhibitor. Qualitatively similar results have been obtained with puromycin, anisomycin, emetin and tylocerebrine.     

Effect of Loading Doses of l-Valine on Relative Contributions of Valine Derived from Protein Degradation and Plasma to the Precursor Pool for Protein Synthesis in Rat Brain

"Flooding" amino acid pools with high doses of labeled amino acids of low specific activity has been proposed to minimize the effects of recycling of amino acids derived from protein degradation on the specific activity of the amino acid precursor pool for protein synthesis. We have examined the influence of recycling on the precursor pool for protein synthesis under conditions in which plasma valine concentrations were normal (0.19 mM) and "flooded" (10-28 mM) by comparing the steady-state specific activity of the tRNA-bound valine with that of the plasma valine. Under normal and "flooding" conditions, the relative contributions of valine from protein degradation to the precursor pool were 63 and 26%, respectively; "flooding" with a plasma level of 28 mM raised the brain acid-soluble pool level to 3.1 mM but was no more effective in decreasing the relative contribution of valine from protein degradation to the precursor pool than "flooding" with a plasma level of 17 mM valine, which raised the brain acid-soluble level only to 2.3 mM. The results of these studies show that "flooding" amino acid pools does indeed reduce the effect of recycling on the precursor amino acid pool for protein synthesis, but it does not totally eliminate it.     

Amino Acid Metabolism of Lemna minor L. : I. Responses to Methionine Sulfoximine

When Lemna minor L. is supplied with the potent inhibitor of glutamine synthetase, methionine sulfoximine, rapid changes in free amino acid levels occur. Glutamine, glutamate, asparagine, aspartate, alanine, and serine levels decline concomitantly with ammonia accumulation. However, not all free amino acid pools deplete in response to this inhibitor. Several free amino acids including proline, valine, leucine, isoleucine, threonine, lysine, phenylalanine, tyrosine, histidine, and methionine exhibit severalfold accumulations within 24 hours of methionine sulfoximine treatment. To investigate whether these latter amino acid accumulations result from de novo synthesis via a methionine sulfoximine insensitive pathway of ammonia assimilation (e.g. glutamate dehydrogenase) or from protein turnover, fronds of Lemna minor were prelabeled with [¹⁵N]H₄⁺ prior to supplying the inhibitor. Analyses of the ¹⁵N abundance of free amino acids suggest that protein turnover is the major source of these methionine sulfoximine induced amino acid accumulations. Thus, the pools of valine, leucine, isoleucine, proline, and threonine accumulated in response to the inhibitor in the presence of [¹⁵N]H₄⁺, are ¹⁴N enriched and are not apparently derived from ¹⁵N-labeled precursors. To account for the selective accumulation of amino acids, such as valine, leucine, isoleucine, proline, and threonine, it is necessary to envisage that these free amino acids are relatively poorly catabolized in vivo. The amino acids which deplete in response to methionine sulfoximine (i.e. glutamate, glutamine, alanine, aspartate, asparagine, and serine) are all presumably rapidly catabolized to ammonia, either in the photorespiratory pathway or by alternative routes.     

Quantitative gas chromatographic analysis of amino acids on a short glass capillary column

A study of the quantitative gas chromatographic analysis of protein amino acids as their N-heptafluorobutyric amino acid n-propyl esters on a glass capillary column has been made. The analysis is completed within 35 min with good separation of the common protein amino acids in a single-column run.Hydrolyzed peptides have been analyzed. The analyses were performed with a precision varying between 1 and 6% (mean relative standard deviation) depending on the number of amino acid residues in the peptide. The amount taken for analysis was 20–300 μg. The results agree with the known sequences of the peptides and with the analyses by ion-exchange chromatography except for cysteine. This amino acid can be analyzed after modification as S-methylated cysteine.     

Topograph,a Software Platform for Precursor Enrichment Corrected Global Protein Turnover Measurements

Defects in protein turnover have been implicated in a broad range of diseases, but current proteomics methods of measuring protein turnover are limited by the software tools available. Conventional methods require indirect approaches to differentiate newly synthesized protein when synthesized from partially labeled precursor pools. To address this, we have developed Topograph, a software platform which calculates the fraction of peptides that are from newly synthesized proteins and their turnover rates. A unique feature of Topograph is the ability to calculate amino acid precursor pool enrichment levels which allows for accurate calculations when the precursor pool is not fully labeled, and the approach used by Topograph is applicable regardless of the stable isotope label used. We validate the Topograph algorithms using data acquired from a mouse labeling experiment and demonstrate the influence that precursor pool corrections can have on protein turnover measurements.Methods of measuring protein synthesis and degradation using stable or radioactive isotope labels have existed for decades. The isotope label is introduced in the form of a labeled amino acid or amino acid precursor, and the incorporation or removal of that label from protein is used to estimate average protein turnover rates (1, 2). Historically, the amount of stable isotope label incorporated into a protein is measured by enriching for the protein (e.g. affinity chromatography, gel electrophoresis, and other biochemical methods), hydrolyzing the protein to amino acids, derivatizing the amino acids, and measuring the labeled amino acid by gas chromatography-mass spectrometry or gas chromatography-combustion-isotope ratio mass spectrometry (3, 4). More recently, proteomics methods have been developed that measure the labeled amino acid on the peptide level, eliminating the need for a protein enrichment step and enabling the monitoring of many proteins in a single experiment (5).Proteomics approaches to measuring protein turnover rates in mice have been accomplished by the introduction of a ¹⁵N stable isotope label. The labeled diets were created by supplementing a protein-free diet with a ¹⁵N enriched protein source. Price et al. (6) generated ¹⁵N-labeled protein from the alga, Spirulina platensis and Zhang et al. (7) introduced ¹⁵N-label in the form of lysate from the bacterium, Ralstonia eutropha. An advantage of using complete ¹⁵N labeling is the rapid incorporation of ¹⁵N and separation of isotope distributions between labeled and natural isotope abundance peptides, which reduces the need to deconvolute the two distributions. However, current methods require that the dietary protein content be derived from bacterial or alga lysate, a diet that is not normally fed to laboratory mice. As a result, measurements of protein turnover may not reflect conventional mouse model systems because of effects of diet on protein and amino acid metabolism. A more recent work by Claydon et al. (8) demonstrated a stable isotope labeling method by supplementing labeled valine into a standard mouse diet.The complex data generated from these analyses creates a data processing and analysis challenge; exemplified by recent software platforms that have been developed. Guan et al. (9) and Hoopmann et al. (10) demonstrated data analysis pipelines for ¹⁵N labeled SILAM and SILAC experiments. Here we describe the software platform, Topograph, we have developed for the analysis of liquid chromatography-tandem MS (LC-MS/MS) data from samples with isotopic labels. Topograph is able to deconvolute the complex spectra that may result from overlapping isotope distributions, regardless of the isotope label used. More uniquely, Topograph is able to calculate the relative isotope abundance (RIA)¹ of the amino acid precursor pool, which is necessary to correctly determine the amount of newly synthesized peptide and to subsequently calculate peptide and protein turnover rates.     

Amino Acid Metabolism of Lemna minor L. : III. Responses to Aminooxyacetate

Aminooxyacetate, a known inhibitor of transaminase reactions and glycine decarboxylase, promotes rapid depletion of the free pools of serine and aspartate in nitrate grown Lemna minor L. This compound markedly inhibits the methionine sulfoximine-induced accumulation of free ammonium ions and greatly restricts the methionine sulfoximine-induced depletion of amino acids such as glutamate, alanine, and asparagine. These results suggest that glutamate, alanine, and asparagine are normally catabolized to ammonia by transaminase-dependent pathways rather than via dehydrogenase or amidohydrolase reactions. Aminooxyacetate does not inhibit the methionine sulfoximine-induced irreversible deactivation of glutamine synthetase in vivo, indicating that these effects cannot be simply ascribed to inhibition of methionine sulfoximine uptake by amino-oxyacetate. This transaminase inhibitor promotes extensive accumulation of several amino acids including valine, leucine, isoleucine, alanine, glycine, threonine, proline, phenylalanine, lysine, and tyrosine. Since the aminooxyacetate induced accumulations of valine, leucine, and isoleucine are not inhibited by the branched-chain amino acid biosynthesis inhibitor, chlorsulfuron, these amino acid accumulations most probably involve protein turnover. Depletions of soluble protein bound amino acids are shown to be approximately stoichiometric with the free amino acid pool accumulations induced by aminooxyacetate. Aminooxyacetate is demonstrated to inhibit the chlorsulfuron-induced accumulation of α-amino-n-butyrate in L. minor, supporting the notion that this amino acid is derived from transamination of 2-oxobutyrate.     

Hind leg muscle amino acid balances in cold-exposed rats

The changes in hind leg tissue (muscle and skin) amono acid pool size and arteriovenous balance were measured in rats subjected to 0–90 min of cold exposure (4°C). Tissue free amino acid pools presented a different composition pattern from protein amino acids. Muscle rapidly reacted to cold exposure by releasing small amounts of some amino acids (alanine, aspartate), with only small changes in pool size during the first 30 min. Amino acid oxidation was very limited during the whole period of cold exposure, since at all times tested there was either nil ammonia efflux or net absorption of ammonia and glutamine; i.e. the muscle was in positive nitrogen balance throughout the period studied. Thus most of the amino acid nitrogen taken up from the blood and not found in the free amino pools must have been incorporated into protein, since it was not oxidized, as shown by the glutamine and ammonia blance. The data on amino acid incorporation into proteins indicate that hind leg protein turnover is rapidly and widely modulated from a low initial setting upon cold exposure to a higher protein synthesis rate immediately afterwards, suggesting that protein turnover may have an important role in short-term events in cold-exposed muscle, in addition to its influence in long-term adaptation.     

Regulation of intestinal protein metabolism by amino acids

Gut homeostasis plays a major role in health and may be regulated by quantitative and qualitative food intake. In the intestinal mucosa, an intense renewal of proteins occurs, at approximately 50 % per day in humans. In some pathophysiological conditions, protein turnover is altered and may contribute to intestinal or systemic diseases. Amino acids are key effectors of gut protein turnover, both as constituents of proteins and as regulatory molecules limiting intestinal injury and maintaining intestinal functions. Many studies have focused on two amino acids: glutamine, known as the preferential substrate of rapidly dividing cells, and arginine, another conditionally essential amino acid. The effects of glutamine and arginine on protein synthesis appear to be model and condition dependent, as are the involved signaling pathways. The regulation of gut protein degradation by amino acids has been minimally documented until now. This review will examine recent data, helping to better understand how amino acids regulate intestinal protein metabolism, and will explore perspectives for future studies.     

Structure and expression of the gene encoding ribosomal protein S1 from the cyanobacterium Synechococcus sp. strain PCC 6301: striking sequence similarity to the chloroplast ribosomal protein CS1

We isolated a 38 kDa ssDNA-binding protein from the unicellular cyanobacterium Synechococcus sp. strain PCC 6301 and determined its N-terminal amino acid sequence. A genomic clone encoding the 38 kDa protein was isolated by using a degenerate oligonucleotide probe based on the amino acid sequence. The nucleotide sequence and predicted amino acid sequence revealed that the 38 kDa protein is 306 amino acids long and homologous to the nuclear-encoded 370 amino acid chloroplast ribosomal protein CS1 of spinach (48% identity), therefore identifying it as ribosomal protein (r-protein) S1. Cyanobacterial and chloroplast S1 proteins differ in size from Escherichia coli r-protein S1 (557 amino acids). This provides an additional evidence that cyanobacteria are closely related to chloroplasts. The Synechococcus gene rps1 encoding S1 is located 1.1 kb downstream from psbB, which encodes the photosystem 11 P680 chlorophyll a apoprotein. An open reading frame encoding a potential protein of 168 amino acids is present between psbB and rps1 and its deduced amino acid sequence is similar to that of E. coli hypothetical 17.2 kDa protein. Northern blot analysis showed that rps1 is transcribed as a monocistronic mRNA.     

Dual-Label Radioisotope Method for Simultaneously Measuring Bacterial Production and Metabolism in Natural Waters

Bacterial production and amino acid metabolism in aquatic systems can be estimated by simultaneous incubation of water samples with both tritiated methyl-thymidine and ¹⁴C-labeled amino acids. This dual-label method not only saves time, labor, and materials, but also allows determination of these two parameters in the same microbial subcommunity. Both organic carbon incorporation and respiration can be estimated. The results obtained with the dual-label technique are not significantly different from single-radiolabel methods over a wide range of bacterial activity. The method is particularly suitable for large-scale field programs and has been used successfully with eutrophic estuarine samples as well as with oligotrophic oceanic water. In the mesohaline portion of Chesapeake Bay, thymidine incorporation ranged seasonally from 2 to 635 pmol liter⁻¹ h⁻¹ and amino acid turnover rates ranged from 0.01 to 28.4% h⁻¹. Comparison of thymidine incorporation with amino acid turnover measurements made at a deep, midbay station in 1985 suggested a close coupling between bacterial production and amino acid metabolism during most of the year. However, production-specific amino acid turnover rates increased dramatically in deep bay waters during the spring phytoplankton bloom, indicating transient decoupling of bacterial production from metabolism. Ecological features such as this are readily detectable with the dual-label method.     

Protein turnover and amino acid transport kinetics in end-stage renal disease

Protein and amino acid metabolism is abnormal in end-stage renal disease (ESRD). Protein turnover is influenced by transmembrane amino acid transport. The effect of ESRD and hemodialysis (HD) on intracellular amino acid transport kinetics is unknown. We studied intracellular amino acid transport kinetics and protein turnover by use of stable isotopes of phenylalanine, leucine, lysine, alanine, and glutamine before and during HD in six ESRD patients. Data obtained from amino acid concentrations and enrichment in the artery, vein, and muscle compartments were used to calculate intracellular amino acid transport and muscle protein synthesis and catabolism. Fractional muscle protein synthesis (FSR) was estimated by the precursor product approach. Despite a significant decrease in the plasma concentrations of amino acids in the artery and vein during HD, the intracellular concentrations remained stable. Outward transport of the amino acids was significantly higher than the inward transport during HD. FSR increased during HD (0.0521 +/- 0.0043 vs. 0.0772 +/- 0.0055%/h, P < 0.01). Results derived from compartmental modeling indicated that both protein synthesis (118.3 +/- 20.6 vs. 146.5 +/- 20.6 nmol.min-1.100 ml leg-1, P < 0.01) and catabolism (119.8 +/- 18.0 vs. 174.0 +/- 14.2 nmol.min-1.100 ml leg-1, P < 0.01) increased during HD. However, the intradialytic increase in catabolism exceeded that of synthesis (57.8 +/- 13.8 vs. 28.0 +/- 8.5%, P < 0.05). Thus HD alters amino acid transport kinetics and increases protein turnover, with net increase in protein catabolism.     

三角鲂和长春鳊肌肉营养成分分析与品质评价

用常规方法测定、分析了三角鲂(Megalobrama tarminalis)和长春鳊(Parabramis pekinensis肌肉中营养成分组成与含量.结果显示,三角鲂肌肉蛋白质、脂肪含量分别为18.19％和3.06％,长春鳊肌肉蛋白质、脂肪含量分别为19.38％和2.89％.三角鲂和长春鳊肌肉中均检测出18种氨基酸,其中包括了8种人体必需氨基酸.三角鲂肌肉中氨基酸总量为76.27％,其中,8种人体必需氨基酸含量为32.17％,占氨基酸总量的42.18％;长春鳊肌肉中氨基酸总量为77.60％,其中,8种人体必需氨基酸含量为31.70％,占氨基酸总量的40.85％.必需氨基酸的构成比例基本符合FAO/WHO的标准.三角鲂肌肉中限制性氨基酸主要为甲硫氨酸加胱氨酸,必需氨基酸指数为63.55,4种呈味氨基酸为氨基酸总量的32.81％;长春鳊肌肉中限制性氨基酸主要为色氨酸,必需氨基酸指数为66.81,4种呈味氨基酸为氨基酸总量的33.80％.脂肪酸中二十碳五烯酸(EPA)与二十二碳六烯酸(DHA)含量均较高,三角鲂为7.96％,长春鳊为3.11％.矿物元素比值合理.以上分析表明,三角鲂和长春鳊均为营养价值、经济价值都较高的优质鱼类,相比而言,三角鲂肌肉脂肪、脂肪酸含量和质量更优,而长春鳊肌肉在蛋白质、氨基酸组成与含量方面更优.     

Uptake of Dissolved Organic Carbon by Gammaproteobacterial Subgroups in Coastal Waters of the West Antarctic Peninsula

Heterotrophic bacteria are well known to be key players in the turnover of dissolved organic material (DOM) in the oceans, but the relationship between DOM uptake and bacterial clades is still not well understood. Here we explore the turnover and single-cell use of glucose, an amino acid mixture, N-acetylglucosamine (NAG), and protein by gammaproteobacterial clades in coastal waters of the West Antarctic Peninsula in summer and fall. More than 60% of the cells within two closely related gammaproteobacterial clades, Ant4D3 and Arctic96B-16, were active in using the amino acid mixture, protein, and NAG. In contrast, an average of only 7% of all SAR86 cells used amino acids and protein even in summer when DOM use was high. In addition to DOM uptake within a group, we explored the contribution of the three gammaproteobacterial groups to total community uptake of a compound. SAR86 contributed 5- to 10-fold less than the other gammaproteobacterial subgroups to the uptake of all compounds. We found that the overall contribution of the Ant4D3 clade to DOM uptake was highest, whereas the SAR86 clade contributed the least to DOM turnover in West Antarctic Peninsula waters. Our results suggest that the low growth activity of a bacterial clade leads to low abundance, fewer active cells and a low contribution to the turnover of DOM components.     

Rapid Cycling of Organic Nitrogen in Taiga Forest Ecosystems

ABSTRACT We examined the dynamics of organic nitrogen (N) turnover in situ across a primary successional sequence in interior Alaska, USA, in an attempt to understand the magnitude of these fluxes in cold, seasonally frozen soils. Through a combination of soil extraction procedures and measurements of ¹³C-enriched CO₂ efflux from soils amended in the field with ¹³C-labeled amino acids, we were able to trace the fate of this N form. Amino acid turnover in situ at soil temperatures of 10°C or below show that amino acids represent a highly dynamic soil N pool with turnover times of approximately 3–6 h. The rapid turnover of free amino acids is associated with high soil proteolytic activity, which in turn is tightly correlated with soil protein concentration. Moreover, these estimates of soil amino acid turnover in the field correspond well with measurements of amino acid turnover under equivalent temperatures in the laboratory. The gross flux of amino acid-N over the growing season greatly exceeded the annual vegetation N requirement, suggesting that microbial biomass represent a significant sink for this organic N. Depending on the strength of this sink, N flow via free soil amino acids can potentially account for the entire N demand of vegetation in the absence of net N mineralization. These relationships underscore the important biogeochemical role of labile DON fractions in high-latitude forest ecosystems.     

A static model to analyze carbon and nitrogen partitioning in the mammary gland of lactating sows

Quantitative estimates of mammary nutrient inputs, outputs and metabolism in sows are scarce, despite being critical elements to identify parameters controlling milk synthesis central for the feeding of lactating sows. The objective of this study was to quantify the mammary gland input and output of nutrients as well as the intramammary partitioning of carbon and nitrogen with the purpose to identify mechanisms controlling mammary nutrient inputs, metabolism and milk production in lactating sows. A data set was assembled by integration of results from four studies. The data set included data on litter performance, mammary arterial-venous concentration differences (AV-difference) of energy metabolites and amino acids, and the contents of lactose, fat and amino acids in milk. Milk yield was estimated based on average litter size and litter gain, and mammary plasma flow (MPF) was estimated using the sum of phenylalanine and tyrosine as internal flow markers. The yield and composition of milk were used to estimate mammary nutrient output in milk, and MPF and AV-difference were used to estimate net mammary input of carbon and nitrogen and output of CO₂. Carbon and nitrogen used for the synthesis of lactose, fat and protein in milk and CO₂-yielding processes were represented in a static nutrient partitioning model. The origin of mammary CO₂ output was calculated using theoretical estimates of carbon released in processes supporting mammary synthesis of de novo fat, protein and lactose in milk, mammary tissue protein turnover and transport of glucose and amino acids. Results indicated that total input of carbon from glucose and lactate was partitioned into lactose (36%), fat (31%) and CO₂-yielding processes (34%). Theoretical CO₂ estimates indicated that de novo fat synthesis, milk protein synthesis and mammary tissue protein turnover were the main processes related to mammary CO₂ production. More than 90% of mammary gland amino acid input was used for milk protein. The quadratic relationship between AV-difference and mammary input of essential amino acids indicated that both changes in AV-difference and MPF contributed to the regulation of mammary input of essential amino acids. The impact of the arterial supply of amino acids on mammary input may be greater for the branched-chain amino acids, arginine and phenylalanine than for other essential amino acids. In conclusion, relationships between input and output parameters indicate that AV-difference and MPF regulate mammary nutrient input to match the supply and demand of nutrients for the mammary gland.     

DNA polymerase ι: The long and the short of it!

The cDNA encoding human DNA polymerase ι (POLI) was cloned in 1999. At that time, it was believed that the POLI gene encoded a protein of 715 amino acids. Advances in DNA sequencing technologies led to the realization that there is an upstream, in-frame initiation codon that would encode a DNA polymerase ι (polι) protein of 740 amino acids. The extra 25 amino acid region is rich in acidic residues (11/25) and is reasonably conserved in eukaryotes ranging from fish to humans. As a consequence, the curated Reference Sequence (RefSeq) database identified polι as a 740 amino acid protein. However, the existence of the 740 amino acid polι has never been shown experimentally. Using highly specific antibodies to the 25 N-terminal amino acids of polι, we were unable to detect the longer 740 amino acid (ι-long) isoform in western blots. However, trace amounts of the ι-long isoform were detected after enrichment by immunoprecipitation. One might argue that the longer isoform may have a distinct biological function, if it exhibits significant differences in its enzymatic properties from the shorter, well-characterized 715 amino acid polι. We therefore purified and characterized recombinant full-length (740 amino acid) polι-long and compared it to full-length (715 amino acid) polι-short in vitro. The metal ion requirements for optimal catalytic activity differ slightly between ι-long and ι-short, but under optimal conditions, both isoforms exhibit indistinguishable enzymatic properties in vitro. We also report that like ι-short, the ι-long isoform can be monoubiquitinated and polyubiuquitinated in vivo, as well as form damage induced foci in vivo. We conclude that the predominant isoform of DNA polι in human cells is the shorter 715 amino acid protein and that if, or when, expressed, the longer 740 amino acid isoform has identical properties to the considerably more abundant shorter isoform.     

Inclusion of nonproteinous amino acids in thermally prepared models for prebiotic protein

Sixteen nonproteinous amino acids (those not coded for in contemporary protein biosynthesis) were incorporated during the thermal formation of polyamino acids under postulated prebiotic conditions, although not all into a single polyamino acid. The copresence of proteinous or even α-amino acids was not required. (Norleucine color equivalents and elution times on a Beckman model 120C amino acid analyzer were determined for these nonproteinous amino acids). The results suggest that prebiotically available nonproteinous amino acids would have been constituents of prebiotic protein if the latter were formed thermally. Some differences in properties of the polyamino acids could be attributed to particular nonproteinous amino acid residues; however, the tested properties did not suggest a means for evolutionary selection against nonproteinous amino acids as a group. Selection against this class of amino acids in toto was likely a later, biotic, event.