Nitrogen metabolism and excretion in the aquatic chinese soft-shelled turtle, Pelodiscus sinensis, exposed to a progressive increase in ambient salinity

This study aimed to determine effects of 6-day progressive increase in salinity from 1 per thousand to 15 per thousand on nitrogen metabolism and excretion in the soft-shelled turtle, Pelodiscus sinensis. For turtles exposed to 15 per thousand water on day 6, the plasma osmolality and concentrations of Na+, Cl- and urea increased significantly, which presumably decreased the osmotic loss of water. Simultaneously, there were significant increases in contents of urea, certain free amino acids (FAAs) and water-soluble proteins that were involved in cell volume regulation in various tissues. There was an apparent increase in proteolysis, releasing FAAs as osmolytes. In addition, there might be an increase in catabolism of certain amino acids, producing more ammonia. The excess ammonia was retained as indicated by a significant decrease in the rate of ammonia excretion on day 4 in 15 per thousand water, and a major portion of it was converted to urea. The rate of urea synthesis increased 1.4-fold during the 6-day period, although the capacity of the hepatic ornithine urea cycle remained unchanged. Urea was retained for osmoregulation because there was a significant decrease in urea excretion on day 4. Increased protein degradation and urea synthesis implies greater metabolic demands, and indeed turtles exposed to 15 per thousand water had significantly higher O2 consumption rate than the freshwater (FW) control. When turtles were returned from 15 per thousand water to FW on day 7, there were significant increases in ammonia (probably released through increased amino acid catabolism) and urea excretion, confirming that FAAs and urea were retained for osmoregulatory purposes in brackish water.     

Long-term effectiveness of high-dosed ornithine-aspartate on urea synthesis rate and portal hypertension in human liver cirrhosis

Summary The effectiveness of ammonia reducing amino acids on hyperammonemia and hepatic encephalopathy is well known in patients suffering from liver cirrhosis. Data concerning long-term therapy on hepatic function and urea synthesis rate (UNSR) are still lacking. According to Vilstrup/Poulsen it is a good standard for functioning liver mass. Therefore, 25 patients with histologically proven liver cirrhosis and distinct portal hypertension were treated daily with 9 gr. ornithinasparte over 13 years (8–20 years). Shunt operations, esophageal varicosis sclerosis, or portal pressure reducing medication were not applied. Rigorous alcohol abstinence and 60 gr protein/day were prescribed. During the investigation, 3 laparoscopies and 4 liver biopsies were performed, on the average, on each individual. Significant improvements of clinical and biochemical results (Child-Pugh-Index; Composite Clinical and Laboratory Index) were obtained during the long-term therapy with ornithine-aspartate. Esophageal varicosis II–III was either reduced to 0-I or totally eliminated. Also significant was an increased urea synthesis rate and a decreased hyperammonemia.A plausible explanation for the long-term therapy effectiveness with ornithine-aspartate is the possible recovery of the functioning mass without hepatic size increase. Also important is the rigorous alcohol abstinence. It leads to a significant reduction of portal hypertension in patients suffering from alcohol induced liver cirrhosis (Reynolds, own observations).Additional favorable factors are intensive muscle training and absence of gastrointestinal bleeds.     

Transport of amino acids in the splanchnic bed by blood plasma and blood in the preruminant calf]

Three preruminant calves were fitted with catheters in portal and hepatic veins and in a mesenteric artery. Two electromagnetic flowmeter probes were clipped around the portal vein and the hepatic artery. The calves were fed either a diet with a low (L) or a high (R) abomasal emptying rate for dietary proteins. Blood flow and free amino acid levels in plasma (P) and blood (S) were determined before the morning meal and during the following 7 h. In the portal vein, for most amino acids P/S ratios were correlated to the net amino acid balance of the digestive tract measured in plasma. By contrast in the hepatic vein, these ratios were mainly correlated to hepatic balance measured in whole blood. Correlations between digestive tract and hepatic balance calculated using either plasma or whole blood pool were different for some amino acids. This suggests that amino acid exchange between plasma and blood cells is low and absorbed amino acids are mainly transported to the liver by plasma, whereas whole blood rather than plasma is concerned in amino acid exchanges in the liver.     

Rat intestinal amino acid balances after the administration of an oral protein load.

The intestinal amino acid balances in rats given an oral load of protein were measured three hours after the gavage. During that period, about one half of the nitrogen from the protein given appeared as net balance in the portal blood. The release of amino acids was not a continuous process, since two peaks of maximal intestinal efflux were found at 1 and 2.5 hours after gavage. This pattern followed that of portal blood flow with a 30 minute delay. Under prandial conditions, the intestine showed a net uptake of glutamine, aspartate, serine, threonine, phenylalanine, tyrosine, leucine, isoleucine and valine. It also showed a net production of alanine, glutamate, ornithine skeleton (arginine + citrulline + ornithine) and ammonia. There was also a surge of taurine, attributed to reabsorption of secreted taurine conjugates. There was a net unchanged absorption of glycine, proline, lysine and histidine, with respect to their proportions in the protein administered. The results suggest that the amino acid metabolism in the intestine under prandial conditions is much less passive than is generally assumed. The intestinal action upon the luminal amino acids is not limited to absorption, but is directly implied in their transformation to complement the ensuing homeostatic action of the liver upon them.     

Hepatic and portal-drained viscera balances of amino acids, insulin, glucagon and gastrin in the pig after ingestion of casein or rapeseed proteins

Hepatic and intestinal balances of amino acids, insulin, glucagon and gastrin were studied in 6 non-anaesthetized Large White pigs (mean body weight 64 +/- 4.8 kg) after ingestion of casein or rapeseed proteins. The animals were fitted with permanent catheters in the portal vein, the brachiocephalic artery and the right hepatic vein. In addition, 2 electromagnetic flow probes were implanted, one around the portal vein and the other around the hepatic artery. After a preliminary adaptation to each diet the animals received at 1-wk intervals and according to a double latin square design, 3 test meals of 800 g each, one containing 23.2% of rapeseed concentrate (diet RA 12) and the others 13.9 or 27.8% of hydrochloric casein (diets CA 12 and CA 24). Each observation period lasted 12 h. Amino acids from all diets were very well absorbed. In 12 h, the absorption of total amino acids as a percentage of the ingested quantities was 99% for CA 12, 102% for CA 24 and 104% for RA 12. Hepatic uptake of total amino acids in 12 h expressed as a percentage of the absorbed quantities was 13% for CA 12, 66% for CA 24 and 25% for RA 12. Differences in the hepatic extraction rate of essential amino acids appeared between the 2 levels of casein ingestion and for Arg between the 2 protein sources. Whatever the nature of the ingested protein or the level of casein, the liver showed a net production of Asp and Glu. The production and hepatic balance of insulin were the lowest after ingestion of RA 12. No differences were noted in the same parameters for glucagon and gastrin. Independently of the nutritional situation, the hepatic extraction rate of insulin appeared to be higher than those of glucagon and gastrin. Our results showed that the nature as well as the level of dietary proteins have large effects on the sequence and volume of absorptive phenomena, the hepatic metabolism of nutrients, the production of gastrointestinal hormones and the non-hepatic tissue disposal of absorbed nutrients.     

Effect of adding dietary L-lysine, L-threonine and L-methionine to a low gluten diet on urea synthesis in rats

Summary. We have shown that urinary urea excretion increased in rats fed a low quality protein. The purpose of present study was to determine whether an addition of dietary limiting amino acids affected urea synthesis in rats fed a low gluten diet. Experiments were done on three groups of rats given diets containing 10% gluten, 10% gluten+0.5% L-lysine or 10% gluten+0.5% L-lysine, 0.2% L-threonine and 0.2% L-methionine for 10d. The urinary excretion of urea, and the liver concentrations of serine and ornithine decreased with the addition of dietary L-lysine, L-threonine and L-methionine. The fractional and absolute rates of protein synthesis in tissues increased with the treatment of limiting amino acids. The activities of hepatic urea-cycle enzymes was not related to the urea excretion. These results suggest that the addition of limiting amino acids for the low gluten diet controls the protein synthesis in tissues and hepatic ornithine and decline urea synthesis.     

Key role of L-alanine in the control of hepatic protein synthesis.

We investigated the effects of administration of single amino acids to starved rats on the regulation of protein synthesis in the liver. Of all the amino acids tested, only alanine, ornithine and proline promoted statistically significant increases in the extent of hepatic polyribosome aggregation. The most effective of these was alanine, whose effect of promoting polyribosomal aggregation was accompanied by a decrease in the polypeptide-chain elongation time. The following observations indicate that alanine plays an important physiological role in the regulation of hepatic protein synthesis. Alanine was the amino acid showing the largest decrease in hepatic content in the transition from high (fed) to low (starved) rates of protein synthesis. The administration of glucose or pyruvate is also effective in increasing liver protein synthesis in starved rats, and their effects were accompanied by an increased hepatic alanine content. An increase in hepatic ornithine content does not lead to an increased protein synthesis, unless it is accompanied by an increase of alanine. The effect of alanine is observed either in vivo, in rats pretreated with cycloserine to prevent its transamination, or in isolated liver cells under conditions in which its metabolic transformation is fully impeded.     

Splanchnic and peripheral uptake of amino acids in relation to the gut

There are many complexities and theoretical aspects to consider for studies of amino acid absorption and metabolism by the gut, liver, and peripheral tissues. The experimental approach must vary depending on the amino acid. Whether to sample whole blood or plasma has to be considered carefully. Also, a specific blood vessel has to be chosen for taking samples. A jugular vein can be the poorest sampling site for many studies. The amounts of individual amino acids appearing in portal blood are different from amounts disappearing from the gut lumen. Some are absorbed in amounts equal to that disappearing but most are absorbed in lesser quantities because of intestinal metabolism. Further, the liver removes absorbed amino acids and synthesizes plasma proteins, urea, and glucose. Peripheral tissues, of course, exchange amino acids with protein for normal turnover but also use amino acids for oxidation and transamination. Alanine, glutamine, glycine, and arginine are important in transporting nitrogen out of peripheral tissues in a nontoxic form. Branched-chain amino acids are removed by both liver and peripheral tissues mainly for plasma protein and ketoacid formation, respectively. During fasting, however, muscle releases branched-chain amino acids while removal by liver is maintained.     

Regulation of hepatic phosphoenolpyruvate carboxykinase (GTP) Role of dietary proteins and amino acids in vivo and in the isolated perfused rat liver

The effect of protein feeding and the addition of amino acids on the activity of hepatic phosphoenolpyruvate carboxykinase (GTP: oxalacetate carboxylyase (transphosphorylating), EC 4.1.1.32) was investigated in vivo and in the isolated perfused rat liver. Protein feeding resulted in a considerable increase in phosphoenolpyruvate carboxykinase activity within 6 h. This rise was independent of the presence of glucocorticoids.In the isolated perfused liver system amino acids per se had a small effect on phosphoenolpyruvate carboxykinase activity and led to an increase by 20% when glucocorticoids were present, but resulted in a rise by 100% when glucocorticoids plus dibutyryl cyclic AMP were added to the perfusion medium. The effect of amino acids in the presence of dibutyryl cyclic AMP could also be observed in the liver of glucocorticoid-deprived rats.Cycloheximide, a translational inhibitor, totally blocked all effects of amino acids on enzyme activity.These results indicate that the concentration of amino acids in the portal vein modify the regulation of phosphoenolpyruvate carboxykinase by cyclic AMP.     

Amino acid catabolism by perfused rat hindquarter. The metabolic fates of valine.

Hindquarters from starved rats were perfused with plasma concentrations of amino acids, but without other added substrates. Release of amino acids was similar to that previously reported, but, if total amino acid changes were recorded, alanine and glutamine were not formed in excess of their occurrence in muscle proteins. In protein balance (excess insulin) there was no net formation of either alanine or glutamine, even though the branched-chain amino acids and methionine were consumed. If [U-14C]valine was present, radiolabelled 3-hydroxyisobutyrate and, to a lesser extent, 2-oxo-3-methylbutyrate accumulated and radiolabel was incorporated into citrate-cycle intermediates and metabolites closely associated with the citrate cycle (glutamine and glutamate, and, to a smaller extent, lactate and alanine). If a 2-chloro-4-methylvalerate was present to stimulate the branched-chain oxo acid dehydrogenase, flux through this step was accelerated, resulting in increased accumulation of 3-hydroxyisobutyrate, decreased accumulation of 2-oxo-3-methylbutyrate, and markedly increased incorporation of radiolabel (specific and total) into all measured metabolites formed after 3-hydroxyisobutyrate. It is concluded that: amino acid catabolism by skeletal muscle is confined to degradation of the branched-chain amino acids, methionine and those that are interconvertible with the citrate cycle; amino acid catabolism is relatively minor in supplying carbon for net synthesis of alanine and glutamine; and partial degradation products of the branched-chain amino acids are quantitatively significant substrates released from muscle for hepatic gluconeogenesis. For valine, 3-hydroxyisobutyrate appears to be quantitatively the most important intermediate released from muscle. A side path for inter-organ disposition of the branched-chain amino acids is proposed.     

Radioautographic study on the localization of an anti-allergic agent, tranilast, in the rat liver

In order to demonstrate the localization associated with metabolism of an anti-allergic agent, Tranilast, in the liver, light microscopic radioautography of the liver was performed. Rats were administrated orally with 3H-Tranilast, and were sacrificed at 15 minutes to 24 hours after the administration. The livers were taken out and fixed, embedded and processed for light microscopic radioautography. 3H-Tranilast was absorbed rapidly, and the radioactivity in the liver increased and decreased within several hours. The number of radioautographic silver grains reached a maximum 3 hours after the administration. From 1 to 6 hours after the administration, the silver grains decreased from the portal area toward the central area. Seventy to 80% of all silver grains on the hepatocytes were retained in the cytoplasms of the hepatocytes at any experimental period. From these results, it was concluded that the localization of radioautographic silver grains was associated with Tranilast uptake of hepatocytes in each hepatic lobular compartment and that the metabolic process from uptake to excretion of Tranilast took part in the hepatocytes in each hepatic lobular compartment.     

An isotope dilution model for partitioning leucine uptake by the liver of the lactating dairy cow.

An isotope dilution model for partitioning leucine uptake by the liver of the lactating dairy cow is constructed and solved in the steady state. If assumptions ae made, model solution permits calculation of the rate of leucine uptake from portal and hepatic arterial blood supply, leucine export into the hepatic vein, leucine oxidation and transamination, and synthesis and degradation of hepatic constitutive and export proteins. The model requires the measurement of plasma flow rate through the liver in combination with leucine concentrations and plateau isotopic enrichments in arterial, portal and hepatic plasma during a constant infusion of [1-13C]leucine tracer. The model can be applied to other amino acids with similar metabolic fates and will provide a means for assessing the impact of hepatic metabolism on amino acid availability to peripheral tissues. This is of particular importance when considering the dairy cow and the requirements of the mammary gland for milk protein synthesis.     

Effects of cycloheximide on protein degradation and gluconeogenesis in the perfused rat liver.

Cycloheximide at concentrations above 18 muM produced a 93% inhibition of total protein synthesis measured by valine incorporation in the perfused rat liver. Rates of protein degradation were estimated by perfusing livers prelabeled in vivo with L-[1-14C]valine with medium containing 15 mM L-valine. Thus labeled valine released from liver protein during perfusion was greatly diluted and reincorporation of label was minimized. Cycloheximide at 18 muM inhibited protein degradation by over 60%, after a delay of 15-20 min. Associated with these effects were dose-dependent increases in the rates of glucose and urea production. Glucose production increased 3 fold, from 0.54 +/- 0.07 in control to 1.85 +/- 0.24 mumol/min/100 g rat in cycloheximide-treated livers. Urea production increased from 0.24 +/- 0.02 to 0.62 +/- 0.06 mumol/min/100 g rat. No changes in liver glycogen or cyclic AMP content were seen. The data suggest that inhibition of protein synthesis provides an increased availability of intra-cellular amino acids and that many of these are rapidly degraded, yielding urea and glucose. This is supported by the fact that intracellular alanine levels were significantly increased following cycloheximide treatment. It is possible that the inhibition of protein degradation by cycloheximide is due to altered intra-cellular pools of amino acids or their metabolites.     

Absorption of a protein gavage in Zucker lean rats. Influence of protein content in the diet.

The rate of protein absorption was measured in Zucker lean rats. Rats were fed with a bolus that contained ca. 300 mg of 14C-labelled protein at the beginning of the light cycle. Blood was extracted from the portal vein at intervals up to 9 hours after gavage. Label incorporation into tissue protein was monitored. The digestion and absorption of protein was slow, and 9 hours after the gavage, 20% of the bolus remained in the stomach. Forty percent of the protein was absorbed in the first hour. This was followed first by a linear absorption process, then by the amino acid incorporation into tissue proteins. The appearance of label in the portal vein increased progressively for up to four hours, shifting to a progressive decrease that coincides with the maintenance of this label in the tissues. The skin, the striated muscle and the liver showed the highest amounts of labelled proteins. The application of this model to animals fed low-(LP) or high-protein (HP) content diets showed that the HP group digested the protein faster than the LP group, and that catabolism of the amino acids was higher in the HP group. The LP group digested protein much more slowly than the RD (control) group, but protein accretion was more efficient.     

Shifting patterns of nitrogen excretion and amino acid catabolism capacity during the life cycle of the sea lamprey (Petromyzon marinus)

The jawless fish, the sea lamprey (Petromyzon marinus), spends part of its life as a burrow-dwelling, suspension-feeding larva (ammocoete) before undergoing a metamorphosis into a free swimming, parasitic juvenile that feeds on the blood of fishes. We predicted that animals in this juvenile, parasitic stage have a great capacity for catabolizing amino acids when large quantities of protein-rich blood are ingested. The sixfold to 20-fold greater ammonia excretion rates (J(Amm)) in postmetamorphic (nonfeeding) and parasitic lampreys compared with ammocoetes suggested that basal rates of amino acid catabolism increased following metamorphosis. This was likely due to a greater basal amino acid catabolizing capacity in which there was a sixfold higher hepatic glutamate dehydrogenase (GDH) activity in parasitic lampreys compared with ammocoetes. Immunoblotting also revealed that GDH quantity was 10-fold and threefold greater in parasitic lampreys than in ammocoetes and upstream migrant lampreys, respectively. Higher hepatic alanine and aspartate aminotransferase activities in the parasitic lampreys also suggested an enhanced amino acid catabolizing capacity in this life stage. In contrast to parasitic lampreys, the twofold larger free amino acid pool in the muscle of upstream migrant lampreys confirmed that this period of natural starvation is accompanied by a prominent proteolysis. Carbamoyl phosphate synthetase III was detected at low levels in the liver of parasitic and upstream migrant lampreys, but there was no evidence of extrahepatic (muscle, intestine) urea production via the ornithine urea cycle. However, detection of arginase activity and high concentrations of arginine in the liver at all life stages examined infers that arginine hydrolysis is an important source of urea. We conclude that metamorphosis is accompanied by a metabolic reorganization that increases the capacity of parasitic sea lampreys to catabolize intermittently large amino acid loads arising from the ingestion of protein rich blood from their prey/hosts. The subsequent generation of energy-rich carbon skeletons can then be oxidized or retained for glycogen and fatty acid synthesis, which are essential fuels for the upstream migratory and spawning phases of the sea lamprey's life cycle.     

Control of proteolysis in perifused rat hepatocytes

The mechanism by means of which amino acids inhibit intrahepatic protein degradation has been studied in perifused rat hepatocytes. Proteolysis was extremely sensitive to inhibition by low concentrations of amino acids. A mixture of 0.5 mM leucine and 1-2 mM alanine, concentrations found in the portal vein of the rat after feeding, inhibited proteolysis to the same extent as a complete physiological mixture of amino acids. Inhibition by these two amino acids was accompanied by a rise in the intracellular concentrations of glutamate and aspartate, and was largely prevented by addition of glucagon, by addition of the transaminase inhibitor aminooxyacetate, or by omission of K+. Acceleration of proteolysis by K+ depletion was accompanied by a fall in intracellular glutamate caused by an increased rate of transport of this amino acid to the extracellular fluid. It is concluded that intracellular leucine, glutamate and aspartate are important elements in the control of hepatic protein degradation.     

Portal-drained viscera and hepatic fluxes of branched-chain amino acids do not account for differences in circulating branched-chain amino acids in rats fed arginine-deficient diets

Summary Concentrations and fluxes of amino acids across the portal-drained viscera (PDV) and liver were assessed in rats fed a meal of one of three arginine-deficient diets containing either alanine or the arginine precursors, ornithine or citrulline. A previous report included findings of seven arginine-related amino acids and indicated that only the citrulline-containing diet protected blood arginine concentrations. In the present report we extend these findings and note that the concentrations and fluxes of the non-arginine-related amino acids showed remarkable consistency across diet groups. However, total branched-chain amino acid (BCAA) concentrations of arterial blood were higher in rats fed the - Arg/+ Ala and the - Arg/+ Orn diets than in rats fed the control (+ Arg) diet. The elevated BCAA correlated with higher circulating concentrations of other essential amino acids but were inversely correlated with arginine concentrations. PDV and hepatic fluxes of BCAA were not different across diet groups, indicating that amino acid absorption and hepatic utilization of BCAA were generally comparable across diet groups. Hepatic concentrations of 14 of 22 measured amino acids, including total BCAA, were correlated with their arterial concentrations. The circulating concentrations and net PDV and hepatic fluxes of rats fed the control diet were comparable to our previous observations in fed rats and illustrate the role of the liver in utilization of diet-derived essential amino acids.Abbreviations PDV portal-drained viscera - BCAA branched-chain amino acids - SSA 5-sulfosalicylic acid - PBF portal blood flow - HBF hepatic blood flow - SELSM pooled standard errors of least squares means - TAA total amino acids - NEAA nonessential amino acids - EAA essential amino acids - LNAA large neutral amino acids Mention of a trade name, proprietary product or specific equipment does not constitute a guarantee by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

An integrative model of amino acid metabolism in the liver of the lactating dairy cow

The objective of this work was to construct a dynamic model of hepatic amino acid metabolism in the lactating dairy cow that could be parameterized using net flow data from in vivo experiments. The model considers 22 amino acids, ammonia, urea, and 13 energetic metabolites, and was parameterized using a steady-state balance model and two in vivo, net flow experiments conducted with mid-lactation dairy cows. Extracellular flows were derived directly from the observed data. An optimization routine was used to derive nine intracellular flows. The resulting dynamic model was found to be stable across a range of inputs suggesting that it can be perturbed and applied to other physiological states. Although nitrogen was generally in balance, leucine was in slight deficit compared to predicted needs for export protein synthesis, suggesting that an alternative source of leucine (e.g. peptides) was utilized. Simulations of varying glucagon concentrations indicated that an additional 5 mol/d of glucose could be synthesized at the reference substrate concentrations and blood flows. The increased glucose production was supported by increased removal from blood of lactate, glutamate, aspartate, alanine, asparagine, and glutamine. As glucose output increased, ketone body and acetate release increased while CO(2) release declined. The pattern of amino acids appearing in hepatic vein blood was affected by changes in amino acid concentration in portal vein blood, portal blood flow rate and glucagon concentration, with methionine and phenylalanine being the most affected of essential amino acids. Experimental evidence is insufficient to determine whether essential amino acids are affected by varying gluconeogenic demands.     

Molecular genetics of bacteriophage P22 scaffolding protein's functional domains

The assembly intermediates of the Salmonella bacteriophage P22 are well defined but the molecular interactions between the subunits that participate in its assembly are not. The first stable intermediate in the assembly of the P22 virion is the procapsid, a preformed protein shell into which the viral genome is packaged. The procapsid consists of an icosahedrally symmetric shell of 415 molecules of coat protein, a dodecameric ring of portal protein at one of the icosahedral vertices through which the DNA enters, and approximately 250 molecules of scaffolding protein in the interior. Scaffolding protein is required for assembly of the procapsid but is not present in the mature virion. In order to define regions of scaffolding protein that contribute to the different aspects of its function, truncation mutants of the scaffolding protein were expressed during infection with scaffolding deficient phage P22, and the products of assembly were analyzed. Scaffolding protein amino acids 1-20 are not essential, since a mutant missing them is able to fully complement scaffolding deficient phage. Mutants lacking 57 N-terminal amino acids support the assembly of DNA containing virion-like particles; however, these particles have at least three differences from wild-type virions: (i) a less than normal complement of the gene 16 protein, which is required for DNA injection from the virion, (ii) a fraction of the truncated scaffolding protein was retained within the virions, and (iii) the encapsidated DNA molecule is shorter than the wild-type genome. Procapsids assembled in the presence of a scaffolding protein mutant consisting of only the C-terminal 75 amino acids contained the portal protein, but procapsids assembled with the C-terminal 66 did not, suggesting portal recruitment function for the region about 75 amino acids from the C terminus. Finally, scaffolding protein amino acids 280 through 294 constitute its minimal coat protein binding site.