ATP-dependent proteolysis of hemoglobin alpha chains in beta-thalassemic hemolysates is ubiquitin-dependent

beta-Thalassemia is an inherited human disorder which is characterized by a deficient production of hemoglobin beta chains and an attendant accumulation of structurally normal alpha chains in the erythropoietic cells. The objective of this work is to understand the mechanism of intracellular proteolysis of these excess alpha chains. Dialyzed stroma-free hemolysates (32 mg/ml hemoglobin) of blood reticulocytes from four individuals with beta-thalassemia intermedia were incubated with human hemoglobin 3H-alpha chains (0.13 mg/ml) at 37 degrees C in a reaction mixture supporting protein degradation. In the presence of ATP and an ATP-generating system, the fraction of alpha chain 3H radioactivity made acid-soluble after 4 h ranged from 4 to 12% among the different hemolysates; in the absence of ATP or when hemolysates of normal human erythrocytes were used, only 1 to 2% of the 3H-alpha chains were degraded. It is likely that the ATP-dependent proteolysis of 3H-alpha chains in the beta-thalassemic hemolysates corresponds to the ATP-dependent turnover of newly synthesized soluble alpha chains in intact beta-thalassemic reticulocytes observed previously (Shaeffer, J. (1983) J. Biol. Chem. 258, 13172-13177) because of the following similarities between the two systems: (a) free 3H-alpha chains, but not 3H-labeled tetrameric hemoglobins, were readily degraded; (b) the rate of 3H-alpha chain proteolysis in the cell-free system was at least one-half of that observed for the turnover of newly synthesized alpha chains (t1/2 approximately 6 h) in intact cells; and (c) the ATP-dependent proteolytic activity of both systems was inhibited substantially by certain chemical agents (orthovanadate, N-ethylmaleimide, o-phenanthroline, and phenylmethylsulfonyl fluoride) but only slightly, if at all, by others (epsilon-aminocaproic acid and leupeptin). When excess human erythrocyte ubiquitin was added to the beta-thalassemic cell-free systems, a stimulation in ATP-dependent proteolysis of 3H-alpha chains ranging from 30 to 58% was observed. Conversely, addition of from 1.25 to 2.50 mg/ml affinity-purified rabbit antiubiquitin inhibited almost all (greater than 90%) of the ATP-dependent 3H-alpha chain proteolysis; in control experiments, antiubiquitin neutralized with excess ubiquitin inhibited only 13 to 30% of the total (including ubiquitin-stimulated) ATP-dependent proteolysis.(ABSTRACT TRUNCATED AT 400 WORDS)     

Structure-activity relations of the molluscan neuropeptide FMRFamide on some molluscan muscles

Analogs of the molluscan neuropeptide FMRFamide were tested on four different molluscan muscle preparations which show qualitatively different responses to the peptide; the structure-activity relations are basically similar, but not identical. The C-terminal amide and the Arg³ residue are critical for FMRFamide-like activity on all four preparations. In contrast, analogs extended at the N-terminal or with conservative substitutions for the Phe¹ or Met² residue are approximately equipotent to FMRFamide. These structural requirements parallel those for the C-terminal tetrapeptide amide of gastrin.     

Alanine metabolism in the perfused rat liver. Studies with (15)N.

We have utilized [(15)N]alanine or (15)NH(3) as metabolic tracers in order to identify sources of nitrogen for hepatic ureagenesis in a liver perfusion system. Studies were done in the presence and absence of physiologic concentrations of portal venous ammonia in order to test the hypothesis that, when the NH(4)(+):aspartate ratio is >1, increased hepatic proteolysis provides cytoplasmic aspartate in order to support ureagenesis. When 1 mm [(15)N]alanine was the sole nitrogen source, the amino group was incorporated into both nitrogens of urea and both nitrogens of glutamine. However, when studies were done with 1 mm alanine and 0.3 mm NH(4)Cl, alanine failed to provide aspartate at a rate that would have detoxified all administered ammonia. Under these circumstances, the presence of ammonia at a physiologic concentration stimulated hepatic proteolysis. In perfusions with alanine alone, approximately 400 nmol of nitrogen/min/g liver was needed to satisfy the balance between nitrogen intake and nitrogen output. When the model included alanine and NH(4)Cl, 1000 nmol of nitrogen/min/g liver were formed from an intra-hepatic source, presumably proteolysis. In this manner, the internal pool provided the cytoplasmic aspartate that allowed the liver to dispose of mitochondrial carbamyl phosphate that was rapidly produced from external ammonia. This information may be relevant to those clinical situations (renal failure, cirrhosis, starvation, low protein diet, and malignancy) when portal venous NH(4)(+) greatly exceeds the concentration of aspartate. Under these circumstances, the liver must summon internal pools of protein in order to accommodate the ammonia burden.     

Identification of a GM1-Binding Protein on the Surface of Murine Neuroblastoma Cells

S20Y murine neuroblastoma cells appear to express a protein component(s) able to adhere specifically to the oligosaccharide portion of GM1 (oligo-GM1). To identify proteins with which the oligo-GM1 becomes closely associated, a radiolabeled (125I), photoactivatable derivative of oligo-GM1 was prepared. This was accomplished by reductive amination of the glucosyl moiety of oligo-GM1 to 1-deoxy-1-aminoglucitol, followed by reaction of the amine with sulfosuccinimidyl 2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (SASD). Crosslinking studies using the photoactivatable probe indicated that it came in close proximity to a protein with an apparent molecular mass of approximately 71 kDa. In competition experiments, as little as a 10-fold molar excess of oligo-GM1 resulted in a selective reduction in labeling of this protein; preincubation with a 200-fold molar excess of siayllactose was necessary to observe the same change in the labeling pattern, lending additional support to the hypothesis that the approximately 71-kDa protein specifically associates with oligo-GM1. Cell surface location of the oligo-GM1 binding protein was confirmed using subcellular fractionation and morphological analyses.     

Hemoglobin Dallas (alpha 97(G4)Asn-->Lys): functional characterization of a high oxygen affinity natural mutant.

Hemoglobin Dallas, an alpha-chain variant with a substitution of lysine for asparagine at position 97(G4), was found to have increased oxygen affinity (p1/2 = 1 mmHg at pH 7.3 and 20 degrees C), diminished cooperativity (n, the Hill coefficient = 1.7) and reduced Bohr effect (about 50%). Addition of allosteric effectors (such as 2,3-diphosphoglycerate, inositol hexakisphosphate and bezafibrate) led to a decrease in oxygen affinity and increase in cooperative energy. Kinetic studies at pH 7.0 and 20 degrees C revealed that (i), the overall rate of oxygen dissociation is 1.4-fold slower than that for HbA and (ii), the carbon monoxide dissociation rate is unaffected. The abnormal properties of this hemoglobin variant can be attributed to a more 'relaxed' T-state.     

Circulating immune complexes in the pathogenesis of recurrent erysipelas

The dynamics of circulating immune complexes (CIC) in comparison with the level of SH-groups of serum deproteinate and other characteristics of cell-mediated and humoral immunity (the reaction of the inhibition of antibodies, the levels of T-cells and their main subpopulations) was studied in 103 erysipelas patients and in 46 persons having had the disease at the acute period of this infection and at the periods between relapses. The elevated levels of CIC and SH-groups of serum deproteinate were found to be directly correlated with the inhibition index. The study showed that, as a rule, in patients with the elevated level of CIC the frequently relapsing form of erysipelas, accompanied by the formation of relative hypersuppressor-type secondary immunodeficiency and by a decrease in the functional activity of dermal macrophages, was observed.     

Binding of lipoproteins to inert materials revisited with computer-assisted analysis

A number of studies conducted in the last decade showed that saturable ('specific') binding, by itself, does not necessarily imply biological significance. That is, biological ligands were shown to bind to inert materials as well as to biological receptors in a saturable manner. In these studies specific binding was operationally defined as binding that was displaceable by excess concentrations of unlabeled ligand. This method of measuring specific binding is now no longer considered optimal. To investigate whether optimal (computer-assisted) techniques of measuring specific binding--namely, nonlinear least-squares curve fitting of total binding data, with mathematical separation of the total binding into its various components--might ensure biological significance of measured specific binding, we studied the binding of high-density lipoproteins (HDL3) to tissue culture dishes as an example of binding without biological significance. This binding closely followed the paradigm of a ligand interacting with a class of homogeneous, saturable sites and with a class of relatively unsaturable sites, just as it would have if the HDL3 were interacting with an unpurified biological receptor. This finding indicates that computer-assisted analysis, while most accurately describing binding data, nevertheless does not ensure that measured specific binding has biological significance. Saturability is such a nonselective feature of equilibrium binding data that it should probably no longer be considered one of the criteria for deciding whether or not a defined binding site is a receptor.     

Round robin investigation of methods for recovering human enteric viruses from sludge

To select a tentative standard method for detection of viruses in sludge the American Society for Testing and Materials D19:24:04:04 Subcommittee Task Group initiated round robin comparative testing of two procedures that, after initial screening of several methodologies, were found to meet the basic criteria considered essential by the task group. Eight task group member laboratories agreed to perform round robin testing of the two candidate methods, namely, The Environmental Protection Agency or low pH-AlCl3 method and the Glass or sonication-extraction method. Five different types of sludge were tested. For each particular type of sludge, a single laboratory was designated to collect the sludge in a single sampling, make samples, and ship it to the participating laboratories. In most cases, participating laboratories completed all the tests within 48 h of sample arrival. To establish the reproducibility of the methods, each laboratory tested each sludge sample in triplicate for the two candidate virus methods. Each processed sludge sample was quantitatively assayed for viruses by the procedures of each individual round robin laboratory. To attain a more uniform standard of comparison, a sample of each processed sample from all laboratories was reassayed with one cell line and passage number by a single laboratory (Environmental Protection Agency Environmental Monitoring and Support Laboratory, Cincinnati, Ohio). When the data were statistically analyzed, the Environmental Protection Agency method was found to yield slightly higher virus recoveries for all sludge types, except the dewatered sludge. The precisions of both methods were not significantly different.(ABSTRACT TRUNCATED AT 250 WORDS)