Vinculin Proteolysis Unmasks an ActA Homolog for Actin-based Shigella Motility

In polarized Madin-Darby canine kidney (MDCK) cells, the transferrin receptor (TR) is selectively delivered to the basolateral surface, where it internalizes transferrin via clathrin-coated pits and recycles back to the basolateral border. Mutant tailless receptors are sorted randomly in both the biosynthetic and endocytic pathways, indicating that the basolateral sorting of TR is dependent upon a signal located within the 61–amino acid cytoplasmic domain. To identify the basolateral sorting signal of TR, we have analyzed a series of mutant human TR expressed in MDCK cells. We find that residues 19–41 are sufficient for basolateral sorting from both the biosynthetic and endocytic pathways and that this is the only region of the TR cytoplasmic tail containing basolateral sorting information. The basolateral sorting signal is distinct from the YTRF internalization signal contained within this region and is not tyrosine based. Detailed functional analyses of the mutant TR indicate that residues 29–35 are the most important for basolateral sorting from the biosynthetic pathway. The structural requirements for basolateral sorting of internalized receptors from the endocytic pathway are not identical. The most striking difference is that alteration of G₃₁DNS₃₄ to YTRF impairs basolateral sorting of newly synthesized receptors from the biosynthetic pathway but not internalized receptors from the endocytic pathway. Also, mutations have been identified that selectively impair basolateral sorting of internalized TRs from the endocytic pathway without affecting basolateral sorting of newly synthesized receptors. These results imply that there are subtle differences in the recognition of the TR basolateral sorting signal by separate sorting machinery located within the biosynthetic and endocytic pathways.     

The MUC5AC gene: RFLP analysis with the Jer58 probe

We have recently obtained evidence that the locus corresponding to three groups of partial tracheobronchial cDNAs (A=Jer47, B=Jer57, C=Jer58) which mapped to chromosome 11p15 and was given the symbol MUC5 corresponds to two distinct genes which we have provisionally called MUC5B and MUC5AC. Here we describe the detection, using the Jer58 probe, which contains a 24-bp tandem repeat, of polymorphism in the MUC5AC gene with seven different restriction enzymes.     

Interaction of human alpha 1-antichymotrypsin with chymotrypsin

Human alpha 1-antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time-course studies, performed on mixtures containing an excess of native inhibitor and kept at 0 degree C or at 25 degrees C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified alpha 1-antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native alpha 1-antichymotrypsin.     

Effect of lymphatic cannula outflow height on lung microvascular permeability estimations

Estimates of the pulmonary microvascular membrane reflection coefficient (sigma) and permeability-surface area product (PS) are frequently made with the assumption that a percent change in transmicrovascular fluid flux (Jv) will be represented by an equal percent change in the lymph flow rate (QL) from a single cannulated lung lymph vessel. To test this, we measured QL in seven anesthetized dogs with the outflow end of the lymph cannula set at several heights (H) above and below the lung hilus. The left atrial pressure was then elevated to increase Jv, and QL was again measured at several H's. The percent increase in QL at elevated left atrial pressure depended on H. We used the QL data and lymph and plasma protein concentrations to estimate sigma and PS with a modified form of the Kedem and Katchalsky equations. The calculated values varied considerably with H. Our results indicate that changes in Jv are not represented by equal changes in QL. Therefore, techniques for estimating permeability that depend upon QL as an estimate of Jv may lead to erroneous estimates of sigma and PS.     

Structure determination of the glycans of human-serum alpha 1-antichymotrypsin using 1H-NMR spectroscopy and deglycosylation by N-glycanase

alpha 1-Antichymotrypsin purified from normal human serum was separated by affinity chromatography into th ree microheterogeneous forms on a concanavalin-A-Sepharose column: a pass-through (peak 1), a retarded (peak 2) and a bound form (peaks 3 + 4). For each form the asparagine-linked carbohydrate chains were liberated as oligosaccharides by hydrazinolysis, submitted to reduction with NaBH4 after re-N-acetylation and further separated by affinity chromatography on a concanavalin-A-Sepharose column. The complete primary structure of the glycans was determined by high-resolution 1H-NMR spectroscopy. The results indicated the presence of disialyl diantennary and of trisialyl triantennary type glycanic structures, the latter being accompanied by traces of disialylated triantennary oligosaccharide. The N-glycanase was used for the deglycosylation of the unfractionated alpha 1-antichymotrypsin; the successive removal of the N-linked complex-type oligosaccharide side chains of alpha 1-antichymotrypsin was studied in the presence of detergents. From these experiments it is concluded that alpha 1-antichymotrypsin carries four oligosaccharide side chains. Moreover our results show that the peak 1 contains four triantennary glycans, the peak 2 three triantennary and one diantennary glycans while the bound peaks 3 + 4 possess, on average, about one triantennary and three diantennary glycans per molecule. Since we showed that the peak 4 contains mostly diantennary glycans, it can be deduced that in peak 3 there are molecules carrying two triantennary and two diantennary glycans and others carrying one triantennary and three diantennary glycans.     

Identification of the protein responsible for hepatic system N amino acid transport activity.

In the liver, glutamine utilization may be limited by the rate of transport across the plasma membrane by the System N carrier. System N-mediated transport activity has been solubilized from rat liver plasma membrane, partially purified, and then reconstituted into proteoliposomes. To identify the System N carrier protein, monoclonal antibodies were generated against the protein fraction enriched for System N activity. Two antibodies , 3E1-2 and 1E7-3, inhibited System N activity in hepatocytes. These antibodies also immunoprecipitated System N activity from a mixture of solubilized proteins and were specific for antigen recognition in that neither immunoprecipitated System A activity. The antibody recognized a single protein of molecular size 100 kDa by immunoblot analysis. Recognition of this protein by the antibody increased in parallel with the enrichment of System N activity in solubilized membrane fractions. These data suggest that a 100-kDa plasma membrane protein mediates System N transport activity in rat hepatocytes.     

Enhancement of detection for partially methylated alditol acetates by chemical ionization mass spectrometry

Treatment of washed, intact platelets with Bolton-Hunter reagent is a satisfactory method for ¹²⁵I-labeling of many platelet proteins. Analysis by two dimensional polyacrylamide gel electrophoresis and autoradiography shows that the major platelet cytoskeletal proteins and at least four surface-exposed proteins are labeled. The method allows the identification of these labeled proteins in amounts that are below the limits of detection by Coomassie blue staining. Two granule proteins, thrombospondin and fibrinogen, are slightly labeled. Conditions of labeling do not appear to affect platelet structure or function, as assessed by phase-contrast microscopy, ⁵¹CrO₄^2? release, and aggregation in response to thrombin or fibrinogen/adenosine-5′-diphosphate.     

Oligosaccharides accumulated in the kidney of a goat with β-mannosidosis: Mass spectrometry of intact permethylated derivatives

Two oligosaccharides accumulate in the kidney of a goat with β-mannosidosis. These oligosaccharides were isolated and purified from kidney extracts by Bio-Gel P2 gel permeation column chromatography. Their structures were characterized as Manβ1 → 4GlcNAc and Manβ1 → 4G1cNAcβ1 → 4G1cNAc by mass spectrometry of the permethylated intact oligosaccharide alcohols and permethylated native oligosaccharides. Carbohydrate composition analysis, methylation linkage studies, and enzymatic hydrolysis were also performed. Stored in 1 g of kidney were 1.6 μmol of disaccharide and 7.6 μmol of trisaccharide, which was three times that found in the brain of this affected animal (M. Z. Jones and R. A. Laine, 1981, J. Biol. Chem., 256, 5181–5184). In both the brain and kidney of the affected goat, oligosaccharide accumulation was evidently represented by membrane-bound, electron-lucent vacuoles in numerous cell types. While lesions in the brain were associated with profound neurological deficits, functional impairment of the kidney was not apparent. Similar oligosaccharides excreted in urine may be derived from those stored in the kidney. The mass spectrometric methods utilized in this investigation will facilitate comparison of oligosaccharide composition in different tissues and biological samples in β-mannosidosis and other disorders of glycoprotein catabolism.     

Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato

Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH₄ at 100°C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium.