Phosphorylation pattern of a 25 Kdalton stress protein from rat myoblasts

Phosphorylation of a 25 Kdalton nuclear stress protein from rat muscle was examined by two-dimensional gel electrophoresis and one-dimensional peptide mapping. These studies show that three 25 Kdalton stress proteins found in stressed rat myoblasts are actually the same protein with charge variation brought about by multiple phosphorylations. Furthermore, the predominant charge variant of 25 Kdalton protein found in cells is dependent on the intensity of the stress applied to cells.     

Distribution and biosynthesis of aminopeptidase N and dipeptidyl aminopeptidase IV in rat small intestine

The regional, cellular and subcellular distribution patterns of aminopeptidase N and dipeptidyl aminopeptidase IV were examined in rat small intestine. Aminopeptidase N of brush border membrane had maximal activity in the upper and middle intestine, while dipeptidyl aminopeptidase IV had a more uniform distribution profile with relatively high activity in the ileum. Along the villus and crypt cell gradient, the activity of both enzymes was maximally expressed in the mid-villus cells. However there was substantial dipeptidyl aminopeptidase IV activity in the crypt cells. Both enzymes were primarily associated with brush border membranes in all segments, however, in the proximal intestine, a significant amount of dipeptidyl aminopeptidase IV activity was associated with the cytosol fraction. The cytosol and brush border membrane forms of dipeptidyl aminopeptidase IV were immunologically identical and had the same electrophoretic mobility on disc gels. In contrast, the soluble and brush border membrane-bound forms of aminopeptidase N were immunologically distinct. When the total amount of aminopeptidase N and dipeptidyl aminopeptidase IV was determined by competitive radioimmunoassay, there were no regional or cellular differences in specific activity (enzyme activity/mg of enzyme protein) of either enzyme in brush border membrane and homogenate. The specific activity of both enzymes in a purified Golgi membrane fraction as measured by radioimmunoassay was about half that of the brush border membrane fraction. These results suggest that (1) aminopeptidase N and dipeptidyl aminopeptidase IV have different regional, cellular and subcellular distribution patterns; (2) there are enzymatically inactive forms of both enzymes present in a constant proportion to active molecules and that (3) a two-fold activation of precursor enzyme forms occurs during transfer from the Golgi membranes to the brush border membranes.     

Strategy for trapping intermediates in the folding of ribonuclease and for using 1H-nmr to determine their structures

The major unfolded form of ribonuclease A is known to show well-populated structural intermediates transiently during folding at 0°–10°C. We describe here how the exchange reaction between D₂O and peptide NH protons can be used to trap folding intermediates. The protons protected from exchange during folding can be characterized by ¹H-nmr after folding is complete. The feasibility of using ¹H-nmr to resolve a set of protected peptide protons is demonstrated by using a specially prepared sample of ribonuclease S in D₂O in which only the peptide protons of residues 7–14 are in the ¹H-form. All eight of these protected peptide protons are H-bonded. Resonance assignments made on isolated peptides containing these residues have been used to identify the protected protons. Other sets of protected protons trapped in the ¹H-form can also be isolated by differential exchange, using either ribonuclease A or S. Earlier model compound studies have indicated that H-bonded folding intermediates should be unstable in water unless stabilized by additional interactions. Nevertheless, peptides derived from ribonuclease A that contain residues 3–13 do show partial helix formation in water at low temperatures. We discuss the possibility that specific interactions between side chains can stabilize short α-helixes by nucleating the helix, and that specific interactions may also define the helix boundaries at early stages in folding.     

Influence of cholesterol content on red cell membrane viscoelasticity and fluidity.

The purpose of this investigation was to correlate the viscoelastic properties and lipid fluidity of the red blood cell membrane to its lipid composition. The viscoelastic properties of human red cells that had been enriched or depleted in cholesterol were determined by the micropipette technique. The lipid fluidity of the outer and inner leaflets of the erythrocyte membrane was concurrently assessed by steady state fluorescence depolarization. The elastic modulus and the viscosity moduli of the erythrocyte membrane showed no significant differences between the cholesterol-modified and the control cells. Cholesterol enrichment decreased the lipid fluidity of the outer membrane leaflet alone, and cholesterol depletion increased the fluidity mainly of the inner leaflet.     

A mitochondrial protein essential for the formation of the cytochrome c1-c complex. Isolation, purification, and properties

A new mitochondrial protein was isolated to pure form. This protein was indispensable for the formation of the cytochrome c1-c complex; hence, it was provisionally named the hinge protein for formation of the cytochrome c1-c complex, or for simplicity, merely called the hinge protein. The simplest method for the preparation of the pure protein involved essentially pH 5.5 treatment of high purity of "two-band" cytochrome c1 prepared from an improved method. The use of two band cytochrome c1 prepared by an improved method was preferred because the improved method apparently yielded less tight bonding between the heme-containing and colorless protein entities than that from the original methods (King, T. E. (1978) Methods Enzymol. 53, 181-191). The c1-c complex comprised 1 molar equivalent each of the hinge protein, "one-band" cytochrome c1 and cytochrome c. It was demonstrated by gel filtration chromatography that in the absence of the hinge protein, there was no complex formation between cytochromes c and one-band c1. In titration of the complex formed between one-band cytochrome c1 and cytochrome c with the hinge protein present by using the increase of the Soret-Cotton effect as a criterion (Chiang, Y. L., Kaminsky, L. S., and King, T. E. (1976) J. Biol. Chem. 251, 29-36), a sharp break was observed which showed the three species to be present in equivalent amounts. The hinge protein showed low extinction in the 280 nm region and exhibited poor color value and diffuse character of the band in sodium dodecyl sulfate-polyacrylamide gel electrophoresis after staining with Coomassie brilliant blue. The molecular weight was found to be (i) 9,800 from sedimentation equilibrium, (ii) 11,000 from sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iii) 23,000 with a Stokes radius of 22.4 A from gel filtration chromatography estimated from a standard curve with proteins of known molecular parameters. The disparities in these data from the actual value of 9,175 from calculations based on amino acid sequence, as previously reported (Wakabayashi, S., Takeda, H., Matsubara, H., Kim, C. H., and King, T. E. (1982) J. Biochem. (Tokyo) 91, 2077-2085), have been discussed.     

Yeast mannans inhibit binding and phagocytosis of zymosan by mouse peritoneal macrophages

We have examined the effects of various mannans, glycoproteins, oligosaccharides, monosaccharides, and sugar phosphates on the binding and phagocytosis of yeast cell walls (zymosan) by mouse peritoneal macrophages. A phosphonomannan (PO(4):mannose ratio = 1:8:6) from kloeckera brevis was the most potent inhibitor tested; it inhibited binding and phagocytosis by 50 percent at concentrations of approximately 3-5 μg/ml and 10 μg/ml, respectively. Removal of the phosphate from this mannan by mild acid and alkaline phosphatase treatment did not appreciably reduce its capacity to inhibit zymosan phagocytosis. The mannan from saccharomyces cerevisiae mutant LB301 inhibits phagocytosis by 50 percent at 0.3 mg/ml, and a neutral exocellular glucomannan from pichia pinus inhibited phagocytosis by 50 percent at 1 mg/ml. Cell wall mannans from wild type S. cervisiae X2180, its mnn2 mutant which contains mannan with predominantly 1(arrow)6- linked mannose residues, yeast exocellular mannans and O-phosphonomannans were less efficient inhibitors requiring concentrations of 1-5 mg/ml to achieve 50 percent reduction in phagocytosis. Horseradish peroxidase, which contains high-mannose type oligosaccharides, was also inhibitory. Mannan is a specific inhibitor of zymosan binding and phagocytosis. The binding and ingestion of zymosan but not of IgG- or complement-coated erythrocytes can be obliterated by plating macrophages on substrates coated with poly-L-lysin (PLL)-mannan. Zymosan uptake was completely abolished by trypsin treatment of the macrophages and reduced by 50-60 percent in the presence of 10 mM EGTA. Pretreatment of the macrophages with chloroquine inhibited zymosan binding and ingestion. These results support the proposal that the macrophage mannose/N-acetylglucosamine receptor (P. Stahl, J.S. Rodman, M.J. Miller, and P.H. Schlesinger, 1978, Proc. Natl. Acad. Sci. U.S.A. 75:1399-1403, mediates the phagocytosis of zymosan particles.     

A monoclonal antibody from infected mice to a Schistosoma mansoni egg proteinase

A number of monoclonal antibodies were obtained by fusion of SP2/0 myeloma cells and spleen lymphocytes from mice infected with Schistosoma mansoni. These antibodies were tested for their ability to inhibit acidic, thiol-dependent proteinases previously isolated from Schistosoma mansoni eggs and adult worms. One of the monoclonal antibodies isolated inhibits egg proteinase activity measured in vitro with the use of a low m.w. synthetic substrate. This antibody, which is an IgG1 isotype, does not appreciably inhibit an acidic, thiol-dependent proteinase obtained from the adult stage of Schistosoma mansoni. Immunocytochemical methods with the monoclonal antibody have been used to localize the egg proteinase within a set of "penetration" glands in the unhatched miracidium.     

Ethnic Elders and American Health Care—A Physician's Perspective

The aging process is a fugue composed of innumerable themes; the theme of “ethnicity” is by far one of its more dominant. Due to the increasing incidence of chronic, progressive infirmity and acute, catastrophic illness, the elderly are thrust into direct contact with the health care systems of their society. The experiences of ethnic elders in American health care situations are fraught with conflict and mutual dissatisfaction with the physician-patient relationship. Both providers and consumers of health care services harbor differing culture-bound perceptions of health, illness and the healing process; these cultural beliefs define personal and professional needs and expectations and notions of how those needs are to be met by others. Both physicians and patients can enhance their communication and their compassion for one another by acknowledgment of cultural differences and by increased willingness to interpret motives and behavior within native context.It behooves us in medicine to examine the cultural traditions underlying our own attitudes, beliefs and values about the aged in a universal sense, as well as in a culturally specific sense, that we may gain insight that will be helpful in serving elderly persons more effectively, and in solving some of the problems inherent in the aging process.     

Melting order of successively longer yeast phenylalanine-accepting transfer ribonucleic acid fragments with a common 5' end

Temperature-jump methods were used to study the kinetics of the helix to coil transition in three fragments of yeast tRNAPhe that share a common 5' terminus (the 5' end of the mature tRNA). Correlation of the extrapolated helix dissociation time constants with NMR exchange broadening results allows assignment of the structural basis of the optical melting transition in the fragments. The results confirm nuclear magnetic resonance findings on these fragments: the 5' 1/4 fragment has no helical structure; the 5' 1/2 fragment contains the D stem; and the 5' 3/5 fragment contains the D stem and the anticodon stem. These are the structures expected if sequential folding of the tRNA during biosynthesis were to occur. The D stem is the last helix to melt in the 5' 3/5 fragment. We suggest that structural elements in addition to the four Watson-Crick base pairs of the D-stem helix are responsible for the anomalously high Tm of that hairpin.