alpha-chymotrypsin and sodium-borohydride

Fourier transform infrared and UV fourth-derivative spectroscopies were used to study the secondary structure of bacteriorhodopsin and its chymotryptic and one of the sodium borohydride fragments dissolved in chloroform-methanol (1:1, v/v), 0.1 M LiClO4. The C1 fragment (helices C, D, E, F, and G) showed an alpha-helical content of about 53%, whereas C2 (helices A and B) had about 60%, and B2 (helices F and G) about 65% alpha-helix. The infrared main band indicated differences in alpha-helical properties between these fragments. These techniques were also used to obtain information on the interactions among helices. According to the results obtained from the hydrogen/deuterium exchange kinetics, about 40% of the amide protons of C2 are particularly protected against exchange, whereas for the C1 fragment this process is unexpectedly fast. UV fourth-derivative spectra of these samples were used to obtain information about the environment of Trp side chains. The results showed that the Trp residues of C2 are more shielded from the solvent than those of C1 or B2. The results of this work indicate that the specific interactions existing between the transmembrane segments induce different types of helical conformations in native bacteriorhodopsin.

Topics: Bacteriorhodopsins; Borohydrides; Chloroform; Chymotrypsin; Deuterium; Halobacterium; Hydrogen; Lithium Compounds; Methanol; Models, Molecular; Peptide Fragments; Perchlorates; Protein Structure, Secondary; Solutions; Spectrophotometry, Ultraviolet; Spectroscopy, Fourier Transform Infrared

Because of the conflicting conclusions that have been reached regarding the location of the two putative membrane-spanning segments from cysteine 911 through isoleucine 929 and from isoleucine 946 through cysteine 964 in the alpha subunit of native ovine Na+/K(+)-transporting ATPase, the disposition of lysine 943 with respect to the plane of the lipid bilayer was investigated. Sealed, right-side-out vesicles were modified with pyridoxal phosphate and Na[3H]BH4 in the presence and absence of saponin, a reagent that creates holes in the membranes. Modified alpha polypeptide was isolated, and digested with trypsin and chymotrypsin to release the desired peptides, QQGMK and QQGMK([3H]pyr)NK (where [3H]pyr designates the modification on lysine 943). These peptides, after cyclization of their amino-terminal glutamines, were isolated with an immunoadsorbent specific for the amino-terminal sequence pyroglutamyl-QGM-followed by high-pressure liquid chromatography on a C-18 reverse phase column. Comparisons were made of the extent of incorporation of radioactivity into lysine 943 between sealed vesicles and sealed vesicles pretreated with saponin. An increase in incorporation into lysine 943 of 5-fold to 18-fold was seen in vesicles pretreated with saponin prior to the modification with pyridoxal phosphate. This increase in incorporation is consistent with a cytoplasmic location for lysine 943. This conclusion places the residues on the carboxy-terminal side of the putative membrane-spanning segment from cysteine 911 through isoleucine 929 and the amino-terminal side of the putative membrane-spanning segment from isoleucine 946 through cysteine 964 in the ovine alpha subunit on the cytoplasmic side of the membrane.

Topics: Amino Acid Sequence; Animals; Borohydrides; Cell Membrane; Chromatography, High Pressure Liquid; Chymotrypsin; Immunosorbent Techniques; Indicators and Reagents; Isoleucine; Kidney Medulla; Lipid Bilayers; Lysine; Macromolecular Substances; Membrane Lipids; Molecular Sequence Data; Oligopeptides; Peptide Fragments; Pyridoxal Phosphate; Saponins; Sheep; Sodium-Potassium-Exchanging ATPase; Trypsin

A Mr = 32,000 membrane glycoprotein can be uniquely labeled by galactose oxidase/[3H]sodium borohydride on rat caudal, but not caput, epididymal sperm. It has been suggested that this protein is related to a Mr = 32,000 galactose oxidase-sensitive glycoprotein present in rat caudal epididymal fluid. The tritiated membrane glycoprotein was solubilized and its hydrodynamic properties were determined by conventional gel filtration, high performance gel filtration, sedimentation rate determination in linear sucrose gradients prepared in H2O and D2O, and equilibrium isopycnic centrifugations in CsCl. The Stokes radius and sedimentation coefficient were 4.87 +/- 0.07 nm and 1.73 +/- 0.08 S, respectively. The sedimentation profile in CsCl gradients was asymmetric with a major peak occurring at a density of 1.081 g/cm3 (v = 0.92 cm3/g) and a shoulder at 1.108 g/cm3 (v = 0.90 cm3/g). The glycoprotein did not enter a 5 to 20% linear sucrose gradient prepared in D2O and could be extracted from the intact sperm into acidic chloroform:methanol solutions. These data are consistent with a protein which binds substantial amounts of detergent and/or lipid and has exposed hydrophobic regions. Two-dimensional gel electrophoresis indicated that the membrane protein exhibits charge heterogeneity, with the major components having pI values of 5.4 and 4.9. The fluid glycoprotein was monodisperse on two-dimensional gel electrophoresis having a pI of 3.8. Binding studies failed to demonstrate specific binding of the Mr = 32,000 caudal fluid glycoprotein to caput cells. Moreover, "Western blots" of electrophoretically resolved caput and caudal fluid proteins, followed by immunolabeling with antibodies raised against unfractionated caudal fluid, demonstrated the presence of a Mr = 32,000 protein in caudal fluid which was absent from caput epididymal fluid. Using the same technique, it was shown that antibodies raised against caudal fluid proteins did not cross-react with a Mr = 32,000 caudal membrane glycoprotein. Our data do not support the view that the Mr = 32,000 fluid and membrane proteins are identical.

Topics: Animals; Borohydrides; Cell Membrane; Chymotrypsin; Epididymis; Galactose Oxidase; Glycoproteins; Male; Molecular Weight; Rats; Rats, Inbred Strains; Spermatozoa; Trypsin

We report a study of HEMPAS erythrocyte membrane glycoproteins in relation to proteolytic digestion and surface labelling with galactose-oxidase/NaB[3H]4. The proteolytic digestion of band 3, the major intrinsic glycoprotein of the human erythrocyte membrane, reveals an abnormality in the outer glycosylated segment of this protein. 3H incorporation in band 3 and band 4.5 glycoproteins after treatment with galactose-oxidase/NaB[3H]4 is reduced in HEMPAS red cells suggesting a defective glycosylation of these proteins. These findings together with the persistence of i antigen and the normal presence of I antigen lead us to conclude that erythroblastic membrane features may persist in HEMPAS erythrocytes.

Topics: Adult; Anemia, Dyserythropoietic, Congenital; Anemia, Hemolytic, Congenital; Borohydrides; Child; Chymotrypsin; Electrophoresis, Polyacrylamide Gel; Erythrocyte Membrane; Erythrocytes; Galactose Oxidase; Glycoproteins; Humans; Tritium; Trypsin

Spontaneous rosette formation (SRF) of pig and guinea pig thymocytes (Th) and thymus derived peripheral blood lymphocytes (PTL) was tested under different experimental conditions. Treatment with different proteases (trypsin, chymotrypsin, protease type VII) showed that SRF of pig Th and PTL is inhibited to a much higher extent than that of guinea pig Th and PTL. Cytochalasin B (CB) inhibited SRF of PTL of both species to a higher degree as compared to Th. Different carbohydrates neither influenced SRF of Th nor of PTL in both species indicating that simple carbohydrates are not the recognized component of SRF receptors. Treatment with oxidising and reducing agents did not influence SRF.

Topics: Animals; Borohydrides; Chymotrypsin; Cytochalasin B; Guinea Pigs; Neuraminidase; Peptide Hydrolases; Periodic Acid; Rosette Formation; Species Specificity; Swine; T-Lymphocytes; Thymus Gland; Trypsin

Topics: Borohydrides; Boron; Chymotrypsin; Chymotrypsinogen; Disulfides; Sulfhydryl Compounds