Proton translocation by cytochrome oxidase vesicles catalyzing the peroxidatic oxidation of ferrocytochrome c

Coupled with the peroxidatic oxidation of ferrocytochrome c under anaerobic conditions, proteoliposomes reconstituted with a purified preparation of bovine heart cytochrome oxidase ejected protons into the external medium with an apparent H+/e- ratio of 0.9. At the same time, protons in the intravesicular space were consumed. Dicyclohexylcarbodiimide significantly inhibited the proton translocation. Cyanide (0.14 mM) completely inhibited both the peroxidase and proton translocating activities. On the contrary, in the presence of 1 mM CO the proton ejection was abolished almost completely, but 50% of the peroxidase activity persisted. This result suggests the operation of multiple mechanisms in the peroxidase reaction and that the CO-sensitive one is coupled to the proton translocation.     

Purified protein kinase C phosphorylates microtubule-associated protein 2

We have investigated actions of purified protein kinase C on microtubule- and microfilament-related proteins. Among the cytoskeletal proteins examined, microtubule-associated protein 2 (MAP2) was found to serve as a good substrate. Other cytoskeletal proteins, tubulin, fodrin, cofilin, tropomyosin, and 53,000-Da protein, were very poorly phosphorylated. The amino acid residues of MAP2 that were phosphorylated by the protein kinase C were almost exclusively serine. The peptide mapping analysis indicated that protein kinase C and cAMP-dependent protein kinase phosphorylate MAP2 differently. The ability of MAP2 to interact with actin was markedly reduced by this protein kinase C-mediated phosphorylation. These data raise the possibility that phosphorylation of MAP2 by activated protein kinase C may be involved in cell-surface signal transduction.     

A kinetic description of antifreeze glycoprotein activity

The antifreeze glycoproteins (AFGP) of polar fish have the ability to depress the freezing temperature of water approximately 500 times the amount expected based on the number of AFGP molecules in solution; yet AFGP solutions have a purely colligative melting point depression. The difference of solution melting and freezing temperatures is the antifreeze activity of AFGP. One characteristic of AFGP activity that requires further examination is the effect of concentration on antifreeze activity, especially whether the activity saturates at high concentrations or the measured activity increases ad infinitum. This study first surveys the activity of the various antifreeze components from both Pagothenia borchgrevinki and the Arg-containing antifreeze glycoprotein from Eleginus gracilis (EgAF). It was found that all AFGP components examined have a plateau in activity at high concentration, but the actual value of the plateau activity differs between the different length AFGP components and between AFGP and EgAF. While the low molecular weight components of both AFGP and EgAF lose activity at deep supercooling, at high concentration activity is restored. The activity data is then shown to fit a reversible kinetic model of AFGP activity, and the coefficients obtained are used to compare the activity differences between AFGP components and between AFGP and EgAF. The model is also shown to describe the activity of the antifreeze protein of the fish Pseudopleuronectes americanus and the thermal hysteresis protein of the insect, Tenebrio molitor.     

Selective oxidation and reduction of methionine residues in peptides and proteins by oxygen exchange between sulfoxide and sulfide

Treatment of amino acids, peptides, and proteins with aqueous solution of dimethyl sulfoxide (Me2SO) and hydrochloric acid (HCl) resulted in the oxidation of methionine to methionine sulfoxide. In addition to methionine, SH groups are also oxidized, but this reaction proceeds after a lag period of 2 h. Other amino acids are not modified by aqueous Me2SO/HCl. The reaction is strongly pH-dependent. Optimal conditions are 1.0 M HCl, 0.1 M Me2SO, at 22 degrees C. The reaction exhibits pseudo-first order kinetics with Kobs = 0.23 +/- 0.015 M-1 min-1 at 22 degrees C. Incubation of methionine sulfoxide with dimethyl sulfide and HCl resulted in the conversion of methionine sulfoxide to methionine. This reaction is fast (t1/2 = 4 min at room temperature) and quantitative at relatively anhydrous condition (i.e. at H2O:concentrated HCl:dimethyl sulfide ratio of 2:20:1). Quantitative conversions of methionine sulfoxide back to methionine are obtained in peptides and proteins as well, with no observable other side reactions in amino acids and proteins. The wide applications of this selective oxidation and reduction of methionine residues are demonstrated and discussed.     

Alpha 1 type IV collagen gene evolved differently from fibrillar collagen genes

Type IV collagen is a major structural component in basement membranes. It is considerably different from the fibrillar collagens, types I-III. For example, unlike fibrillar collagens, the triple helical domain of type IV collagen is frequently interrupted by nonhelical regions. In this report, we demonstrate several overlapping genomic clones which cover most of the mouse alpha 1(IV) chain. Electron microscopic analysis of R-loops revealed that there were at least 28 exons within 35 kilobases of the gene segment. The sizes of six exons were determined by DNA sequence analysis to be 81, 178, 134, 73, 129, and 213 base pairs. These sizes do not appear to be related to the 54-base pair coding unit which is characteristic of fibrillar collagen exons, suggesting that the alpha 1 type IV collagen gene evolved differently from the fibrillar collagen genes.     

Large complex globular domains of type VII procollagen contribute to the structure of anchoring fibrils

Type VII collagen, in the form of an antiparallel dimer, is a major protein component of anchoring fibrils. The ultrastructural appearance of these fibrils suggests that they may serve to anchor the basement membrane zone to the underlying connective tissue matrix. We report here the identification and initial characterization of Type VII procollagen, recovered from the media of epidermoid carcinoma cell cultures. Immunoblotting using monospecific antibodies to Type VII procollagen identifies a single, homogeneous band of at least Mr 320,000 following disulfide bond reduction. This chain contains 170 kDa of collagen triple helix and 150 kDa of non-helical domain at the carboxyl terminus. Pepsin digestion of this material yields Type VII collagen identical to that isolated from whole tissue and a series of quasi-stable peptides derived from the carboxyl-terminal region. Cell extracts contain procollagen chains identical in size to those secreted into the media. There is no evidence for processing of this material in cell culture. Partial purification by velocity sedimentation and transmission electron microscopic observation following rotary shadowing reveals both monomers (426 nm) and dimers (785 nm). Dimers are antiparallel and interact through 60-nm overlap, with amino-terminal globular domains present at the ends of the overlap. The multi-domain carboxyl-terminal region appears as three similar arms originating from a centralized globular region adjacent to the collagen helix. The carboxyl globular domain is present in whole tissue and may participate in the unique fibril form of this collagen. The amino-terminal globule may function in the antiparallel assembly of dimers.     

Developmental appearance of matrix GLA protein during calcification in the rat

A marked dissociation has been observed between the timed accumulation in calcified tissues of two related vitamin K-dependent proteins, bone Gla protein (BGP) and the recently discovered matrix Gla protein (MGP). In long bone diaphyses, total levels of MGP were essentially equivalent in newborn, juvenile, and adult rats. In agreement with previous studies, BGP levels were only 5% of adult levels in newborn rat bones and increased to 90% of adult levels by 19 days of age. Similar results were obtained from the analysis of the longitudinal distribution of MGP and BGP in 14-day-old rat tibia, a bone in which new mineral is added rapidly at both growth plates. Again, MGP was essentially at the same level in the regions nearest the growth plates as in the midshaft while BGP levels were 10-fold lower in the regions nearest the growth plates. These differences in the timed accumulation of MGP and BGP in calcifying tissues indicate that MGP could function earlier in bone formation than does BGP. To further characterize the MGP antigen in bone, extracts from newborn and adult rat bones were chromatographed by gel filtration over Sephacryl S-200. All of the antigen extracted by formic acid and most of the antigen subsequently extracted by guanidine HCI emerged at the position expected for the 79-residue MGP. There was a significant difference in the fraction of total MGP which was extracted by guanidine HCI in newborn (50%) and adult (20%) bone. The radioimmunoassay for rat MGP which was developed for these studies employs rabbit antibody directed against calf MGP and rat MGP for standards and radioiodinated tracer. This assay has a sensitivity of 0.1 ng and does not detect rat or calf BGP.     

Reactivity of a sulfhydryl group of the ras oncogene product p21 modulated by GTP binding

We have studied the sensitivity of sulfhydryl groups of a highly purified p21 protein of the v-rasH oncogene to a thiol-specific reagent, N-ethylmaleimide (NEM). Approximately 70% of GTP binding and autokinase activities of p21 were inactivated by NEM, and excessive amounts of GTP or GDP protected p21 activities. Thiol titration revealed the presence of one fast reactive cysteine residue, the susceptibility of which is modulated by GTP binding. A total of 4 and 6 residues, respectively, became titratable upon denaturation and reduction, suggesting the presence of a disulfide bond. This GTP-modulated sulfhydryl group was identified as Cys-80 in the following tryptic peptide sequence: NH2-Thr-Gly-Glu-Gly-Phe-Leu-Cys-Val-Phe-Ala-Ile-Asn-Asn-Thr-Lys-COOH. This is based on the comparative tryptic peptide mapping of [14C]NEM-modified p21 in the presence and absence of GTP. The GTP-modulated peptide co-chromatographed with a synthetic peptide of the predicted sequence. Amino acid analysis of the purified [14C]NEM-modified peptide from tryptic digests of p21 also confirmed its identity. This region of p21 shares an extensive sequence homology with various G-proteins and appears to be in the vicinity of the GTP-binding domain of these proteins.     

Effect of depletion of bicarbonate or phosphate ions on insulin action in rat adipocytes. Further characterization of the receptor-effector system

In the preceding paper (Shechter, Y., and Ron, A. (1986) J. Biol. Chem. 261, 14945-14950) we have shown that in fat cells, prepared and maintained in an isotonic buffer (pH 7.4) containing neither phosphate nor bicarbonate anions (Buffer A), the dose-response curve to insulin shifted to the right by about 2 logarithms and insulin binding affinity or capacity was only slightly decreased. In the current paper we demonstrate that progressive loss of insulin binding, either by treatment with trypsin or preincubating the cells with isoproterenol, correlates well with the reduced ability of the cells to elicit maximal lipogenesis in response to insulin. We further demonstrate in the "new" system that: the dissociation of labeled insulin from fat cells is not accelerated by the inclusion of unlabeled insulin in the medium; termination of lipogenesis in Buffer A occurs immediately; ligand-induced receptor internalization is grossly defective; and insulin is unable to stimulate lipogenesis at 15 degrees C. The data support the hypothesis that in the new experimental system all measurable binding sites are linked to a coupling mechanism. Each site behaves as an independent, separate entity and there are no site to site interactions. This leads to a linear relationship between binding and bioactivation, lack of negative or positive cooperatively, accelerated rate of termination, defective internalization, a shift to the right in the dose-response curve to insulin, and a lack of insulin response at a lower temperature. In more general terms, the study indicates that all measurable insulin receptors are chemically homogeneous in their potential capability to be coupled to an insulin effector (biologically relevant) system, and they do so under particular experimental conditions.