Phosphorylation of laminin-1 by protein kinase C

Several extracellular proteins have been reported to be phosphorylated. Previous studies of our laboratory indicated that laminin-1 can be phosphorylated by protein kinase A (PKA). Moreover, it has been reported that protein kinase C (PKC), although known to be intracellular, can phosphorylate extracellular proteins in the case of cellular damage and/or platelet activation. In the present study we examined the possibility of laminin-1 serving as a substrate of PKC. Amino acid analysis revealed that laminin-1 is phosphorylated by this enzyme on serine residues. Self assembly, heparin binding, and cell attachment on the phosphorylated molecule were then studied. Phosphorylated laminin-1 showed an increased and more rapid self assembly than the non-phosphorylated molecule. Heparin binding and cell attachment experiments indicated enhanced heparin and cell binding capacity of the phosphorylated molecule in comparison to the non- phosphorylated control. These results indicate that laminin-1 can be phosphorylated by protein kinase C. Furthermore, phosphorylation by protein kinase C seems to alter several properties of the molecule, though, the in vivo significance of this phenomenon remains to be studied.     

Nitric-oxide reductase. Structure and properties of the catalytic site from resonance Raman scattering

We have applied resonance Raman spectroscopy to investigate the properties of the dinuclear center of oxidized, reduced, and NO-bound nitric-oxide reductase from Paracoccus denitrificans. The spectra of the oxidized enzyme show two distinct nu(as)(Fe-O-Fe) modes at 815 and 833 cm(-1) of the heme/non-heme diiron center. The splitting of the Fe-O-Fe mode suggests that two different conformations (open and closed) are present in the catalytic site of the enzyme. We find evidence from deuterium exchange experiments that in the dominant conformation (833 cm(-1) mode, closed), the Fe-O-Fe unit is hydrogen-bonded to distal residue(s). The ferric nitrosyl complex of nitric-oxide reductase exhibits the nu(Fe(3+)-NO) and nu(N-O) at 594 and 1904 cm(-1), respectively. The nitrosyl species we detect is photolabile and can be photolyzed to generate a new form of oxidized enzyme in which the proximal histidine is ligated to heme b(3), in contrast to the resting form. Photodissociation of the NO ligand yields a five-coordinate high-spin heme b(3). Based on the findings reported here, the structure and properties of the dinuclear center of nitric- oxide reductase in the oxidized, reduced, and NO-bound form as well as its photoproduct can be described with certainty.     

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR spectroscopies reveal a unique active site in cytochrome caa3 oxidase from Thermus thermophilus

Fourier transform infrared (FTIR) and step-scan time-resolved FTIR difference spectra are reported for the [carbonmonoxy]cytochrome caa(3) from Thermus thermophilus. A major C-O mode of heme a(3) at 1958 cm(-1) and two minor modes at 1967 and 1975 cm(-1) (7:1:1) have been identified at room temperature and remained unchanged in H(2)O/D(2)O exchange. The observed C-O frequencies are 10 cm(-1) higher than those obtained previously at 21 K (Einarsdóttir, O., Killough, P. M., Fee, J. A., and Woodruff, W. H. (1989) J. Biol. Chem. 264, 2405-2408). The time-resolved FTIR data indicate that the transient Cu(B)(1+)-CO complex is formed at room temperature as revealed by the CO stretching mode at 2062 cm(-1). Therefore, the caa(3) enzyme is the only documented member of the heme-copper superfamily whose binuclear center consists of an a(3)-type heme of a beta-form and a Cu(B) atom of an alpha-form. These results illustrate that the properties of the binuclear center in other oxidases resulting in the alpha-form are not required for enzymatic activity. Dissociation of the transient Cu(B)(1+)-CO complex is biphasic. The rate of decay is 2.3 x 10(4) s(-1) (fast phase, 35%) and 36.3 s(-1) (slow phase, 65%). The observed rate of rebinding to heme a(3) is 34.1 s(-1). The implications of these results with respect to the molecular motions that are general to the photodynamics of the binuclear center in heme-copper oxidases are discussed.     

Proteome Analysis of Enriched Heterocysts from Two Hydrogenase Mutants from Anabaena sp. PCC 7120

Cyanobacteria are oxygenic photosynthetic prokaryotes and play a crucial role in the Earth's carbon and nitrogen cycles. The photoautotrophic cyanobacterium Anabaena sp. PCC 7120 has the ability to fix atmospheric nitrogen in heterocysts and produce hydrogen as a byproduct through a nitrogenase. In order to improve hydrogen production, mutants from Anabaena sp. PCC 7120 are constructed by inactivation of the uptake hydrogenase (ΔhupL) and the bidirectional hydrogenase (ΔhoxH) in previous studies. Here the proteomic differences of enriched heterocysts between these mutants cultured in N₂‐fixing conditions are investigated. Using a label‐free quantitative proteomics approach, a total of 2728 proteins are identified and it is found that 79 proteins are differentially expressed in the ΔhupL and 117 proteins in the ΔhoxH variant. The results provide for the first time comprehensive information on proteome regulation of the uptake hydrogenase and the bidirectional hydrogenase, as well as systematic data on the hydrogen related metabolism in Anabaena sp. PCC 7120.     

Protein Dynamics of the Sensor Protein HemAT as Probed by Time-Resolved Step-Scan FTIR Spectroscopy

The heme-based aerotactic transducer (HemAT) is an oxygen-sensor protein consisting of a sensor and a signaling domain in the N- and C-terminal regions, respectively. Time-resolved step-scan FTIR spectroscopy was employed to characterize protein intermediate states obtained by photolysis of the carbon monoxide complexes of sensor-domain, full-length HemAT, and the Y70F (B-helix), L92A (E-helix), T95A (E-helix), and Y133F (G-helix) HemAT mutants. We assign the spectral components to discrete substructures, which originate from a helical structure that is solvated (1638 cm^?1) and a native helix that is protected from solvation by interhelix tertiary interactions (1654 cm^?1). The full-length protein is characterized by an additional amide I absorbance at 1661 cm^?1, which is attributed to disordered structure suggesting that further protein conformational changes occur in the presence of the signaling domain in the full-length protein. The kinetics monitored within the amide I absorbance of the polypeptide backbone in the sensor domain exhibit two distinct relaxation phases (t₁ = 24 and t₂ = 694 μs), whereas that of the full-length protein exhibits monophasic behavior for all substructures in a time range of t = 1253–2090 μs. These observations can be instrumental in monitoring helix motion and the role of specific mutants in controlling the dynamics in the communication pathway from the sensor to the signaling domain. The kinetics observed for the amide I relaxation for the full-length protein indicate that the discrete substructures within full-length HemAT, unlike those of the sensor domain, relax independently.     

The Protein Effect in the Structure of Two Ferryl-Oxo Intermediates at the Same Oxidation Level in the Heme Copper Binuclear Center of Cytochrome c Oxidase

Identification of the intermediates and determination of their structures in the reduction of dioxygen to water by cytochrome c oxidase (CcO) are particularly important to understanding both O₂ activation and proton pumping by the enzyme. In this work, we report the products of the rapid reaction of O₂ with the mixed valence form (Cu_A²⁺, heme a³⁺, heme a₃²⁺-Cu_B¹⁺) of the enzyme. The resonance Raman results show the formation of two ferryl-oxo species with characteristic Fe(IV)=O stretching modes at 790 and 804 cm⁻¹ at the peroxy oxidation level (P_M). Density functional theory calculations show that the protein environment of the proximal H-bonded His-411 determines the strength of the distal Fe(IV)=O bond. In contrast to previous proposals, the P_M intermediate is also formed in the reaction of Y167F with O₂. These results suggest that in the fully reduced enzyme, the proton pumping ν_Fe(IV)=O = 804 cm⁻¹ to ν_Fe(IV)=O = 790 cm⁻¹ transition (P→F, where P is peroxy and F is ferryl) is triggered not only by electron transfer from heme a to heme a₃ but also by the formation of the H-bonded form of the His-411-Fe(IV)=O conformer in the proximal site of heme a₃. The implications of these results with respect to the role of an O=Fe(IV)-His-411-H-bonded form to the ring A propionate of heme a₃-Asp-399-H₂O site and, thus, to the exit/output proton channel (H₂O) pool during the proton pumping P→F transition are discussed. We propose that the environment proximal to the heme a₃ controls the spectroscopic properties of the ferryl intermediates in cytochrome oxidases.     

The origin of the FeIV = O intermediates in cytochrome aa3 oxidase

The dioxygen reduction mechanism in cytochrome oxidases relies on proton control of the electron transfer events that drive the process. Proton delivery and proton channels in the protein that are relevant to substrate reduction and proton pumping are considered, and the current status of this area is summarized. We propose a mechanism in which the coupling of the oxygen reduction chemistry to proton translocation (P → F transition) is related to the properties of two groups of highly conserved residues, namely, His411/G386-T389 and the heme a₃–propionateA–D399–H403 chain. This article is part of a Special Issue entitled: Respiratory Oxidases.     

The pathogenetic influence of I-parathyroid hormone on slipped capital femoral epiphysis. Towards a new etiologic approach?

The displacement of the femoral head along the upper femoral physis that occurs during adolescence or slipped capital femoral epiphysis (SCFE) is not a very common traumatic entity. Ever since Muller1 first described it in 1888, its symptoms, clinical manifestations, diagnosis, treatment and complications have been thoroughly described and studied. Nevertheless little progress has been accomplished as far as its etiology is concerned. In order to assess the potential pathologic influence of any parathyroid hormone (PTH) disturbances on the development of SCFE, we conducted a prospective clinical study with 14 patients, 7 boys and 7 girls (16 hips), suffering from SCFE (Group A). Another 5 patients who had been treated for SCFE a few years before the study, were used as a control group (Group B). We measured the level of I-PTH along with serum calcium (Ca) and phosphorus (P) levels. Furthermore, we checked all the necessary anthropometric characteristics of the patients (i.e., age, height, weight and sexual maturation). Each patient of Group A was categorized from grade I to grade V according to the progress of the slipping. The results showed an increased incidence of serum PTH level abnormalities (both decrease and increase) in Group A while Group B patients had normal results. The detected I-PTH serum level abnormalities were not in any pattern related to the Ca and P serum levels. We believe that a temporary parathyroid hormone disorder during the early years of adolescence may play a potentially significant role (along with other etiologic factors) in the development of SCFE.     

Severe total hepatic ischemia and reperfusion: relationship between very high alpha-tocopherol uptake and lipid peroxidation.

Reperfusion injury of the liver occurs in liver transplantation and in major hepatectomies. It triggers a severe oxidative stress that leads to increased lipid peroxidation. In our study we examined the effect of parenteral supranutritional administration of alpha-tocopherol, a vitamin that plays a key role in the endogenous antioxidant system, to rats subjected to severe ischemia/reperfusion (I/R) injury of the liver. alpha-Tocopherol was administered to the animals at doses of 30 and 300 mg/kg bw, whereas total hepatic ischemia was induced for 60 min followed by 120 min reperfusion. Tissue and blood samples were collected for malonyldialdehyde (MDA) and serum alpha-tocopherol assay, respectively. In the sham operation group, mean MDA level in liver was 1.14 nmole/g wet tissue in the control subgroup, and 1.01 or 0.74 nmole/g wet tissue in the subgroups given 30 or 300 mg/kg alpha-tocopherol. In the I/R group, mean MDA level was 1.57 nmole/g wet tissue in the control subgroup, and 0.97 and 0.77 nmole/g wet tissue in the subgroups given 30 or 300 mg/kg alpha-tocopherol. Mean levels of alpha-tocopherol in serum (mumole/l) were 10.20 and 1.80 in the control subgroups, 25.28 and 11.25 in the subgroups treated with 30 and 300 mg/kg bw of alpha-tocopherol, and 31.00 and 13.02 in the subgroups treated with 30 and 300 mg/kg bw of alpha-tocopherol, within the sham-operation and I/R groups, respectively. A significant decrease of MDA accompanied by a significant increase of serum alpha-tocopherol was documented in the alpha-tocopherol-treated rats within both groups. Ischemia/reperfusion triggered a significant increase of the MDA level in the liver of the rats not treated with alpha-tocopherol as compares with the treated animals.