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PubMed1987

Biosynthesis and secretion of functional protein S by a human megakaryoblastic cell line (MEG-01).

A human megakaryoblastic cell line (MEG-01) was investigated for the presence of protein S in culture medium and cell lysates using a specific enzyme-linked immunoassay (ELISA) and a functional assay. When 5 X 10(5) MEG-01 cells/mL was subcultured in RPMI 1640 medium with 10% fetal calf serum (FCS), the concentration of protein S antigen in the culture medium increased progressively with time from less than 8 ng/mL on day 0 to 105.6 +/- 6.0 ng/mL on day 13. Vitamin K2(1 microgram/mL) increased the production of functional protein S, whereas warfarin (1 microgram/mL) profoundly decreased the quantity and the specific activity of secreted protein S. By an indirect immunofluorescent technique, protein S antigen was detected in both MEG-01 cells and human bone marrow megakaryocytes. Immunoblot analysis of culture medium revealed two distinct bands (mol wt 84,000 and 78,000) that are identical to the doublets of purified plasma protein S. De novo synthesis of protein S was demonstrated by the presence of specific immunoprecipitable radioactivity in the medium after 5 hours of labeling of the cells with [35S]-methionine as a 84,000 mol wt protein. Plasma protein S levels of nine patients with severe aplastic anemia were not significantly different from those of normal controls. These results suggest that megakaryocytes produce functional protein S and contain the enzymes required for the carboxylation of selected glutamic acid residues, and that protein S synthesized by megakaryocytes does not represent a main source of plasma protein S.

Anemia, Aplastic↗

PubMed1992

Molecular cloning of a cDNA encoding chicken T-protein of the glycine cleavage system and expression of the functional protein in Escherichia coli. Effect of mRNA secondary structure in the translational initiation region on expression.

DNA clones encoding chicken T-protein of the glycine cleavage system were isolated from chicken liver lambda gt10 cDNA libraries. Three overlapping clones provided an open reading frame of 1176 nucleotides that predicts a polypeptide of 392 amino acids (M(r) 42,056) comprised of a 16-residue mitochondrial targeting sequence and a 376-residue mature protein (M(r) 40,292). The amino acid sequence predicted for the mature protein showed 67% identity with that of bovine T-protein. A cDNA encoding mature T-protein was constructed, and the nucleotide sequence just downstream of the initiation codon was modified without amino acid substitution to reduce the free energy of formation for the folded mRNA. Expression plasmids containing these cDNA variants produced large amounts of T-protein in Escherichia coli, while very low expression was observed with a plasmid containing wild type cDNA. Enzymatically active T-protein was obtained when the expression was conducted at 30 degrees C with 25 microM isopropyl-1-thio-beta-D-galactopyranoside. Under the full inducing condition (at 37 degrees C and 1 mM inducer), the expressed T-protein was recovered as insoluble and inactive protein. The recombinant T-protein was purified to near homogeneity with a yield of about 30%. Apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis is approximately 40,000, similar to the size of T-protein purified from chicken liver. NH2-terminal amino acid sequence analysis (9 residues) revealed 100% identity with chicken T-protein determined chemically. The kinetic properties of the recombinant T-protein resembled those of the native chicken T-protein.

Amino Acid Sequence↗

PubMed1991

Renal function, protein excretion and pathology of Balkan endemic nephropathy. I. Renal function.

Renal function studies were performed on 59 patients who had the clinical criteria for Balkan endemic nephropathy (BEN). They were divided into three groups according to DTPA clearance (DTPA). Group 1, 11 individuals, had a mean age of 41.6 years and DTPA greater than 100 ml/min. Group 2, 20 persons, had a mean age of 49 years and DTPA of 60 to 100 ml/min. Group 3 was made up of 28 people with a DTPA less than 60 ml/min and an average age of 50.4 years. No distinguishing specific or characteristic symptoms of BEN were found in any of the three groups. Anemia was not found to be an early indicator when compared to other forms of progressive renal disease. Proteinuria was minimal and intermittent in all three groups. Maximum concentrating ability was significantly reduced only in the third group. These features do not allow the clinical differentiation of BEN from other chronic progress tubulointerstitial nephropathies. The geographic isolation and familial nature of the disease, associated with minimal proteinuria make BEN a unique entity. Kidney size by ultrasound was decreased in all three groups, suggesting that this may be another early and characteristic feature to BEN.

Adult↗

PubMed1991

G protein function in the ischaemic myocardium.

The activity of adenylyl cyclase (AC) is controlled by its interaction with receptor-regulated G proteins. The efficiency to form cyclic AMP is strongly influenced by the amount, the subspecies and function of these regulatory proteins. An impairment of AC function has been shown to occur in sarcolemmal preparations (SL) of hearts exposed to either local or global ischaemia. To examine the contribution of G protein function to this phenomenon, cholera toxin (CT)-catalysed ADP-ribosylation of Gs and pertussis toxin (PT)-catalysed ADP-ribosylation of G proteins have been investigated in SL of porcine hearts exposed to global ischaemia for 15-45 min. ADP-ribosylation by CT of an approximately 45 kDa polypeptide was 0.46 +/- 0.06 and ADP-ribosylation by PT of three 39-41 kDa polypeptides was 4.77 +/- 0.77 pmol mg-1 protein in SL of non-ischaemic myocardium. Whereas no change was observed in CT-catalyzed ribosylation after 30 min of ischaemia, there was a reduction in PT-catalyzed ADP-ribosylation to 3.7 +/- 0.35 pmol mg-1 protein after 30 min of ischaemia. Prolongation of ischaemia to 45 min did not reduce further ADP-ribosylation capacity. Quantitative immunoblotting of PT-sensitive G proteins suggests that the diminution of ADP-ribosylation occurred because of a loss of alpha-subunits of G0, Gi-1, and Gi-2 from sarcolemmal membranes.

Adenosine Diphosphate Ribose↗

PubMed1990

Chaperonin assisted polypeptide folding and assembly: implications for the production of functional proteins in bacteria.

Production of biologically active foreign proteins with correct three-dimensional structures is often difficult in bacteria. Recent advances demonstrate that, for some proteins at least, their correct folding and assembly is facilitated by a class of proteins known as molecular chaperones. An understanding of the function of molecular chaperones may assist in the synthesis in bacteria of functional foreign proteins produced by recombinant techniques.

Bacteria↗

PubMed1992

Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.

The retinoblastoma susceptibility gene (RB) product, the retinoblastoma protein (pRb), functions as a regulator of cell proliferation. Introduction of the RB gene into SAOS-2 osteosarcoma cells, which lack functional pRb, prevents cell cycle progression. Such growth-suppressive functions can be modulated by phosphorylation of pRb, which occurs via cell cycle-regulated kinases. We show that constitutively expressed cyclins A and E can overcome pRb-mediated suppression of proliferation. pRb becomes hyperphosphorylated in cells overexpressing these cyclins, and this phosphorylation is essential for cyclin A- and cyclin E-mediated rescue of pRb-blocked cells. This suggests that G1 and S phase cyclins can act as regulators of pRb function in the cell cycle by promoting pRb phosphorylation.

Cell Nucleus↗

PubMed1991

Frequencies of codons in histones, tubulins and fibrinogen: bias due to interference between transcription signals and protein function.

The distribution of codons was studied in 65 proteins: 48 histones, 14 tubulins, and three fibrinogens, With the methodology used, (1) we confirmed that the preterminator state of a codon has no detectable effect on codon bias. (2) The well-known effect of CG suppression was visible. We also found that (3) some codons which are very rare, are equal to parts of known transcription signals. Thus, we advanced that to avoid signal interference, the use of these codons is suppressed when a synonymous codon is available. In addition we found that in the whole series of codons, transcription signals are less frequent than in a random sequence of equal composition. Finally we observed (4) that tryptophan is absent in histones. This absence was related not to the TGG codon itself, but to characteristics of the amino acid. We conclude that the functional constraints of a protein can influence, at least for synonymous codon usage, the evolution of its own coding sequence.

Animals↗

PubMed1990

A human ubiquitin carboxyl extension protein functions in yeast.

The ability of the human ubiquitin carboxyl extension protein (HUBCEP80) to functionally replace its yeast homolog was determined in a ubi3 mutant of Saccharomyces cerevisiae. Expression of HUBCEP80 in ubi3 mutants resulted in processing of the fusion protein to produce free ubiquitin and extension protein, the latter of which localized specifically with the 40 S ribosomal subunit. Furthermore, expression of the human fusion protein completely alleviated the phenotypic deficiencies found in ubi3 mutants, including slow growth, abnormal ribosomal RNA processing, and correspondingly low levels of 40 S ribosomal subunits. Finally, expression of the extension protein alone was much less efficient in complementing the ubi3 mutant phenotype as compared with expression of the normal ubiquitin-fused extension protein. In the latter case, cells were found to contain at least 5-fold more extension protein, suggesting that ubiquitin either increased translational efficiency of the HUBCEP80 transcript or increased the stability of the processed extension protein.

Chromosome Deletion↗

PubMed1991

Human genome protein function database.

A database which focuses on the normal functions of the currently-known protein products of the Human Genome was constructed. Information is stored as text, figures, tables, and diagrams. The program contains built-in functions to modify, update, categorize, hypertext, search, create reports, and establish links to other databases. The semi-automated categorization feature of the database program was used to classify these proteins in terms of biomedical functions.

Databases, Factual↗

PubMed1989

The role of oligosaccharides in modifying protein function.

It has been proposed that protein-bound oligosaccharides interact with the protein to which they are attached to up- or down-regulate the bioactivity of the 'composite' glycoprotein. Oligosaccharide analyses of the glycoproteins Thy-1, tissue plasminogen activator and immunoglobulin G are presented. Correlations between particular glycoforms and enzymic activities are demonstrated for tissue plasminogen activator. The change in the prevalence of particular immunoglobulin G glycoforms is shown to correlate with disease activity in rheumatoid activity.

Animals↗

PubMed1986

Pharmacological modulation of cardiac and vascular contractile protein function.

The Ca2+-dependent regulation of contractile protein interactions in cardiac and vascular smooth muscle involves structurally related but distinct Ca2+ binding proteins. In vascular smooth muscle, Ca2+ binds to calmodulin, and Ca2+-calmodulin activates myosin light chain (MLC) kinase with ultimate stimulation of MLC phosphorylation and actin-myosin interactions. The largest class of inhibitors of vascular contractile protein interactions are the calmodulin antagonists which include certain Ca2+ entry blockers. Pharmacologically, some of these agents can be distinguished from pure Ca2+ entry blockers by being more effective vs. vasoconstrictor agents in vitro, less cardiac depressant, and more effective as platelet aggregation inhibitors. An even greater distinction from Ca2+ entry blockers is evident with another series of agents, isoquinolinesulfonamides, which directly inhibit protein kinase activity. Cardiac muscle myofibrillar regulation involves Ca2+ binding to troponin C (TnC). Some cardiotonics, such as Vardax and APP 201-533, increase the Ca2+ sensitivity of cardiac myofibrillar ATPase activity with a concomitant increase in Ca2+ binding to TnC. Several calmodulin antagonists, Ca2+ blockers, and structurally related agents differentially affect cardiac myofibrillar ATPase activity. Potency and efficacy of some of these stimulating agents is markedly greater than Vardax or APP 201-533. Mechanistically, all agents do not affect cardiac MLC phosphorylation, but directly enhance the Ca2+ sensitivity of ATPase activity. However, differential effects on basal and maximum ATPase activity by some agents suggest more complex or additional effects which are related to the type of agent as well as the species (dog vs. hamster). A major subcellular defect in congestive heart failure in various small animal models is a depressed maximum ATPase activity. Thus, a desired goal would be a pharmacological modulator which increases maximum ATPase activity, not necessarily Ca2+ sensitivity. In sum, it is possible to identify agents, Ca2+ binding protein modulators, which directly inhibit vascular smooth muscle and stimulate cardiac muscle contractile protein interactions. The potential advantages/disadvantages of this approach for vasodilator/cardiotonic drug development will have to await future development of novel compounds targeted specifically for these cellular regulatory processes.

Animals↗

PubMed2025

Properties Governing Native State Entanglements and Relationships to Protein Function.

Non-covalent lasso entanglements are structural motifs found in a majority of globular proteins, and their misfolding has been linked to a range of biological consequences. Here, we characterize these motifs' structural and physicochemical properties, sequence biases, functional site correlations, and universal features across E. coli, S. cerevisiae, and H. sapiens. We find that the crossing residues, which pierce the plane of the entanglement loop, are 11-times more likely to be a β-strand than an α-helix or random coil, and that around this position the protein sequence is 2.5-times more likely to be composed of a stretch of all hydrophobic residues (most often Val, Ile, or Phe) compared to other sequence motifs. Functionally, crossing residues are enriched at enzyme active sites in S. cerevisiae and small molecule binding residues across all species to degrees greater than expected by random chance. Metal binding residues are enriched in these entanglements in H. sapiens. Increasing statistical power by pooling together these species data, we find RNA-binding residues are enriched in these entanglement components. On the other hand, there is a spatial depletion of crossing residues at sites involved in protein binding. Using machine learning, we identified eight robust features predictive of these entanglements, achieving AUROC scores of 0.8 across species. These results are significant because they suggest a direct role for components of native entanglements in particular protein functions, as well as identifying strong secondary structure and sequence preferences in native entanglements.

Humans↗

PubMed1990

Diagnosis of protein C deficiency in patients on oral anticoagulant treatment: comparison of three different functional protein C assays.

The efficacy of three different protein C activity assays and of protein C antigen determination for the diagnosis of protein C deficiency was studied in 13 protein C deficient patients (11 with type I, 2 with type II deficiency) and in 51 presumably non-deficient patients (control group), both groups being on oral anticoagulant (OAC) treatment. For protein C activity measurement (1) the assay according to Francis (slightly modified) with thrombin activation and measurement of activated protein C in the aPTT system, (2) an assay using Protac activation and chromogenic substrate (Protac-CS) and (3) an assay using Protac activation and the aPTT system (Protac-PTT) were used. Protein C antigen was determined by Laurell immunoelectrophoresis. The three activity assays gave different results, with the highest values obtained by the Protac-CS assay and the lowest values by the Protac-PTT assay. The Francis assay gave intermediate results. Protein C activity and antigen values were significantly lower in protein C deficient patients compared to the control group. Protein C activity tests had a higher discriminative power than the antigen determination. After taking into account the intensity of treatment, by the Francis assay all deficient and non-deficient patients were correctly classified, by the Protac-CS and the Protac-PTT assay 2 and 4 patients, respectively, were misclassified and by the antigen assay 8 patients were misclassified. Calculation of the ratios of protein C activity to factor II activity was of high discriminative power. We conclude that for diagnosis of protein C deficiency protein C activity tests are superior to antigen determination not only in type II but also in type I deficient patients.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Hydroxycoumarins↗

PubMed1979

Purification of cytoplasmic tubulin and microtubule organizing center proteins functioning in microtubule initiation from the alga Polytomella.

Cytoplasmic tubulin and the microtubule organizing centers (MTOCs) for the cytoskeletal microtubule system of the flagellate Polytomella have been isolated. The isolated MTOCs serve as sites for the in vitro assembly of the purified tubulin protein. The major proteins (four polypeptides of molecular weights 190,000-210,000) functioning in this assembly have been extracted from the MTOCs and purified. Kinetic studies and experiments with in vivo 35S-labeled MTOC proteins (or 35S-labeled tubulin) demonstrate that these proteins function specifically in microtubule initiation and do not contribute to microtubule elongation. The results indicate that microtubule assembly in vivo is controlled by microtubule initiating proteins associated with the organelles termed MTOCs.

Carrier Proteins↗

PubMed1990

A Drosophila ribosomal protein functions in mammalian cells.

A cDNA expression vector encoding Drosophila ribosomal protein S14 was transfected into cultured Chinese hamster ovary (CHO) cells that harbor a recessive RPS14 emetine resistance mutation. Transformants synthesized the insect mRNA and polypeptide and consequently displayed an emetine-sensitive phenotype. These observations indicate that the insect protein was accurately expressed and correctly assembled into functional mammalian 40S ribosomal subunits.

Animals↗

PubMed1989

The human mannose-binding protein functions as an opsonin.

The human mannose-binding protein (MBP) is a multimeric serum protein that is divided into three domains: a cysteine-rich NH2-terminal domain that stabilizes the alpha-helix of the second collagen-like domain, and a third COOH-terminal carbohydrate binding region. The function of MBP is unknown, although a role in host defense is suggested by its ability to bind yeast mannans. In this report we show that native and recombinant human MBP can serve in an opsonic role in serum and thereby enhance clearance of mannose rich pathogens by phagocytes. MBP binds to wild-type virulent Salmonella montevideo that express a mannose-rich O-polysaccharide. Interaction of MBP with these organisms results in attachment, uptake, and killing of the opsonized bacteria by phagocytes. These results demonstrate that MBP plays a role in first line host defense against certain pathogenic organisms.

Antigens, Bacterial↗

PubMed1992

Yeast SNF2/SWI2, SNF5, and SNF6 proteins function coordinately with the gene-specific transcriptional activators GAL4 and Bicoid.

The SNF2 (SWI2), SNF5, and SNF6 genes are required for transcription of many diversely regulated genes in Saccharomyces cerevisiae. Previously, we showed that SNF2, SNF5, and SNF6 function interdependently in transcriptional activation, possibly forming a heteromeric complex. Here, we present evidence that SNF6 has a more direct role in stimulating transcription than SNF2 and SNF5. The global effects of mutations in SNF2, SNF5, and SNF6 suggested that these SNF proteins may function coordinately with many gene-specific activators. We show that LexA-GAL4 and LexA-Bicoid fusion proteins are both dependent on SNF2, SNF5, and SNF6 for activation of target genes containing one or multiple lexA operators. The stringency of the requirement for the SNF proteins varies with the activator, the number of binding sites for the activator, and the target promoter. Thus, these SNF proteins appear to represent a class of intermediary proteins that facilitate transcriptional activation by gene-specific regulatory proteins.

Adenosine Triphosphatases↗

PubMed1992

Spatial specificity of chromophore assisted laser inactivation of protein function.

Chromophore assisted laser inactivation (CALI) is a new technique that selectively inactivates proteins of interest to elucidate their in vivo functions. This method has application to a wide array of biological questions. An understanding of aspects of the mechanism of CALI is required for its judicious application. A critical concern for CALI is its spatial specificity because nonspecific inactivation of neighboring unbound proteins by CALI is a possibility. We show here that CALI is very dependent on the distance between the chromophore and the protein such that there is no significant effect beyond 60 A. CALI using antibodies can inactivate other proteins through a complex but its efficacy decreases approximately fourfold for each intervening protein. These data imply that CALI is spatially specific and damage to neighboring proteins is unlikely.

Acetylcholinesterase↗