The MAS-encoded processing protease of yeast mitochondria. Interaction of the purified enzyme with signal peptides and a purified precursor protein

The matrix of yeast mitochondria contains a chelator-sensitive protease that removes matrix-targeting signals from most precursor proteins transported into this compartment. The enzyme consists of two nonidentical subunits that are encoded by the nuclear genes MAS1 and MAS2. With the aid of these cloned genes, we have now overexpressed the active holoenzyme in yeast, purified it in milligram amounts, and studied its biochemical and physical properties. Atomic absorption analysis shows that the purified enzyme lacks significant amounts of zinc, manganese, or cobalt; if none of these metal ions is added during the assay, the enzyme is catalytically inactive but can still cleave substoichiometric amounts of substrate. The amino-terminal sequences of the two mature subunits were determined; comparison with the deduced amino acid sequences of the corresponding precursors revealed that the MAS1 and MAS2 subunits are synthesized with prepeptides composed of 19 and 13 residues, respectively, which have similar sequences. The enzyme is inhibited competitively by chemically synthesized matrix-targeting peptides; the degree of inhibition correlates with the peptides' targeting efficacy. Matrix-targeting peptides containing the cleavage site of the corresponding authentic precursor protein are cleaved correctly by the purified enzyme. A purified artificial precursor protein bound to the holoenzyme can be photocross-linked to the MAS2 subunit.     

Identification and analysis of discrete functional domains in the pro region of pre-pro-transforming growth factor beta 1

A series of site-specific insertion and deletion mutants was prepared in the pro domain of transforming growth factor beta 1 (TGF beta 1) encoded by simian TGF beta 1 cDNA. These mutants were transiently expressed in COS-1 cells and the ability of each to be properly processed, folded correctly, and secreted was determined by immunoblot analysis of cells and culture supernatants. Insertions in regions corresponding to amino acid residues 50, 154, and 170 blocked secretion; culture supernatants from COS-1 cells showed no immunologically reactive proteins, whereas intact cells contained high levels of the mutant polypeptides. Insertions in the middle portion of the pro domain at residues 81, 85, and 144 affected disulfide maturation of the mature TGF beta 1. An insertion at residue 110, on the other hand, appeared to destabilize the mature TGF beta 1 polypeptide, resulting in degraded growth factor. Relatively small (10 amino acids) to large (125 amino acids) deletion mutations in the pro domain of TGF beta 1, when expressed as the full-length pre-pro-TGF beta 1, appeared to block secretion. By contrast, if the pro domain (designated beta 1-latency-associated peptide [beta 1-LAP]) was expressed independently, deletion mutants in the region 40-110 were readily secreted by the COS-1 cells, whereas deletions in residues 110-210 either destabilized the structure of the protein or blocked its intracellular transport. Cross-linking assays employing radioiodinated TGF beta 1 and biological assays indicate that residues 50-85 of beta 1-LAP are required for association with mature TGF beta 1.     

Apparent convergence (at 2-monoacylglycerol level) of phosphatidic acid and 2-monoacylglycerol pathways of synthesis of chylomicron triacylglycerols

Dietary fats are converted into chylomicron triacylglycerols via the 2-monoacylglycerol and phosphatidic acid pathways of acylglycerol formation. In view of the known positional and fatty acid specificity of the acyltransferases, the triacylglycerol structures resulting from the two pathways would be expected to differ, but this has not been demonstrated. We have performed stereospecific analyses on the chylomicron triacylglycerols from rats fed menhaden oil and the corresponding fatty acid alkyl esters, which would be expected to be assimilated via the monoacylglycerol and the phosphatidic acid pathways, respectively. The results show a remarkable similarity between the two triacylglycerol types in the fatty acid composition of the sn-1 and sn-3 positions, along with marked differences in the composition of the sn-2 positions. The triacylglycerols from rats fed oil retained about 85% of the original fatty acids in the sn-2 position, including a high proportion of the long chain polyunsaturates (e.g., 5-7% 20:5 and 4-5% 22:6). The triacylglycerols from rats fed the alkyl ester contained large amounts of endogenous fatty acids in the sn-2 position (e.g., 18% 16:1, 14% 18:1, 14% 18:2, and 2.5% 20:4), which approximated the composition of the sn-2 position of the presumed phosphatidic acid intermediates. The sn-1 position contained a much higher proportion of polyunsatured fatty acids (e.g., 12-13% 20:5, 5-6% 22:6) than the sn-2 position (e.g. 2-3% 20:5, 0-0.6% 22:6) of triacylglycerols from rats fed the ester. We conclude that the chylomicron triacylglycerols arising via the 2-monoacylglycerol and the phosphatidic acid pathways differ mainly in the composition of the fatty acids in the sn-2 position. The similarity in the acids of the sn-1 and sn-3 positions of the chylomicron triacylglycerols from rats fed oil or ester is consistent with a hydrolysis of the acylglycerol products of the phosphatidic acid pathway to 2-monoacylglycerols prior to reconversion to triacylglycerols via the monoacylglycerol pathway and secretion as chylomicrons.     

Fulvic acid disturbs processing of procollagen II in articular cartilage of embryonic chicken and may also cause Kashin-Beck disease.

Kashin-Beck disease is an endemic osteoarthropathy in China which may lead to skeletal deformation and dwarfism. We have analysed articular cartilage from two patients and found an accumulation of the precursor molecule, pro-pN-collagen II (pN, peptide attached at the amino-terminus) which was not present in extracts of control fetal cartilage. In addition, collagen II isolated from the same tissue by limited pepsin digestion had a decreased electrophoretic mobility, increased proline hydroxylation and decreased thermal stability. Previously, a genetic defect in pro-pN-collagen-I processing has been described in calf and sheep (dermatosparaxis) and man (Ehlers-Danlos, type VII) which caused an extreme fragility of the skin [Lenaers, A., Ansay, M., Nusgens, B.V. & Lapière, C.M. (1971) Eur. J. Biochem. 23, 533-541; Helle, O. & Nes, N.J. (1972) Acta Vet. Scand. 13, 443-445; Lichtenstein, J.R., Martin, G.R., Kohn, L.D., Byers, P.H. & McKusick, V.A. (1973) Science 182, 298-300]. Accordingly, one may assume that the impaired conversion of pro-pN-collagen II to collagen II and the structural alteration of collagen II, presumably caused by fulvic acid and other environmental factors, play an important role in the pathogenesis of Kashin-Beck disease.     

人源性流行性出血热病毒的形态特征

本文报道流行性出血热病毒(汉坦病毒)H-114株的电镜形态。发现形态发生以内质网膜和胞浆膜芽生为主。病毒颗粒为圆形或卵圆形。具有双层膜结构,大小为90～120nm。提出了汉坦病毒形态发生的理论观点。     

用直接测定基因组DNA序列的方法检测β-地中海贫血基因突变

本文报道一种结合聚合酶链反应(PCR)技术直接测定基因组DNA中单考贝基因片段序列的方法,以及利用这种方法测定两例β-地贫纯合子的β珠蛋白基因序到结果。测定出基因点突变,一例为编码子17(A→T)突变纯合子,另一例为编码子69(G→A)突变纯合子。针对上述两个点突变合成寡核苷酸片段,末端标记~(82)P后为探针进行斑点杂交的结果与测序结果一致。     

Atomic force microscopy of DNA molecules.

DNA-cytochrome c complexes adsorbed on carbon-coated mica surfaces were directly imaged by atomic force microscopy in air using commercially available cantilevers, with a routine resolution of 6 nm. Images of M13 phage DNA and M13-DNA polymerase complex are also shown.     

Inorganic pyrophosphatase from bovine retinal rod outer segments.

Rod outer segments from bovine retina contain a higher level of intracellular inorganic pyrophosphatase (EC 3.6.1.1) activity than has been found in any other mammalian tissue; the specific activity in extracts of soluble outer segment proteins is more than 6-fold higher than in extracts from bovine liver and more than 24-fold higher than in skeletal muscle extracts. This high activity may be necessary to keep inorganic pyrophosphate concentrations low in the face of the high rates of pyrophosphate production that accompany the cGMP flux driving phototransduction. We have begun to explore the role of inorganic pyrophosphatase in photoreceptor cGMP metabolism by 1) studying the kinetic properties of this enzyme and its interactions with divalent metal ions and anionic inhibitors, 2) purifying it and studying its size and subunit composition, and 3) examining the effects of pyrophosphate on rod outer segment guanylyl cyclase. Km for magnesium pyrophosphate was 0.9-1.5 microM, and the purified enzyme hydrolyzed > 885 mumol of PPi min-1 mg-1. The enzyme appears to be a homodimer of 36-kilodalton subunits when analyzed by gel electrophoresis and density gradient centrifugation, implying that kcat = 10(3) s-1, and kcat/Km = 0.7-1 x 10(9) M-1 s-1. The enzyme was inhibited by Ca2+ at submicromolar levels: 28% inhibition was observed at 138 nM [Ca2+], and 53% inhibition at 700 nM [Ca2+]. Imidodiphosphate acted as a competitive inhibitor, with Ki = 1.2 microM, and fluoride inhibited half-maximally approximately 20 microM. Inhibition studies on rod outer segment guanylyl cyclase confirmed previous reports that pyrophosphate inhibits guanylyl cyclase, suggesting an essential role for inorganic pyrophosphatase in maintaining cGMP metabolism.     

Determination of the solution structures of domains II and III of protein G from Streptococcus by 1H nuclear magnetic resonance.

We have used 1H nuclear magnetic resonance spectroscopy to determine the solution structures of two small (61 and 64 residue) immunoglobulin G (IgG)-binding domains from protein G, a cell-surface protein from Streptococcus strain G148. The two domains differ in sequence by four amino acid substitutions, and differ in their affinity for some subclasses of IgG. The structure of domain II was determined using a total of 478 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints; that of domain III was determined using a total of 445 distance restraints, 31 phi and 9 chi 1 dihedral angle restraints. A protocol which involved distance geometry, simulated annealing and restrained molecular dynamics was used to determine ensembles of 40 structures consistent with these restraints. The structures are found to consist of an alpha-helix packed against a four-stranded antiparallel-parallel-antiparallel beta-sheet. The structures of the two domains are compared to each other and to the reported structure of a similar domain from a protein G from a different strain of Streptococcus. We conclude that the difference in affinity of domains II and III for IgG is due to local changes in amino acid side-chains, rather than a more extensive change in conformation, suggesting that one or more of the residues which differ between them are directly involved in interaction with IgG.     

Normal and abnormal pathfinding of facial nerve fibers in the chick embryo.

Development of the facial nerve was studied in normal chicken embryos and after surgical disruption of ingrowing sensory facial nerve fibers at 38-72 h of incubation. Disruption of facial nerve fibers by otocyst removal often induced a rostral deviation of the facial nerve and ganglion to the level of the trigeminal ganglion. Cell bodies of the geniculate ganglion trailed their deviating neurites and occupied an abnormal rostral position adjacent to the trigeminal ganglion. Deviating facial nerve fibers were labeled with the carbocyanine fluorescent tracer DiI in fixed tissue. Labeled fibers penetrated the cranium adjacent to the trigeminal ganglion, but they did not follow the trigeminal nerve fibers into the brain stem. Rather, after entering the cranium, they projected caudally to their usual site of entrance and proceeded towards their normal targets. This rostral deviation of the facial nerve was observed only after surgery at 48-72 h of incubation, but not in cases with early otocyst removal (38-48 h). A rostral deviation of the facial nerve was seen in cases with partial otocyst removal when the vestibular nerve was absent. The facial nerve followed its normal course when the vestibular nerve persisted. We conclude that disruption of the developing facial pathway altered the routes of navigating axons, but did not prevent pathfinding and innervation of the normal targets. Pathfinding abilities may not be restricted to pioneering axons of the facial nerve; later-developing facial nerve fibers also appeared to have positional information. Our findings are consistent with the hypothesis that navigating axons may respond to multiple guidance cues during development. These cues appear to differ as a function of position of the navigating axon.