Proteolytic activity of proteasome on myofibrillar structures

The physiologic function of proteasome remains unclear. Evidence suggests a role in degradation of ubiquitin-protein conjugates, MHC antigen presentation, and some specificity of substrate within certain cell types. To explore further the properties of proteasome we have examined its effect on a well defined structure, the myofibril. We find that despite its large size (20S) proteasome is able to degrade myofibrils and intact, permeabilized muscle fibrils. The proteins degraded showed some specificity because actin, myosin and desmin were degraded faster than -actinin, troponin T and tropomyosin. Changes in ultrastructure were slow and included a general loss of structure with Z and I bands effected before the M band and costameres.     

Cryptic dehalogenase and chloroamidase genes in Pseudomonas putida and the influence of environmental conditions on their expression

Mutants of two strains of Pseudomonas putida expressed two cryptic chloroamidases (C-amidase and Hamidase) and one cryptic dehalogenase (DehII). The mutants were selected on either 2-chloropropionamide (2CPA) or 2-monochloropropionate (2MCPA), developing as papillae in parental colonies growing on a metabolisable support substrate. Mutants expressing C-amidase were selected if 2CPA was utilised as either a carbon or a nitrogen source. H-amidase mutants were selected only if 2CPA was used as a nitrogen source. Growth temperature and pH affected the frequency of papillae production, although different temperatures and pHs did not affect the overall growth characteristics of the parental colonies. Decreasing growth temperature increased the frequency of 2cpa⁺ papillae formation, but decreased the frequency of 2mcpa⁺ papillae formation. Low pH (6.0) prevented the formation of 2mcpa⁺ and 2cpa⁺ papillae. However, in the case of the 2cpa⁺ papillae, decreasing the growth temperature also allowed papillae formation at pH 6.0.Abbreviations CAA Chloroacetamide - 2CPA 2-Chloropropionamide - DCA Dichloroacetic acid - HAA Halogenated alkanoic acid - 2MCPA 2-Monochloropropionic acid     

Molecular basis and PCR-DNA typing of the Fya/fyb blood group polymorphism

Spondyloepiphyseal dysplasia tarda (SEDL) is an X-linked recessive disorder characterized in affected males by short stature resulting from a growth defect of the vertebral bodies. We have extended our earlier studies by analyzing 15 families with newly identified microsatellite DNA markers; analysis of recombination events with these markers indicates that the gene responsible for SEDL is located in Xp22 between DXS 16 and DXS 987 on an interval spanning approximately 2 Mb.     

Kinetic properties of cholinergic desensitization in Aplysia neurons

The kinetic properties of desensitization onset of excitatory cholinergic responses were studied in isolated, voltage-clamped Aplysia neurons. Desensitization of the acetylcholine (ACh)-induced current in response to microperfused acetylcholine occurred in two phases, and was best modelled as the sum of two exponential components plus a constant. Both exponential components were accelerated by increasing ACh dose. At the higher ACh doses the current decline was dominated by the fast exponential component, and the ratio of the plateau-peak current was reduced. Over the range of membrane potentials -50 to -110 mV, no change in the kinetics of desensitization onset was observed. The mean time constants of both exponential components were doubled by cooling from 20 degrees C to 5 degrees C. These results demonstrate that, as at the vertebrate neuromuscular junction, the onset of desensitization of this ACh response involves at least two processes which are dose- and temperature-sensitive. The lack of voltage dependence contrasts with results from vertebrate preparations, and indicates a fundamental difference between the properties of the excitatory ACh response in Aplysia neurons and the vertebrate neuromuscular junction.     

Production of prostacyclin, 6-keto-PGF1α and thromboxane B2 by incubated samples of umbilical vessels: A comparison of methods for expressing the reults on dry weight, wet weight, protein or DNA bases

The production of PG12 (determined by abioassay), and of 6-keto-PGF1α and TXB2 (determined by radioummunoassay) by samples of human umbilical vessels have been measured. The results have been calculated on four bases: dry weigt, wet weight, protein and DNA.There was a higher production of PG12 and 6-keto-PGF1α by umbilical veins than by umbilical arteries; no significant difference in TXB2 production was observed between umbilical veins and arteries. The ratio of 6-keto-PGF1α: TXB2 production was about 100 for the samples of veins and about a40 for the samples of arteries.The best methods of expressing the results were on the bases of protein and DNA, the latter basis being marginally the best. The least satisfactory method for expressing the results was that based on dry weight.The physiological and practical implications of the results are discussed.     

Renin cleavage of a human kidney renin substrate analogous to human angiotensinogen, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, that is human renin specific and is resistant to cathepsin D

A synthetic tetradecapeptide, H-Asp-Arg-Val-Tyr-Ile-His-Pro-Phe-His-Leu-Val-Ile-His-Ser-OH, which corresponds to the 13 amino terminal residues of human angiotensinogen plus a carboxy terminal serine to replace a suggested site of carbohydrate attachment, has been shown to be a good substrate for human kidney renin. At pH 7.2 and 37 degrees C the KM or Michaelis constant was 8.4 +/- 2.9 microM, and the VM or velocity at infinite tetradecapeptide concentration was 11.3 +/- 2.4 mumol angiotensin I made per hour per milligram renin. The tetradecapeptide was highly resistant to cleavage by mouse submaxillary renin. The tetradecapeptide was also slowly cleaved by human liver cathepsin D, by rabbit lung angiotensin-converting enzyme, and by reconstituted human serum, but did not yield angiotensin I. Thus, this synthetic renin substrate should permit more specific measurement of human kidney renin activity.     

Prenatal diagnosis of cystic fibrosis by assay of amniotic fluid microvillar enzymes

Summary Activities of the microvillar enzymes -glutamyl-transpeptidase (GGTP), aminopeptidase M (APM), phosphodiesterase and maltase have been examined in second-trimester amniotic fluid as possible aids to the early prenatal diagnosis of cystic fibrosis (CF). The two peptidases, GGTP and APM, gave best results. If the fifth percentile of the normal range is used as an action line, the sensitivity of a positive test (low GGTP value) is 78% and the predictability 84%. At the tenth percentile the sensitivity is 100% and the predictability 77%. These approximate figures apply only to pregnancies where there has been a previous affected child. Until the primary protein defect in CF is discovered, this may prove an acceptable form of prenatal diagnosis to the high-risk mother.     

Tightly bound nucleotides of the energy-transducing ATPase, and their role in oxidative phosphorylation. II. The beef heart mitochondrial system.

1. Beef heart mitochondrial ATPase, in both the membrane-bound and isolated form, contains tightly bound ATP and ADP. Each mol of ATPase contains about 2.2 mol ATP and 1.3 mol ADP. 2. In the absence of ATPase activity, these nucleotides exchange only slowly with nucleotides in solution. The exchange rate is increased during coupled ATPase activity, but not when the ATPase is uncoupled. 3. Oligomycin and dicyclohexylcarbodiimide inhibit exchange of the bound nucleotides, as does the ATPase inhibitor protein, although in each case some residual exchange occurs. Aurovertin, although inhibiting phosphorylation, does not inhibit the exchange. This is discussed in terms of the reversibility of these inhibitors. 4. The stimulation of exchange seen during coupled ATPase activity requires energisation of the ATPase molecule. Using the exchange reaction as a probe of energisation, it is deduced that energy can be transferred between different ATPase molecules. 5. It is proposed that coupled ATPase activity and phosphorylation in submitochondrial particles involve the tight nucleotide binding sites and the (weak) ATPase site, while uncoupled ATPase activity involves only the weak site.