Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

Effect of cell concentration on the rheology of glucoamylase fermentation broth

During the process optimisation of glucoamylase production by Aspergillus awamori, cell morphology was controlled at such a state that spore aggregation was completely prevented. Samples from five fermentations on complex media using either glucose or starch as carbon source were characterised with a Bohlin CS rheometer. The experimental data were conveniently described in terms of the power law model.     

High affinity specific antiestrogen binding sites are concentrated in rough microsomal membranes of rat liver

Saturation and competitive binding analyses demonstrated the presence of a high affinity (K_D = 0.92 nM), specific antiestrogen binding site (AEBS) in rat liver microsomes and at least 75% of total liver AEBS was recovered in this fraction. When microsomes were further separated into smooth and rough fractions, AEBS was concentrated in the latter. Subsequent dissociation of ribosomes from the rough membranes revealed that AEBS was associated with the membrane and not the ribosomal fraction. Antiestrogen binding activity could not be extracted from membranes with 1 M KCl or 0.5 M acetic acid but could be solubilized with sodium cholate. These data indicate that AEBS is an integral membrane component of the rough microsomal fraction of rat liver.     

Chitin biosynthesis during pupal development of Drosophila melanogaster and the effect of the lethalcryptocephal mutation

Estimation of chitin deposition in the pupal and adult cuticles of adult Drosophila melanogaster during the pupal period is described. The timing of the periods of chitin deposition is compared with that deduced by previous workers using electron microscopy. The hypothesis that lethalcryptocephal mutant homozygotes are unable to evert their cephalic complexes at pupation because of excess chitin deposition is examined. The data obtained show no evidence that the mutation has any effect on chitin deposition.     

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Summary: Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.