An analysis of the organization and evolution of type 4 fimbrial (MePhe) subunit proteins

Summary We have analyzed and compared the amino acid sequences of the type 4 fimbrial subunits fromPseudomonas aeruginosa, Moraxella bovis, M. nonliquefaciens, Bacteroides nodosus, Neisseria gonorrhoeae, andN. meningitidis. We propose a consensus sequence for the highly conserved aminoterminal regions of these proteins. In the variable regions, a domain corresponding to an epitope common toN. gonorrhoeae andN. meningitidis fimbriae is conserved, both in sequence and in environment, in fimbrial subunits fromB. nodosus. The subunits fromM. bovis andP. aeruginosa do not show any homologies to this sequence. In all of the subunits, the carboxy-terminal half of the molecule consists of a series of fairly hydrophobic domains. The last three domains, two of which include the cysteines of the disulfide bridge inN. gonorrhoeae, P. aeruginosa, andM. bovis, are more or less conserved in sequence in all of the proteins including that ofB. nodosus. We propose that these conserved hydrophobic regions, which have the potential to form a series of beta-sheets, form a structural framework around which more variable hydrophilic sequences determining immunological profile are arranged. The evolutionary relationships of the contemporary proteins and the distribution of type 4 fimbriae are also discussed.     

N-Terminal Sequence of Pig Brain Choline Acetyltransferase Purified by a Rapid Procedure

A procedure is reported that allows the purification and amino terminal sequencing of pig brain choline acetyltransferase. The enzyme (present in extremely low amounts in this tissue) is eluted together with its antibody from an affinity column by a mild pH shift and the resulting enzyme-antibody complex separated by gel electrophoresis. The band corresponding to the enzyme is electroeluted from the gel using volatile solutions allowing the direct determination of the amino acid composition and partial sequence. The first 11 residues are: Pro-Ile-Leu-Glu-Lys-Thr-Pro-Pro-Lys-Met-Ala.     

A Phorbol Ester-Sensitive Kinase Catalyzes the Phosphorylation of P0 Glycoprotein in Myelin

The proposed structural protein of peripheral nerve myelin, P0, has been shown to have several covalent modifications. In addition to being glycosylated, sulfated, and acylated, P0 is phosphorylated, with the intracellular site of this latter addition being in question. By employing nerve injury models that exhibit different levels of P0 biosynthesis in the absence and presence of myelin assembly, we have examined the cellular location of P0 phosphorylation. It is demonstrated that there is comparable P0 phosphorylation in both normal and crush-injured adult rat sciatic nerves, although the level of biosynthesis of P0 differs between these myelin maintaining and actively myelinating nerve models, respectively. The glycoprotein does not appear to be phosphorylated readily in the transected adult sciatic nerve, a preparation in which P0 biosynthesis is observed but that lacks myelin membrane. These observations suggest that the modification is not associated with the biosynthesis or maturation of P0 in the endoplasmic reticulum or Golgi, but that it instead occurs after myelin assembly. That P0 phosphorylation occurs in the normal nerve even when translation is inhibited by cycloheximide treatment lends further support to this conclusion. P0 is shown to be phosphorylated on one or more serine residues, with all or most of the phosphate group(s) being labile as evidenced by pulse-chase analysis. Addition of a biologically active phorbol ester, 12-O-tetradecanoylphorbol-13-acetate or 4 beta-phorbol 12,13-dibutyrate, substantially increases the extent of [32P]orthophosphate incorporation into the glycoprotein of normal and crushed nerve but not transected nerve. Biologically inactive 4 alpha-phorbol 12,13-didecanoate has no effect on P0 phosphorylation. Similarly, the addition of the cyclic AMP analog 8-bromo-cyclic AMP causes no appreciable changes in P0 labeling. These findings indicate that the phorbol ester-sensitive enzyme, protein kinase C, may be responsible for the phosphorylation of P0 within the myelin membrane.     

A Ca2+-Dependent Protein Kinase Activity Associated with Serotonin Binding Protein

The endogenous phosphorylation of serotonin binding protein (SBP), a soluble protein found in central and peripheral serotonergic neurons, inhibits the binding of 5-hydroxytryptamine (5-HT, serotonin). A protein kinase activity that copurifies with SBP (SBP-kinase) was partially characterized and compared with calcium/calmodulin-dependent protein kinase II (CAM-PK II). SBP itself is not the enzyme since heating destroyed the protein kinase activity without affecting the capacity of the protein to bind [3H]5-HT. SBP-kinase and CAM-PK II kinase shared the following characteristics: (1) size of the subunits; (2) autophosphorylation in a Ca2+-dependent manner; and (3) affinity for Ca2+. In addition, both forms of protein kinase phosphorylated microtubule-associated proteins well and did not phosphorylate myosin, phosphorylase b, and casein. Phorbol esters or diacylglycerol had no effect on either of the protein kinases. However, substantial differences between SBP-kinase and CAM-PK II were observed: (1) CAM enhanced CAM-PK II activity, but had no effect on SBP-kinase; (2) synapsin I was an excellent substrate for CAM-PK II, but not for SBP-kinase; (3) 5-HT inhibited both the autophosphorylation of SBP-kinase and the phosphorylation of SBP, but had no effect on CAM-PK II. These data indicate that SBP-kinase is different from CAM-PK II. Phosphopeptide maps of SBP and SBP-kinase generated by digestion with S. aureus V8 protease are consistent with the conclusion that these proteins are distinct molecular entities. It is suggested that phosphorylation of SBP may regulate the transport of 5-HT within neurons.     

Enzymatic Protein Carboxyl Methylation in Rat Posterior Pituitary: Neurophysins in Rapid-Turnover Pool Determine Methyl Accepting Capacity

In vitro stimulation of intact rat posterior pituitary by either veratridine or K+ depolarization results in the concomitant release of neurophysins and in a decrease (70-80%) in their carboxyl methylation as measured either with L-[methyl-3H]methionine in the intact lobes after stimulation or in their homogenates with [methyl-3H]S-adenosyl-L-methionine and purified protein carboxyl methyltransferase. A similar reduction in neurophysin methylation (60%) was observed when the arrival of newly synthesized neurophysins at the posterior pituitary was blocked by colchicine. Experimental data indicate that the reduction in neurophysin content of the lobes after 12 h of colchicine treatment (less than 7%) or after in vitro stimulation (about 10%) cannot account for the marked reduction in neurophysin methylation. The results suggest that the granule pool characterized by rapid turnover of neurophysins probably represents the major source of methyl acceptor proteins in the lobe. In spite of the marked reduction in neurophysin methyl accepting capacity observed after stimulation, there was no parallel increase in methyl accepting capacity of the released neurophysins. We propose that a neurophysin subfraction that might be associated with the membrane of releasable granules participates in the methylation reaction in situ.     

Effect of Digestion with Phospholipase A2 on Endogenous Protein Phosphorylation in Particulate Fractions from Rat Brain Synaptosomes

The endogenous phosphorylation of synapsin 1 in cyclic AMP-containing media was greatly decreased by digestion of synaptic vesicles and synaptosomal membranes with phospholipase A2, suggesting that the system is functionally dependent on the membrane structure. Treatment of the synaptic vesicle fraction with phospholipase A2 also caused a small but significant inhibition of the Ca2+/calmodulin-dependent phosphorylation of the same protein. The Ca2+/calmodulin-dependent phosphorylation of other major acceptors, and the basal phosphorylation of a 52-kD acceptor enriched in the vesicle fraction, remained unchanged after cleavage of the membrane phospholipids with phospholipase A2. The significance of the selective effect of phospholipase A2 treatment on endogenous membrane phosphorylation is discussed.     

Effects of starvation,chilling, and injury on endocrine gland function in Galleria mellonella

Starvation, chilling, and injury of last instar Galleria mellonella larvae typically elicit extra larval molts or a delay in pupation. The primary sites of action and the nature of the signals by which these treatments affect development are not known. However, since the connections of the brain to the nerve cord are crucial for the effects of starvation and chilling, these signals apparently affect the brain-centered program of developmental regulation via the nerve cord. Chilling, and occasionally starvation, cause extra larval molts in last instar larvae treated prior to the nervous inhibition of their corpora allata; release of a cerebral allatotropin, which stimulates the production of juvenile hormone, appears to be involved in this effect. After this time, a delay in pupation is the principal effect of starvation and chilling, and is apparently due to a temporal inhibition of the release of the prothoracicotropic hormone. Chilling also appears to inhibit unstimulated ecdysteroid production by the prothoracic glands. The effect of injury is not mediated by the nerve cord, but appears to involve an inhibitory humoral factor that affects either the brain or the prothoracic glands themselves. Injury also stimulates juvenile hormone production, an effect which is enhanced when the brain is separated from the nerve cord and which is evidenced by a delay of ecdysis and the occasional retention of some larval features in the ecdysed insects. None of the effects of these various treatments on the brain and the endocrine glands persist when the brains or glands are implanted into untreated hosts.     

The relationship of calcium and parathyroid hormone in their effect on heart cells

Parathyroid hormone (PTH), the plasma concentration of which is raised in uremia, has been suggested as one of the agents responsible for the myocardial changes commonly seen in uremia. The effect of intact [1–84] PTH on rat heart cells grown in tissue culture has been studied. Addition of the hormone to the media significantly stimulated beating rate. The stimulation was directly proportional to the amount of PTH in the medium. Excessively high concentration of PTH caused immediate cessation of the beating, which was reversed by the addition of calcium to the medium. The extent of stimulation by PTH was inversely proportional to the calcium concentrations in the medium. Isoproterenol and phenylephrine at excessively high concentrations in the medium did not mimic the PTH effect either alone or together with PTH. When beating ceased due to verapamil the effect was not reversed by the addition of calcium to the medium.Calcium added to the myocytes seen after beating ceased reversed the effect and the cells started to beat again. Cells kept for a longer period in the arrested state were not revived by the addition of calcium.     

Environmental control of protein export of Bacillus licheniformis NCIB 6346 reveals two types of extracellular proteins

Teichuronic acid was the major anionic polymer of Bacillus licheniformis NCIB 6346 durign phosphate-limited (P-limited) growth in the chemostat. This polymer was also present in significant quantities when B. licheniformis was grown under carbon-limited (C-limited) or magnesium-limited (Mg-limited) conditions where teichoic acid predominated in the cell wall. However, the cell wall composition was not of significance in protein export and the parameters for the excretion process were found to be environmental. In particular, two types of extracellular proteins were identified: the first type of enzyme, penicillinase, was only weakly catabolite repressed; was maximally synthesized and secreted during P-limited growth; was unaffected by growth in high Na⁺ media but its production was inhibited by gramicidin. The second type of enzyme, -amylase, was strongly catabolite repressed and its export was markedly inhibited during P-limited growth or in the presence of Na⁺ or gramicidin. It is noteworthy that the penicillinase carries a glyceride-cysteine modification at its N-terminus whilst the -amylase does not.     

ReducedS-adenosylmethionine: Protein-lysineN-methyltransferase activity (protein methylase III) in shiverer mutant mouse brain

Mice with the dysmyelinating mutation shiverer were studied by measuring the activity of two protein methylases and myelin marker enzymes in the brain. It was observed thatS-adenosylmethionine: protein-lysineN-methyltransferase (protein methylase III, EC. 2.1.1.43) activity is significantly reduced in phenotypically affected homozygous shiverer (shi/shi) mutant mouse brain compared to the unaffected heterozygous littermate brain. This reduction in enzyme activity is manifested mainly by reduced formation of trimethyllysine during the in vitro methylation of histone. In contrast, myelin marker enzymes such as 2,3-cyclic nucleotide 3-phosphohydrolase and 5-nucleotidase as well asS-adenosyl-methionine: protein-carboxylO-methyltransferase (protein methylase II, EC. 2.1.1.24) activities were not significantly affected in these strains of mice.