7B2 Is a Specific Intracellular Binding Protein of the Prohormone Convertase PC2

Abstract: Biosynthetic pulse-chase analyses have previously demonstrated that the prohormone convertase PC2 is first synthesized as a precursor pro-PC2 and that zymogen activation to PC2 occurs following the slow exit of pro-PC2 from the endoplasmic reticulum (ER) and its concentration within the trans-Golgi network (TGN). The endocrine and neural protein 7B2 is first synthesized as a nonglycosylated precursor (pro-7B2), which is cleaved within the TGN by a furin-like ubiquitous convertase at the RRKRR¹⁵⁵S site to generate 7B2. In this report, we demonstrate that within the ER, pro-7B2 binds pro-PC2 but not any of the other convertases furin, PC1, PACE4, or PC5. This specific binding is Ca²⁺ dependent and does not require an N-glycosylated pro-PC2. Mutagenesis of the RRKRRS sequence demonstrated that the intact hexapeptide is critical for this binding, because the latter was abolished by mutations of the RR¹⁵² and greatly diminished by mutations of either the R¹⁵¹ or S¹⁵⁶ residues of pro-7B2. Once the complex is formed in the ER, it is then transported to the TGN where furin or a furin-like convertase cleaves both precursors, even when present as a complex. We also provide evidence that following zymogen cleavage, 7B2 remains bound to PC2, suggesting the presence of at least one other Ca²⁺-dependent binding site within the 7B2 sequence. Coexpression of 7B2 and PC2, although resulting in an elevation of the level of pro-PC2, did not eliminate the processing of pro-PC2 to PC2. Accordingly, cellular coexpression of 7B2 together with PC2 and proopiomelanocortin only marginally diminished the ability of PC2 to cleave proopiomelanocortin into β-endorphin in constitutive cells and had no effect in regulated cells. These results suggest that in vivo pro-7B2 is a specific PC2-binding protein that only transiently inhibits the processing of pro-PC2 until it reaches the TGN.     

Thermal isoelectric precipitation of alpha-lactalbumin from a whey protein concentrate: Influence of protein-calcium complexation

The selective precipitation of alpha-lactalbumin (alpha-LA) at a pH around its isoelectric point (4.2) under heat treatment is the basis for a fractionation process of whey proteins. As precipitation is a phenomenon dependent on the protein hydrophobicity, and as the release of the tightly bound calcium occurring at pH around 4 modifies the alpha-LA hydrophobicity, the specific role of calcium on isoelectric precipitation is investigated. A study of the extent of alpha-LA precipitation in a whey protein concentrate under various operating conditions of pH, temperature, protein concentration, and calcium content is presented. We propose a mechanism for this phenomenon as a combination of a complexation equilibrium and of an irreversible precipitation, to account for the influence of temperature, alpha-LA concentration total ionic content, and calcium concentration, and also to estimate the complexation equilibrium constant. (c) 1995 John Wiley & Sons, Inc.     

Sporulation: an alternative way to recover recombinant proteins from Bacillus subtilis

A recombinant strain of Bacillus subtilis engineered for endocellular expression of human interleukin-1 receptor antagonist (IL-Ira) was subjected to sporulation. The recombinant protein was recovered from the sporulation supernatant in quantities, purity, and activity comparable with those obtained from a traditional cell lysate. Thus, exploitation of this natural mechanism of autolysis could overcome problems of intact protein recovery related to the cell disruption step. (c) 1995 John Wiley & Sons, Inc.     

Transesterification catalyzed by polystyrene-supported chymotrypsin in toluene: the effect of neutralization of basic or acidic groups attaching to polystyrene resins

Crosslinked polystyrene resins containing a low level of either basic or acidic groups were used for supports of alpha-chymotrypsin (CT), which catalyzed the transesterification of N-acetyl-L-phenylalanine ethyl ester (AcPheOEt) with propanol in toluene. With a minimal amount of water, CT was sorbed to the resins, basic or acidic groups of which were partly or fully neutralized by several soluble acids or bases. With an increasing degree of neutralization of basic resins by free acids, the rate of disappearance of AcPheOEt was decreased, whereas the by-product formation of AcPheOH, due to hydrolysis, was considerably suppressed, compared with the ester-exchange product, AcPheOPr. The pK(a) value of the neutralizing acid was also important for both CT activity and reaction selectivity. AcPheOPr was selectively produced at a certain range of pK(a) values. On the other hand, the neutralization of acidic resins with free amines enhanced the CT activity but a strong base promoted the formation of hydrolysis product. (c) 1995 John Wiley & Sons, Inc.     

THE FOUNDING OF A NEW POPULATION OF DARWIN'S FINCHES

We report the natural colonization of the small Galápagos island Daphne Major by the large ground finch (Geospiza magnirostris). Immigrants of this species were present in every year of a 22-yr study, 1973–1994. Typically they arrived after a breeding season and left at the beginning of the next one. Geospiza magnirostris bred on the island for the first time in the exceptionally wet El Niño year of 1982–1983, and bred in all subsequent years except drought years. In agreement with theoretical expectations the frequency of inbreeding was unusually high. Pronounced fluctuating asymmetry in tarsus length, together with slightly reduced breeding success of inbreeding pairs, suggests a low level of inbreeding depression. Despite this, the population increased from 5 breeding individuals in 1983 to 20 breeding individuals in 1992, and probably more than twice that number in 1993, largely through recruitment of locally born birds.     

Trypsin complexed with α1-proteinase inhibitor has an increased structural flexibility

Mutant rat trypsin Asp¹⁸⁹Ser was prepared and complexed with highly purified human α₁-proteinase inhibitor. The complex formed was purified to homogeneity and studied by N-terminal amino acid sequence analysis and limited proteolysis with bovine trypsin. As compared to uncomplexed mutant trypsin, the mutant enzyme complexed with α₁-proteinase inhibitor showed a highly increased susceptibility to enzymatic digestion. The peptide bond selectively attacked by bovine trypsin was identified as the Arg¹¹⁷-Val¹¹⁸ one of trypsin. The structural and mechanistic relevance of this observation to serine proteinase-substrate and serine proteinase-serpin reactions are discussed.     

The effect of B chromosomes on fitness components inAegilops speltoides Tausch

The effect of B chromosomes (Bs) on various components of vigour and fertility inAegilops speltoides was studied. We designed three types of synthetic populations. In the first type we compared plants from the localities Haifa (which has Bs in nature) and Ashkelon (which has not). Non significant differences were found between the plants from both populations, and between B carriers and non carriers among Haifa individuals. In the second synthetic population we compared 0B, 1B, 2B, 3B and 5B plants from Haifa. We found that vigour variables were not affected by the presence of Bs, while fertility variables were significantly correlated with B number, in such a way that 1B plants produce more grains per plant than any other constitution, while 5Bs produce a remarkable deleterious effect. In the third type of synthetic populations we compared plants with 0B and 1B, 0B and 2B, 0B and 3B, and 0B and 5B. In this case we also found that 1B plants were the most fertile, but the variation of reproductive fitness was higher from plot to plot within the same B class than among B classes. We conclude that the effect of B chromosomes on the fitness variables that we have estimated is close to neutrality in this species. We discuss the relation between the strength of the mechanism of accumulation and the deleterious effects of B chromosomes.     

Neurotoxic Effect of Intranigral Injection of 1-Methyl-4-Phenylpyridinium on GABA-Containing Neurons and Its Relation to Circling Behavior

Abstract: The ionic species 1-methyl-4-phenylpyridinium (MPP⁺) seems to be the metabolite responsible for the damage to dopaminergic neurons occurring after administration of the parkinsonian drug 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. In the present study we show that the unilateral stereotaxic microinjection of MPP⁺ into the substantia nigra pars reticulata in rats produces immediately intense and long-lasting (up to 96 h) contralateral turning behavior in a dose-dependent manner. This behavioral effect was correlated with a dose- and time-dependent decrease (up to 90%) of glutamate decarboxylase activity and with a notable loss of neurons in the injected nigra reticulata. GABA levels in the injected nigra were also decreased, whereas the dopamine concentration in the ipsilateral striatum was not affected at 24 h, when maximal behavioral effects were observed. The circling behavior was prevented by the dopamine carrier blocker nomifensine only during the first 2 h, whereas the dopamine receptor antagonist haloperidol was ineffective. The results indicate that MPP⁺ is toxic for inhibitory GABAergic neurons in the nigra pars reticulata and, furthermore, suggest that disruption of the function of these GABAergic neurons may be involved in the abnormal motor behavior produced by the injection of MPP⁺ in the substantia nigra.     

Adenosine A1 Agonists and the Ca2+ Channel Agonist Bay K 8644 Produce a Synergistic Stimulation of the GTPase Activity of Go in Rat Frontal Cortical Membranes

Abstract: The identity and role of G proteins in coupling adenosine receptors to effectors have been studied to a limited degree. We have identified the G proteins whose GTPase activity is stimulated by adenosine receptor agonists in neuronal membranes. (R)-Phenylisopropyladenosine, 2-chloroadenosine, and N-ethylcarboxamideadenosine produced a concentration-dependent stimulation of GTPase. At 10^?5M, the increase above basal GTPase in frontal cortex was 25 ± 4, 20 ± 3, and 8 ± 1%, respectively, and in the cerebellum 55 ± 2, 41 ± 4, and 22 ± 2%, respectively. The effects of (R)-phenylisopropyladenosine and 2-chloroadenosine were inhibited by (1) A₁ antagonists (76–96% reduction), (2) pretreatment with pertussis toxin (90–100% reduction), and (3) antibodies raised against the α-subunit of G_i and G_o (55–57% reduction by each), suggesting that A₁ receptors interact equally with G_i and G_o. (R)-Phenylisopropyladenosine increased the binding of a nonhydrolyzable analogue of GTP to membranes in a pertussis toxin-sensitive manner, indicative of activation of G_i or G_o. Previously, (±)-Bay K 8644 enhanced GTP hydrolysis by G_o but not G_i. Now we report a profound synergistic stimulation of GTPase in the presence of (R)-phenylisopropyladenosine and (±)-Bay K 8644 (10^?7 to 10^?5M). (±)-Bay K 8644 had no effect on nucleotide exchange and, thus, cannot activate G_o. It appears that a positive cooperative stimulation of G_o occurs when it is first activated by A₁ receptors and subsequently interacts with the L-type Ca²⁺ channel.