Proteolysis in eucaryotic cells: Aminopeptidases and dipeptidyl aminopeptidases of yeast revisited

Using nine different l-aminoacyl-4-nitroanilides and four different dipeptidyl-4-nitroanilides, aminopeptidases and dipeptidyl aminopeptidases active at pH 7.5 and (or) pH 5.5 in logarithmically growing and stationary-phase cells of Saccharomyces cerevisiae were searched for. Ion-exchange chromatography was used to separate the proteins of the soluble cell extract. Besides the three already-characterized aminopeptidases—aminopeptidase I (P. Matile, A. Wiemken, and W. Guyer (1971) Planta (Berlin)96, 43–53; J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41), aminopeptidase II (J. Frey and K. H. Röhm (1978) Biochim. Biophys. Acta527, 31–41; J. Knüver (1982) Thesis, Fachbereich Chemie, Marburg, FRG), and aminopeptidase Co (T. Achstetter, C. Ehmann, and D. H. Wolf (1982) Biochem. Biophys. Res. Commun.109, 341–347)—12 additional aminopeptidase activities are found in soluble cell extracts eluting from the ion-exchange column. These activities differ from the characterized aminopeptidases in one or more of the parameters such as charge, size, substrate specificity, inhibition pattern, pH optimum for activity and regulation. Also, a particulate aminopeptidase, called aminopeptidase P, is found in the nonsoluble fraction of disintegrated cells. Besides the described particulate X-prolyl-dipeptidyl aminopeptidase (M. P. Suarez Rendueles, J. Schwencke, N. Garcia-Alvarez and S. Gascon (1981) FEBS Lett.131, 296–300), three additional dipeptidyl aminopeptidase activities of different substrate specificities are found in the soluble extract.     

Iron-ethylenediaminetetraacetic acid (EDTA)-catalyzed superoxide dismutation revisited: An explanation of why the dismutase activity of Fe-EDTA cannot be detected in the cytochrome c/xanthine oxidase assay system

The recent assertion of J. Diguiseppi and I. Fridovich (1980, Arch. Biochem. Biophys., 203, 145–150) that Fe-EDTA does not catalyze superoxide dismutation is disputed. By directly observing superoxide generated during pulse radiolysis, we have confirmed the results of a previous study (G. J. McClune, J. A. Fee, G. A. McClusky, and J. T. Groves, 1977, J. Amer. Chem. Soc., 99, 5220–5222) which concluded that Fe-EDTA catalyzed superoxide dismutation. We also demonstrate that the reaction of Fe(II)-EDTA, formed during catalyzed superoxide dismutation, with cytochrome c, the probe molecule in the cytochrome c/xanthine oxidase/xanthine assay system for superoxide dismutase activity, is sufficiently rapid (H. L. Hodges, R. A. Holwerda, and H. B. Gray, 1974, J. Amer. Chem. Soc., 96, 3132–3137) to obscure the weak catalysis of superoxide dismutation by Fe-EDTA.     

Trehalose-6-phosphate synthetase activity in extracts of Dictyostelium discoideum.

Trehalose-6-phosphate (T-6-P) synthetase activity in extracts of Dictyostelium discoideum has been reexamined in an effort to resolve discrepancies between the results of previous studies (R. Roth and M. Sussman (1966). Biochim. Biophys. Acta, 122, 225; K. A. Killick and B. E. Wright (1972). J. Biol. Chem., 247, 2967). We find that T-6-P synthetase is not cold sensitive as reported by Killick and Wright (1972), is not present in bacterial-grown vegetative cells (though subject to some modulation by other nutritional conditions), and is not in our hands unmasked or activated by ammonium sulfate fractionation. We conclude that the pattern of T-6-P synthetase accumulation and disappearance during fruiting body construction in D. discoideum is as originally described by R. Roth and M. Sussman (1968). J. Biol. Chem., 243, 5081) and confirmed elsewhere (P. C. Newell et al. (1972). J. Mol. Biol., 63, 373; R. W. Brackenbury et al. (1974). J. Mol. Biol., 90, 529; B. D. Hames and J. M. Ashworth (1974). Biochem. J., 142, 301).     

Investigation of the pH dependence of proton uptake by porcine heart mitochondrial malate dehydrogenase upon binding of NADH

The pH dependence of proton uptake upon binding of NADH to porcine heart mitochondrial malate dehydrogenase (l-malate: NAD⁺ oxidoreductase, EC 1.1.1.37) has been investigated. The enzyme has been shown to exhibit a pH-dependent uptake of protons upon binding NADH at pH values from 6.0 to 8.5. Enzyme in which one histidine residue has been modified per subunit by the reagent iodoacetamide (E. M. Gregory, M. S. Rohrbach, and J. H. Harrison, 1971, Biochim. Biophys. Acta253, 489–497) was used to establish that this specific histidine residue was responsible for the uptake of a proton upon binding of NADH to the native enzyme. It has also been established that while there is no enhancement of the nucleotide fluorescence upon addition of NADH to the iodoacetamide-modified enzyme, NADH is nevertheless binding to the modified enzyme with the same stoichiometry as with native enzyme. The data are discussed in relation to the involvement of the essential histidine residue in the catalytic mechanism of “histidine dehydrogenases” recently proposed by Lodola et al. (A. Lodola, D. M. Parker, R. Jeck, and J. J. Holbrook, 1978, Biochem. J.173, 597–605) and the catalytic mechanism of “malate dehydrogenases” recently proposed by L. H. Bernstein and J. Everse (1978, J. Biol. Chem.253, 8702–8707).     

Specificity of the chlorophyll-to-protein binding in the chlorophyll-protein complexes of the thylakoid

We tried to establish whether the chlorophyll-protein complexes of the thylakoid, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, originate from real entities existing in vivo, or are mere artifacts of the sodium dodecyl sulfate solubilization procedure. Making use of the finding that etiolated leaves exposed to periodic light form selectively the chlorophyll-protein complexes CPI and CPa, while after transfer to continuous light they form in addition the light-harvesting complexes (J. H. Argyroudi-Akoyunoglou, Z. Feleki, and G. Akoyunoglou, 1971, Biochem. Biophys. Res. Commun.45, 606–614; J. H. Argyroudi-Akoyunoglou and G. Akoyunoglou 1979, FEBS Lett.104, 78–84) we tried to see whether the latter complexes contain newly formed chlorophyll. We labeled the chlorophyll a formed in periodic light with δ-[¹⁴C]aminolevulinic acid, and determined the specific radioactivity of chlorophyll in the complexes formed before or after transfer to continuous light. We found that the light-harvesting complexes contain primarily newly formed and nonradioactive chlorophyll. The results suggest that (i) the chlorophyll a of CPI and CPa formed in periodic light does not exchange with that of the light-harvesting complexes formed after transfer to continuous light. (ii) The light-harvesting complexes formed after transfer to continuous light contain primarily newly formed chlorophylls a and b. (iii) The binding of chlorophyll to protein in the complexes is specific and not an artifact of the sodium dodecyl sulfate action. (iv) As the thylakoid membrane grows and differentiates, the chlorophyll synthesized binds on the apoproteins of the complexes in a stepwise manner.     

A note on the sampling theory for infinite alleles and infinite sites models

This note considers sampling theory for a selectively neutral locus where it is supposed that the data provide nucleotide sequences for the genes sampled. It thus anticipates that technical advances will soon provide data of this form in volume approaching that currently obtained from electrophoresis. The assumption made on the nature of the data will require us to use, in the terminology ofKimura (Theor. Pop. Biol.2, 174–208 (1971)), the “infinite sites” model of Karlin and McGregor (Proc. Fifth Berkeley Symp. Math. Statist. Prob.4, 415–438 (1967)) rather that the “infinite alleles” model of Kimura and Crow (Genetics49, 174–738 (1964)). We emphasize that these two models refer not to two different real-world circumstances, but rather to two different assumptions concerning our capacity to investigate the real world. We compare our results where appropriate with corresponding sampling theory of Ewens (Theor. Pop. Biol.3, 87–112 (1972)) for the “infinite alleles” model. Note finally that some of our results depend on an assumption of independence of behavior at individual sites; a parallel paper byWatterson (submitted for publication (1974)) assumes no recombination between sites. Real-world behavior will lie between these two assumptions, closer to the situation assumed by Watterson than in this note. Our analysis provides upper bounds for increased efficiency in using complete nucleotide sequences.     

Pigeon liver malic enzyme: Involvement of an arginyl residue at the binding site for malate and its analogs

Treatment of malic enzyme with arginine-specific reagents phenylglyoxal or 2,3-butanedione results in pseudo-first-order loss of oxidative decarboxylase activity. Inactivation by phenylglyoxal is completely prevented by saturating concentrations of NADP⁺, Mn²⁺, and substrate analog hydroxymalonate. Double log plots of pseudo-first-order rate constant versus concentration yield straight lines with identical slopes of unity for both reagents, suggesting that reaction of one molecule of reagent per active site is associated with activity loss. In parallel experiments, complete inactivation is accompanied by the incorporation of four [¹⁴C]phenylglyoxal molecules, and the loss of two arginyl residues per enzyme subunit, as determined by the colorimetric method of Yamasaki et al (R. B. Yamasaki, D. A. Shimer, and R. E. Feeney (1981) Anal. Biochem., 14, 220–226). These results confirm a 2:1 ratio for the reaction between phenylglyoxal and arginine (K. Takahashi (1968) J. Biol. Chem., 243, 6171–6179) and yield a stoichiometry of two arginine residues reacted per subunit for complete inactivation, of which one is essential for enzyme activity as determined by the statistical method of Tsou (C. L. Tsou (1962) Acta Biochim. Biophys. Sinica, 2, 203–211) and the Ray and Koshland analysis (W. J. Ray and D. E. Koshland (1961) J. Biol. Chem., 236, 1973–1979). Amino acid analysis of butanedione-modified enzyme also shows loss of arginyl residues, without significant decrease in other amino acids. Modification by phenylglyoxal does not significantly affect the affinity of this enzyme for NADPH. Binding of l-malate and its dicarboxylic acid analogs oxalate and tartronate is abolished upon modification, as is binding of the monocarboxylic acid α-hydroxybutyrate. The latter result indicates binding of the C-1 carboxyl group of the substrate to an arginyl residue on the enzyme.     

An assay specific for the active form of liver phosphorylase kinase

An assay specific for the active form of liver phosphorylase kinase (EC 2.7.1.38) has been developed utilizing inhibition of the inactive form of phosphorylase kinase by β-glycerophos, phate and ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid. Following in vitro activation the results compared favorably with those obtained using a less specific assay previously available. (J. R. Vandenheede, S. Keppens, and H. DeWulf, 1977, Biochim. Biophys. Acta.481, 463–470;D. D. Doorneweerd, A. W. H. Tan, and F. Q. Nuttall, 1978, Diabetes27, 474). The in vitro activation of phosphorylase kinase was not associated with the formation of a small-molecular-weight form of the enzyme. The utility of the assay in monitoring in vivo interconversion reactions in response to various physiological stimuli was demonstrated.     

Disruption of the tryptophan binding site in the human serum albumin dimer

Monomer and dimer fractions of human serum albumin (HSA) obtained from charcoal-treated Fraction V HSA have very similar fluorescence and circular dichroism spectra, but the dimer neither binds l-tryptophan nor reacts rapidly with p-nitrophenyl acetate. The latter reaction presumably occurs in a major binding site of the monomer as many strongly bound ligands including l-tryptophan, small fatty acid anions (e.g., S.-W. M. Koh and G. E. Means, 1979, Arch. Biochem. Biophys.192, 73–79), and several drugs (e.g, N. P. Sollenne and G. E. Means, 1979, Mol. Pharmacol.15, 754–757) all decrease rates of reaction in direct proportion to their concentration. Binding data for those ligands indicate the presence of less than one binding site per molecule of charcoal-treated Fraction V HSA, and thus appear to reflect only its content of albumin monomer. The absence of that binding site in the dimer may reflect its inclusion within the dimer interface.     

Liberation of labile sulfur from ferredoxins by alkaline zinc reagent: an appraisal of the methylene blue method.

In disagreement with reported observation by Suhara and her colleagues (K. Suhara, S. Takemori, M. Katagiri, K. Wada, H. Kobayashi, and H. Matsubara, 1975, Anal. Biochem.68, 632–636) we found that more than 90% of labile sulfur was liberated from adrenodoxin within 5 min at 22°C. This rate was faster than those of spinach and clostridial ferredoxins, a result also at variance with Suhara's observation. At low temperature, the reaction was clearly biphasic, and spinach ferredoxin showed a similar profile. In the absence of zinc acetate, activation energies of the decomposition reaction of iron-sulfur center of OH^? were obtained as 39, 26, and 11 kcal/mol for adrenal, spinach, and clostridial ferredoxins, respectively. The adrenal reaction became faster as the dipole moment of the solvent increased. In the presence of 4 m urea and 1 m KCl, the rate was enhanced by approximately 26-fold, relative to the reaction without the addition of urea. In conclusion, the liberation reaction of adrenal labile sulfur with alkaline zinc reagent is fast at 22°C, indicating no need for modification of the original method (T. Kimura and K. Suzuki, 1967, J. Biol. Chem.242, 485–491; P. E. Brumly, R. W. Miller, and V. Massey, 1965, J. Biol. Chem.240, 2222–2228).     

Cotranslational cleavage of immunoglobulin light chain precursors by plasmacytoma microsomes.

Murine plasmacytoma endoplasmic reticulum which has been freed of ribosomes by EDTA treatment is capable of the cotranslational proteolytic processing of representative λ₁,λ₂, and k immunoglobulin light chain precursors. Messenger RNA fractions from the MOPC-104E, MOPC-315, and MOPC-46B tumor lines were used to direct the synthesis of the light chain precursors in a cell-free system derived from Krebs II ascites cells. The precursor cleavage activity of the plasmacytoma membranes is comparable in activity and in characteristics to that of two well-defined membrane preparations: Krebs II ascites intracellular membranes (E. Szczesna and I. Boime, 1976, Proc. Nat. Acad. Sci. USA73, 1179–1183) and EDTA-treated rough endoplasmic reticulum from canine pancreas (34., 35., J. Cell Biol.67, 852–862). The efficiency of the cleavage reaction appears to be dependent upon the precursor being utilized as a substrate. An assay suitable for a preliminary characterization of the plasmacytoma membrane preparations is described.     

Kinship and covariance

Price's (1970) covariance theorem can be used to derive an expression for gene frequency change in kin selection models in which the fitness effect of an act is independent of the genotype of the recipient. This expression defines a coefficient of relatedness which subsumes r(Wright, 1922), b(Hamilton, 1972), ρ (Orlove &; Wood, 1978), and R(Michod &; Hamilton, 1980). The new coefficient extends the domain of Hamilton's rule to models in which the average gene frequency of actors differs from that of recipients.     

Does copper-d-penicillamine catalyze the dismutation of O2−?

It has been reported (M. Younes and U. Weser, 1977, Biochem. Biophys. Res. Commun.78, 1247–1253; E. Lengfelder and E. F. Elstner, 1978, Hoppe-Seyler's Z. Physiol. Chem.359, 751–757) that the complex [Cu(I)₈Cu(II)₆(D-penicillamine)₁₂Cl]^5?-efficiently catalyzes the dismutation of O₂^? and that this activity is resistant to both EDTA and CN^?. However, careful study has demonstrated that this complex is unable to catalyze the dismutation of O₂^?, but that it slowly decomposes to simpler copper complexes which are active. Moreover, the activity which is observed is suppressed by EDTA or by Chelex 100 treatment.     

Quantitative dependence of mitochondrial oxidative phosphorylation on oxygen concentration: a mathematical model.

The model of Wilson and co-workers (2., 3., Arch. Biochem. Biophys. 182, 749–762) for the regulation of mitochondrial oxidative phosphorylation has been extended to include the dependence on oxygen tension. The derived rate expression correctly describes the observed dependence of cellular energy metabolism on oxygen tension, including the oxygen dependence at “normoxic” physiological values. Experimental evidence is presented that oxidative phosphorylation by suspensions of isolated rat liver mitochondria is also dependent on oxygen concentration up to values of at least 100 μM.     

The fine structure of Ameson pulvis (Microspora,Microsporida) and its implications regarding classification and chromosome cycle

Nosema pulvisPerez, 1905, Ameson pulvis (Perez) Sprague, 1977, in muscles of the crabs Carcinus maenas and C. mediterraneus from the coast of France, was observed with the electron microscope. It was found to be structurally similar to the type species A. michaelis (Sprague, 1970). Sprague, 1977, having moniliform sporogonial plasmodia, unikaryotic sporoblasts, and hirsute sporulation stages. It is treated as distinct from A. michaelis because it has slightly smaller spores (by comparison with syntype material of A. michaelis) and appears to have fewer coils in the polar filament. The results require the removal of the genus Ameson from the family Nosematidae Labbé, 1899, where Sprague (1977) had placed it under the erroneous supposition that its sporoblasts are diplokaryotic. Ameson is transferred to family Unikaryonidae Sprague, 1977. Ameson is distinguished from PereziaLéger and Duboscq, 1909, shown by Ormieres et al. to have a similar developmental pattern, by presence of appendages on its sporulation stage. A. nelsoni (Sprague, 1950), the third, and only other species of Ameson, lacks the appendages and is transferred to genus Perezia.     

An autoradiographic analysis of the time of appearance of neurons in the developing chick neural retina

The pattern of incorporation of [³H]thymidine into the chick neural retina has been used to establish the time and order in which different classes of neuroepithelial cells withdraw from the cell cycle and initiate migration and differentiation.The posterior pole of the retina is the first to form during development. In this region most neuroepithelial cells complete mitotic activity between the third and sixth day of incubation. Presumptive ganglion cells initiate the withdrawal process, and they are soon followed by the neuroepithelial precursors of amacrine, horizontal, and receptor cells. Bipolar cell precursors are the last to begin and the last to complete cell cycle activity. It is worthy of note, however, that, in any given region of the retina, neuroepithelial cells of all types cease mitosis in close, overlapping succession.These results are in reasonable agreement with those previously published on the chick retina by Fujita and Horii (1963), and other investigators on the mouse (Mus), killifish (Fundulus), and toad (Xenopus). The present data are also consistent with those proposals of Angevine (1970), Jacobson, 1968a, Jacobson, 1968b, Jacobson, 1970, and others that relate the cessation of mitotic activity of neuroepithelial cells to the determination of neuronal size, axon length, and the specification of neuronal connections.     

Transport and metabolism of N-δ-chloroacetyl-l-ornithine by Saccharomyces cerevisiae

The general amino acid transport system of Saccharomyces cerevisiae functions in the uptake of neutral, basic, and acidic amino acids (M. Grenson, C. Hou, and M. Crabeel, 1970,J. Bacteriol. 103, 770–777; J. Rytka, 1975,J. Bacteriol.121, 562–570; C. Darte and M. Grenson, 1975,Biochem. Biophys. Res. Commun.67, 1028–1033). We have previously demonstrated that this transport system can be inhibited by the amino acid, N-δ-chloroacetyl-l-ornithine (NCAO) (F. S., Larimore and R.J. Roon, 1978,Biochemistry17, 431–436). In the present study radiolabeled NCAO was synthesized and its transport and metabolism studied. Under initial rate conditions: (a) NCAO was transported by the general amino acid transport system with a K_m of 52 μm, a V of 32 nmol/min/mg cells, and a pH optimum of 5.0; (b) the V for NCAO transport in gap mutants, which lack the general amino acid transport system, was approximately 1% of that observed with wild-type cells; (c) the V for NCAO in cells deprived of glucose was less than 5% of that observed when glucose was present. NCAO was transiently concentrated more than 1000-fold by yeast cells when glucose served as an energy source. The internal pool of NCAO was metabolized by the yeast cells and the products were excreted. When 100 μm [¹⁴C]NCAO was incubated with a yeast cell suspension for 8 h, more than 95% of the compound was converted into two ninhydrin-negative excretory products. The effect of NCAO on the growth of yeast cells was determined. Wild-type strains did not grow when 1 mm NCAO was present in the medium. The growth of gap mutants was not inhibited by 1 mm NCAO.     

Refinements in the rapid isolation of glutamine synthetase from Escherichia coli

In an earlier paper, we described a procedure for the isolation of glutamine synthetase and the protein product of the groE gene (pgroE) by polyethyleneimine precipitation and affinity chromatography on a Blue Dextran column (Z. F. Burton and D. Eisenberg, 1980, Arch. Biochem. Biophys.205, 478–488). Subsequently we found that several of the steps can be omitted when isolating glutamine synthetase. Two procedures are described which are very rapid and quantitative for the recovery of glutamine synthetase activity and which are useful for handling quantities of cells at least up to 500 g.     

Cell surface glycosyltransferase activities during normal and mutant (TT) mesenchyme migration

Migrating cells possess surface glycosyltransferase activity toward extracellular substrates, and the appearance of enzyme activity coincides with the onset of cellular migration (Shur, 1977a, Shur, 1977b, Develop. Biol.58, 23–39, 40–55; E. A. Turley and S. Roth, 1979, Cell17, 109–115). In this paper, surface glycosyltransferases were examined during normal and $\text{T}\text{T}$ mutant mesenchyme migration. Of six glycosyltransferases that were assayed, only galactosyltransferase was present at significant levels on the cell surface, despite the presence of a variety of intracellular glycosyltransferases. All controls have been performed to show clearly the enzyme activity was cell surface localized. In both normal and $\text{T}\text{T}$ embryos, surface galactosyltransferase activity was localized, by autoradiography, primarily to migrating mesenchymal cells, and to a lesser degree, to presumptive neural epithelium. During primitive streak formation, putative $\text{T}\text{T}$ embryos were devoid of surface galactosyltransferase activity. However, as development progressed, the $\text{T}\text{T}$ level of activity eventually exceeded wild-type levels by two- to sixfold and was evident in $\text{T}\text{T}$ tissues prior to the onset of microscopic pathology. Other surface enzymes assayed did not show any $\text{T}\text{T}\text{-}\text{dependent}$ increase in activity. The extracellular galactosyl acceptors were not chloroform:methanol soluble, and glycopeptides prepared by exhaustive Pronase digestion were excluded from Sephadex G-50. This large galactosylated glycoconjugate was readily digestable with endo-β-galactosidase, and, therefore, is similar to the poly-N-acetyllactosamine chains previously identified on early embryonic tissues (A. Kapadia, T. Feizi, and M. J. Evans, 1981, Exp. Cell. Res.131, 185–195; T. Muramatsu, G. Gachelin, M. Damonneville, C. Delarbre, and F. Jacob, 1979, Cell18, 183–191; A. Heifetz, W. J. Lennarz, B. Libbus, and Y. -C. Hsu, 1980, Develop. Biol.80, 398–408). These results support an involvement of surface galactosyltransferases in mesenchyme formation and during migration on poly-N-acetyllactosamine substrates.     

Polypeptide composition of acetylcholine receptor purified from teleost and elasmobranch electroplax membranes

Acetylcholine receptor (AcChR) was solubilized and purified from membranes derived from electric organs of the marine fish Torpedo marmorata, Torpedo nobiliana, Narcine brasliensis, and of the freshwater eel, Electrophorus electricus, using techniques originally developed for Torpedo californica (27., 28.Biochem. Biophys. Res. Commun.49, 572–578; 1973, Biochemistry12, 852–856. The conditions used were identical in each case and the goal was to determine the degree of similarity between receptors from each source since conflicting reports have appeared with regard to polypeptide composition. The Torpedo and Narcine preparations were of high specific activity and exhibited four polypeptide components of apparent molecular weights 64, 59, 50, and 40 × 10³ upon polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Two components were observed upon gel electrophoresis in sodium cholate or upon sucrose density gradient centrifugation, representing monomeric and dimeric forms. Eel acetylcholine receptor exhibited three major subunits of apparent molecular weights 57, 49, and 40 × 10³. The amino acid and neutral sugar composition of the purified receptor preparations have been determined. The results support the contention that the receptor is composed of several types of polypeptide.