Enzyme or Electrode?

The process of dissimilatory metal reduction shapes our environment on a global scale by using minerals as terminal acceptors in a biological electron transport chain employed by bacteria under anaerobic conditions. In this issue of Structure, Edwards et?al. present the structure of an extracellular undecaheme cytochrome involved in the step of electron transfer to metal oxides.     

Mean Lifetime and First-Passage Time of the Enzyme Species Involved in an Enzyme Reaction. Application to Unstable Enzyme Systems

Taking as starting point the complete analysis of mean residence times in linear compartmental systems performed by Garcia-Meseguer et al. (Bull. Math. Biol. 65:279–308, 2003) as well as the fact that enzyme systems, in which the interconversions between the different enzyme species involved are of first or pseudofirst order, act as linear compartmental systems, we hereby carry out a complete analysis of the mean lifetime that the enzyme molecules spend as part of the enzyme species, forms, or groups involved in an enzyme reaction mechanism. The formulas to evaluate these times are given as a function of the individual rate constants and the initial concentrations of the involved species at the onset of the reaction. We apply the results to unstable enzyme systems and support the results by using a concrete example of such systems. The practicality of obtaining the mean times and their possible application in a kinetic data analysis is discussed.     

Identification of G Protein α Subunit-Palmitoylating Enzyme

The heterotrimeric G protein α subunit (Gα) is targeted to the cytoplasmic face of the plasma membrane through reversible lipid palmitoylation and relays signals from G-protein-coupled receptors (GPCRs) to its effectors. By screening 23 DHHC motif (Asp-His-His-Cys) palmitoyl acyl-transferases, we identified DHHC3 and DHHC7 as Gα palmitoylating enzymes. DHHC3 and DHHC7 robustly palmitoylated Gα_q, Gα_s, and Gα_i2 in HEK293T cells. Knockdown of DHHC3 and DHHC7 decreased Gα_q/11 palmitoylation and relocalized it from the plasma membrane into the cytoplasm. Photoconversion analysis revealed that Gα_q rapidly shuttles between the plasma membrane and the Golgi apparatus, where DHHC3 specifically localizes. Fluorescence recovery after photobleaching studies showed that DHHC3 and DHHC7 are necessary for this continuous Gα_q shuttling. Furthermore, DHHC3 and DHHC7 knockdown blocked the α_1A-adrenergic receptor/Gα_q/11-mediated signaling pathway. Together, our findings revealed that DHHC3 and DHHC7 regulate GPCR-mediated signal transduction by controlling Gα localization to the plasma membrane.G-protein-coupled receptors (GPCRs) form the largest family of cell surface receptors, consisting of more than 700 members in humans. GPCRs respond to a variety of extracellular signals, including hormones and neurotransmitters, and are involved in various physiologic processes, such as smooth muscle contraction and synaptic transmission (20, 25). Heterotrimeric G proteins, composed of α, β, and γ subunits, transduce signals from GPCRs to their effectors and play a central role in the GPCR signaling pathway (13, 21, 24, 32). Although the Gα subunit seems to localize stably at the cytosolic face of the plasma membrane (PM), a recent report suggested that Gα_o, a Gα isoform, shuttles rapidly between the PM and intracellular membranes (2). The PM targeting of Gα requires both interaction with the Gβγ complex and subsequent lipid palmitoylation of Gα (22). Thus, palmitoylation of Gα is a critical determinant of membrane targeting of the heterotrimer Gαβγ.Protein palmitoylation is a common posttranslational modification with lipid palmitate and regulates protein trafficking and function (7, 18). Gα is a classic and representative palmitoyl substrate (19, 38), and recent studies revealed that protein palmitoylation modifies virtually almost all the components of G-protein signaling, including GPCRs, Gα subunits, several members of the RGS (regulators of G-protein signaling) family of GTPase-activating proteins, GPCR kinase GRK6, and some small GTPases (7, 33). This common lipid modification plays an important role in compartmentalizing G-protein signaling to the specific microdomain, such as membrane caveolae and lipid raft (26). The palmitoyl thioester bond is relatively labile, and palmitates on substrates turn over rapidly, allowing proteins to shuttle between the cytoplasm/intracellular organelles and the PM (2, 3, 27). For example, binding of isoproterenol to the β-adrenergic receptor markedly accelerates the depalmitoylation of the associated Gα_s, shifting Gα_s to the cytoplasm (37). This receptor activation-induced depalmitoylation was also observed in a major postsynaptic PSD-95 scaffold, which anchors the AMPA (alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid)-type glutamate receptor at the excitatory postsynapse through stargazin (6). On glutamate receptor activation, accelerated depalmitoylation of PSD-95 dissociates PSD-95 from postsynaptic sites and causes AMPA receptor endocytosis (6). Thus, palmitate turnover on Gα_s and PSD-95 is accelerated by receptor activation, contributing to downregulation of the signaling pathway. However, the enzymes that add palmitate to proteins (palmitoyl-acyl transferases [PATs]) and those that cleave the thioester bond (palmitoyl-protein thioesterases) were long elusive.Recent genetic studies in Saccharomyces cerevisiae identified Erf2/Erf4 (1, 40) and Akr1 (29) as PATs for yeast Ras and yeast casein kinase 2, respectively. Erf2 and Akr1 have four- to six-pass transmembrane domains and share a common domain, referred to as a DHHC domain, a cysteine-rich domain with a conserved Asp-His-His-Cys signature motif. Because the DHHC domain is essential for the PAT activity, we isolated 23 mammalian DHHC domain-containing proteins (DHHC proteins) and developed a systematic screening method to identify the specific enzyme-substrate pairs (11, 12): DHHC2, -3, -7, and -15 for PSD-95 (11); DHHC21 for endothelial NO synthase (10); and DHHC3 and -7 for GABA_A receptor γ2 subunit (9). Several other groups also reported that DHHC9 with GCP16 mediates palmitoylation toward H- and N-Ras (36) and that DHHC17, also known as HIP14, palmitoylates several neuronal proteins: huntingtin (14), SNAP-25, and CSP (14, 23, 35). However, the existence of PATs for Gα has been controversial because spontaneous palmitoylation of Gα could occur in vitro (4).In this study, we screened the 23 DHHC clones to examine which DHHC proteins can palmitoylate Gα. We found that DHHC3 and -7 specifically and robustly palmitoylate Gα at the Golgi apparatus. Inhibition of DHHC3 and -7 reduces Gα_q/11 palmitoylation levels and delocalizes it from the PM to the cytoplasm in HeLa cells and primary hippocampal neurons. Also, DHHC3 and -7 are necessary for the continuous Gα_q shuttling between the Golgi apparatus and the PM. Finally, blocking DHHC3 and -7 inhibits the α_1A-adrenergic receptor [α_1A-AR]/Gα_q-mediated signaling pathway, indicating that DHHC3 and -7 play an essential role in GPCR signaling by regulating Gα localization.     

Kinetic Studies on α-Aminoisobutyrate Decomposing Enzyme

An enzyme which catalyzes a decomposition of α-aminoisobutyrate (AIB) was purified and its kinetic properties were investigated. Michaelis constants for AIB decomposing reaction are able to be calculated by Ping Pong initial velocity equation. This enzyme catalyzes also l-alanine: α-ketobutyrate transamination as well as AIB decomposing reaction. Approximately equal values of Michaelis constants were obtained for α-ketobutyrate and pyridoxal 5′-phosphate (PLP), which are common substrates of both reactions.

In higher concentration of the enzyme, transamination between PLP and AIB or l-alanine was detected, whereas the reaction between pyridoxamine 5′-phosphate and pyruvate was not observed. These results are probably ascribed to a difference in affinity of two coenzymes for the enzyme.     

Susceptibilities of an Irreversible Michaelis – Menten Enzyme

The nonlinear response of the simplest irreversible Michaelis – Menten enzyme is considered. In the context of metabolic networks, i.e. in vivo, the enzyme is subject to sustained, frequently time-dependent, input fluxes that keep the system out of equilibrium. The connection between the fluxes and the response is investigated by means of a new sensitivity analysis. The kinetics of the enzyme is simple enough to allow for the computations to be carried out analytically. In particular, a set of sensitivities of the response with respect to the substrate influx, the susceptibilities, is derived. The susceptibilities are multivariate functions and thus are suitable for predicting complete progress curves of several variables of biochemical interest, namely, rates and concentrations. This is shown by means of an example. The relationship between the susceptibilities and the stoichiometry of the reaction is also taken into account. Moreover, all the required information comes from decay experiments of initial concentrations, which are common in enzymological setups.     

Soybean Leaf Urease: A Seed Enzyme?

The soybean (Glycine max L. [Merrill]) var Itachi has 0.2 to 0.3% the urease activity found in developing embryos of a normal line, Prize. The hydroxyurea sensitivity and pH preference of this basal seed urease indicate that it represents a unique enzyme rather than an unusually low level of the normal seed urease. Itachi's seed urease is less sensitive to hydroxyurea inhibition (65-80% inhibition) than Prize seed urease (85-95% inhibition) and is more active at pH 6.1 and 8.8 than at 7.4, whereas the normal seed urease is least active at pH 8.8. Both properties of the basal seed urease are in agreement with the behavior of the leaf urease in extracts of Prize and Itachi leaves.

Neither the leaf urease nor the Itachi seed urease is immuneprecipitated by affinity-purified seed urease antibodies. However, when antibody is in excess, Staphylococcus aureus (Cowan) cell walls containing protein A can precipitate soluble antibody-urease complexes (47-68% of total enzyme) from both leaf (Itachi and Prize) and Itachi seed extracts. Under identical conditions, greater than 90% of Prize seed urease is precipitated. At a 100-fold dilution of antibody, 60% of Prize seed urease is still antibody-complexed while the antibody recognition of the leaf or Itachi seed urease is reduced to 2 to 24%.

The cell culture urease also resembles leaf urease by the criteria of pH preference, hydroxyurea sensitivity, and recognition by seed urease antibodies. In the presence of cycloheximide, nickel stimulates cell culture urease levels (14- or 35-fold depending on assay pH) indicating that cell cultures make a preponderance of apourease under nickel-limiting conditions.

Inasmuch as the ureases of leaf, cell culture, and Itachi seeds are more closely related to each other than they are to the abundant (Prize) seed urease, suggests that the three tissues either contain an identical urease or related tissue-specific isozymes. This second form of urease may have an assimilatory role since it is found in both leaf and seed sink tissues and is required for urea assimilation in cell culture (Polacco 1977 Plant Physiol 59: 827-830).

     

Aminotransferase Activity of α-Aminoisobutyric Acid Decomposing Enzyme

The enzyme which decomposes α-aminoisobutyric acid (AIB) to acetone in presence of pyruvate is active to various α-dialkyl-α-amino acids. From relative rates of decomposition of AIB, l-(+)isovaline, 2-ethyl-2-aminobutyrate and d-(?)isovaline, it was suggested that a carbon chain having a configuration of natural d-amino acids was required for the enzyme action.

On the other hand, this enzyme catalyzes the transamination between l-alanine and α-ketobutyrate. The equilibrium constant in the direction of l-α-aminobutyrate formation is 0.62. Electrophoretic migration, α-keto acid specificity and pH dependence of the aminotransferase activity were similar to those of AIB decomposing activity. Moreover, both activities increased in cells incubated by either l-α-aminobutyrate or AIB.     

Conformational “Fingerprint” of the Angiotensin-Converting Enzyme

The angiotensin-converting enzyme (ACE) is a zinc-dependent metalloproteinase widely occurring in the organism; it metabolizes many peptides and plays a key role in blood pressure regulation and vascular remodeling. This enzyme is expressed as a type-1 membrane glycoprotein on the surface of endothelial and epithelial cells, but is also found in a soluble form in biological fluids. In this study, we used purified ACE from lungs, which is mainly produced by endothelial cells of lung capillaries; ACE from heart, produced by endothelial heart cells and, probably, by myofibroblasts; and ACE from seminal fluid, produced by the epithelial cells of the prostate and epididymis. The pattern of binding of a set of 17 mAbs to different conformational epitopes on the surface of two domains of the human ACE significantly differed for ACEs from different organs. This pattern (the conformational “fingerprint” of ACE) reflects the local conformation of the surface of a particular ACE. The differences in the conformational fingerprints of ACEs expressed by different cell types, or even by similar cells but in different organs, can be explained by the posttranslational modification of ACE protein in these organs and, primarily, different glycosylation of N-glycosylation sites Asn25, Asn117, Asn289, Asn666, Asn685, and Asn731. The mass spectrometry of tryptic hydrolyzates of ACEs isolated from different human organs made it possible to reveal, in the composition of different ACEs, N-glycosylation sites that are really occupied by glycans, namely, Asn in positions 82, 117, 416, 648, 666, 685, and 731 in ACE from seminal fluid; Asn in positions 117, 648, 666, and 685 in ACE from lungs; and Asn in positions 117, 480, 666, and 685 in ACE from heart. Differences in the plausible structures of glycans in ACE, in particular, at the Asn666 N-glycosylation site were demonstrated, which can explain the differences in the efficiency of binding of mAbs to ACE from different organs.     

Verteilung und Aktivität hydrolytischer Enzyme in Hypophysenadenomen

Zusammenfassung An 17 Hypophysenadenomen [12 Chromophobe (endokrin-inaktive), 5 Mischtypadenome (endokrin-aktive)] wurde das Verhalten und die Aktivität der hydrolytischen Enzyme -Galaktosidase, -Glucuronidase, -Glykosidase und Arylsulfatase mit histochemischen Methoden geprüft.Die Mischtypadenome zeigen insgesamt eine höhere Aktivität als die Chromophoben. Dabei reagieren -Galaktosidase und -Glykosidase am stärksten, -Glucuronidase etwas schwächer, während Arylsulfatase die niedrigste Aktivität zeigt. Die Befunde werden mit anderen enzymhistochemischen Untersuchungen an Hypophysenadenomen und tierexperimentellen Ergebnissen verglichen. Daraus folgt, daß wahrscheinlich zwischen der Aktivität der untersuchten lysosomalen Enzyme und der endokrinen Aktivität ein Zusammenhang besteht.

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Summary Behaviour and activity of the hydrolytic enzymes -galactosidase, -glucuronidase, -glycosidase and arylsulphatase are tested in 17 adenomas of the hypophysis (12 chromophobic, endocrine-inactive; 5 mixed cell adenomas, endocrine-active).Mixed cell adenomas show an altogether higher activity than chromophobic adenomas. -galactosidase and -glycosidase show the highest, -glucuronidase a slightly lower, and arylsulphatase the lowest activity. The findings are compared with other enzymhistochemical methods and results from animal experiments. The results of this comparison indicate that there is a correlation between the endocrine activity of the lysosomal enzymes in question.

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Der Deutschen Forschungsgemeinschaft danken wir für ihre Unterstützung.

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Fräulein Renate Kott, Fräulein Marianne Lehnen und Fräulein Edith Klasmeier danke ich für ihre technische Unterstützung.     

Is Na + K ATPase a Myelin-Associated Enzyme?

The Na + K ATPase activity associated with purified myelin has been investigated. On the basis of marker enzyme studies, the Na + K ATPase activity of myelin was higher than could be accounted for by microsomal contamination. Fractions prepared from white matter-enriched areas of rat brain showed a threefold enrichment in Na + K ATPase activity in myelin as compared with the white matter homogenate. The ATPase activity in myelin was stimulated fourfold by treatment with sodium deoxycholate, but the activity in the whole brain homogenate and the microsomal fraction was only doubled. This discontinuity temperature for Na + K ATPase activity was significantly higher for the myelin fraction (29 degrees C) than for the microsomal fraction (21 degrees C), but the energies of activation, both above and below the discontinuity temperature, were the same for both fractions, Myelin Na + K ATPase had a lower affinity for strophanthidin than the microsomal enzyme, but both fractions were inhibited to the same extent by 10-3 M-strophanthidin. The evidence thus indicated that much of the ATPase activity of myelin is not the result of microsomal contamination. Although the possibility of axolemmal contamination cannot be ruled out conclusively, indirect evidence suggest that this is not a significant factor and that Na + K ATPase may be a myelin-associated enzyme.     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Decreased Long-Chain Fatty Acyl CoA Elongation Activity in Quaking and Jimpy Mouse Brain: Deficiency in One Enzyme or Multiple Enzyme Activities?

Using long-chain fatty acyl CoAs (arachidoyl CoA and behenoyl CoA), a decrease in overall fatty acid chain elongation activity was observed in the quaking and jimpy mouse brain microsomes relative to controls. Arachidoyl CoA (20:0) and behenoyl CoA (22:0) elongation activities were depressed to about 50% and 80% of control values in quaking and jimpy mice, respectively. Measurement of the individual enzymatic activities of the elongation system revealed a single deficiency in enzyme activity; only the condensation activity was reduced to the same extent as total elongation in both quaking and jimpy mice. The activities of the other three enzymes, beta-ketoacyl CoA reductase, beta-hydroxyacyl CoA dehydrase, and trans-2-enoyl CoA reductase, in both mutants were similar to the activities present in the control mouse. In addition, the activities of these three enzymes were more than two to three orders of magnitude greater than the condensing enzyme activity in all three groups, establishing that the condensing enzyme catalyzes the rate-limiting reaction step of total elongation. When the elongation of palmitoyl CoA was measured, only a 25% decrease in total elongation occurred in both mutants; a similar percent decrease in the condensation of palmitoyl CoA also was observed. The activities of the other three enzymes were unaffected. These results support the concept of either multiple elongation pathways or multiple condensing enzymes.     

Some Characteristics of a New Enzyme “Glycerol Oxidase”

Novel acremolactones B and C were isolated from the acremolactone A-producing Acremonium roseum I4267. The structure of acremolactone B having a phenylpyridyl γ-lactone was elucidated by spectroscopic methods. It showed plant growth inhibitory activity toward Chinese cabbage seedlings. The congener of acremolactone C having a phenylcyclopentenone γ-lactone showed weak activity.