Homologous gene expression in intergeneric fox hybrids (Alopex lagopus x Vulpes vulpes). I. Comparative electrophoretic analysis of blood proteins and enzymes in Arctic and silver foxes]

A comparative study of 27 enzymes and proteins in blue and silver foxes was carried out by means of starch gel electrophoresis. The structure of these enzymes and proteins is determined by about 33 genes. It is shown that a number of blood enzymes and proteins of these species is represented by a single electrophoretic form, while lactate dehydrogenase, carboanhydrase, arylesterase, carboxylesterase, diaphorase, hexokinase and tetrasolium oxidase have several forms. It is also found that these species differ in seven enzymes and proteins: diaphorase, G-6-PD, adenylate kinase, carboxylesterase, albumin, prealbumin, transferrins. Other enzymes and proteins are similar in their electrophoretic mobility. The data obtained afford the evidence that the two species (Vulpes vulpes and Alopex lagopus) differ in a set of enzymes and proteins.     

Sortase enzymes in Gram-positive bacteria

In Gram-positive bacteria proteins are displayed on the cell surface using sortase enzymes. These cysteine transpeptidases join proteins bearing an appropriate sorting signal to strategically positioned amino groups on the cell surface. Working alone, or in concert with other enzymes, sortases either attach proteins to the cross-bridge peptide of the cell wall or they link proteins together to form pili. Because surface proteins play a fundamental role in microbial physiology and are frequently virulence factors, sortase enzymes have been intensely studied since their discovery a little more than a decade ago. Based on their primary sequences and functions sortases can be partitioned into distinct families called class A to F enzymes. Most bacteria elaborate their surfaces using more than one type of sortase that function non-redundantly by recognizing unique sorting signals within their protein substrates. Here we review what is known about the functions of these enzymes and the molecular basis of catalysis. Particular emphasis is placed on 'pilin' specific class C sortases that construct structurally complex pili. Exciting new data have revealed that these enzymes are amazingly promiscuous in the substrates that they can employ and that there is a startling degree of diversity in their mechanism of action. We also review recent data that suggest that sortases are targeted to specific sites on the cell surface where they work with other sortases and accessory factors to properly function.     

Deubiquitinating enzymes--the importance of driving in reverse along the ubiquitin-proteasome pathway

Ubiquitination of proteins is now recognized to target proteins for degradation by the proteasome and for internalization into the lysosomal system, as well as to modify functions of some target proteins. Although much progress has been made in characterizing enzymes that link ubiquitin to proteins, our understanding of deubiquitinating enzymes is less developed. These enzymes are involved in processing the products of ubiquitin genes which all encode fusion proteins, in negatively regulating the functions of ubiquitination (editing), in regenerating free ubiquitin after proteins have been targeted to the proteasome or lysosome (recycling) and in salvaging ubiquitin from possible adducts formed with small molecule nucleophiles in the cell. A large number of genes encode deubiquitinating enzymes suggesting that many have highly specific and regulated functions. Indeed, recent findings provide strong support for the concept that ubiquitination is regulated by both specific pathways of ubiquitination and deubiquitination. Interestingly, many of these enzymes are localized to subcellular structures or to molecular complexes. These localizations play important roles in determining specificity of function and can have major influences on their catalytic activities. Future studies, particularly aimed at characterizing the interacting partners and potential substrates in these complexes as well as at determining the effects of loss of function of specific deubiquitinating enzymes will rapidly advance our understanding of the important roles of these enzymes as biological regulators.     

Enzymes of nucleotide metabolism: the significance of subunit size and polymer size for biological function and regulatory properties

The 72 enzymes in nucleotide metabolism, from all sources, have a distribution of subunit sizes similar to those from other surveys: an average subunit Mr of 47,900, and a median size of 33,300. The same enzyme, from whatever source, usually has the same subunit size (there are exceptions); enzymes having a similar activity (e.g., kinases, deaminases) usually have a similar subunit size. Most simple enzymes in all EC classes (except class 6, ligases/synthetases) have subunit sizes of less than 30,000. Since structural domains defined in proteins tend to be in the Mr range of 5,000 to 30,000, it may be that most simple enzymes are formed as single domains. Multifunctional proteins and ligases have subunits generally much larger than Mr 40,000. Analyses of several well-characterized ligases suggest that they also have two or more distinct catalytic sites, and that ligases therefore are also multifunctional proteins, containing two or more domains. Cooperative kinetics and evidence for allosteric regulation are much more frequently associated with larger enzymes: such complex functions are associated with only 19% of enzymes having a subunit Mr less than or equal to 29,000, and with 86% of all enzymes having a subunit Mr greater than 50,000. In general, larger enzymes have more functions. Only 20% of these enzymes appear to be monomers; the rest are homopolymers and rarely are they heteropolymers. Evidence for the reversible dissociation of homopolymers has been found for 15% of the enzymes. Such changes in quaternary structure are usually mediated by appropriate physiological effectors, and this may serve as a mechanism for their regulation between active and less active forms. There is considerable structural organization of the various pathways: 19 enzymes are found in various multifunctional proteins, and 13 enzymes are found in different types of multienzyme complexes.     

PII signal transduction proteins: sensors of alpha-ketoglutarate that regulate nitrogen metabolism

PII proteins are small homotrimeric signal transduction proteins that regulate the activities of metabolic enzymes and permeases, and control the activities of signal transduction enzymes. The protein family shows high conservation, with examples in eukaryota (plants and eukaryotic algae), archaea, and bacteria. This distribution indicates that PII is one of the most ancient signalling proteins known.     

Patterns of indirect protein interactions suggest a spatial organization to metabolism

It has long been believed that cells organize their cytoplasm so as to efficiently channel metabolites between sequential enzymes. This metabolic channeling has the potential to yield higher metabolic fluxes as well as better regulatory control over metabolism. One mechanism for achieving such channeling is to ensure that sequential enzymes in a pathway are physically close to each other in the cell. We present evidence that indirect protein interactions between related enzymes represent a global mechanism for achieving metabolic channeling; the intuition being that protein interactions between enzymes and non-enzymatic mediator proteins are a powerful means of physically associating enzymes in a modular fashion. By analyzing the metabolic and protein-protein interactions networks of Escherichia coli, yeast and humans, we are able to show that all three species have many more indirect protein interactions linking enzymes that share metabolites than would be expected by chance. Moreover, these interactions are distributed non-randomly in the metabolic network. Our analyses in yeast and E. coli show that reactions possessing such interactions also show higher flux than do those lacking them. On the basis of these observations, we suggest that an important role of protein interactions with mediator proteins is to contribute to the spatial organization of the cell. This hypothesis is supported by the fact that these mediator proteins are also enriched with annotations related to signal transduction, a system where scaffolding proteins are known to limit cross-talk by controlling spatial localization.     

Lysosomal enzymes possess a common antigenic determinant in the cellular slime mold, Dictyostelium discoideum

Antisera have been prepared against two lysosomal enzymes of the cellular slime mold, Dictyostelium discoideum. The two purified enzyme preparations used for immunization, N-acetylglucosaminidase and beta-glucosidase-1, show no cross-contamination with each other and no significant contamination by other lysosomal enzymes. However, antisera raised against either enzyme bind equally well to seven different lysosomal enzymes and show no preference for the enzyme against which they were raised. A total of 10 different antisera have been examined and all show similar results. Preadsorption of antisera with either purified enzyme removes all antibody activity against the other enzyme. Evidence is presented which indicates that the same species of antibodies are responsible for the precipitation of seven lysosomal enzymes. These data are discussed in terms of the proposal that the antigen that is shared by the lysosomal enzymes is a post-translational modification of the enzyme proteins. We have sought to further characterize the distribution of this common antigen among cellular proteins. We show that N-acetylglucosaminidase and beta-glucosidase-1 represent less than 5% of the total common antigen containing proteins in the cell. Precipitation of 35S-labeled cellular proteins from vegetative cells indicates that as much as 15-30% of the total cell protein may possess the common antigen. Preadsorption experiments confirm that all of the proteins immunoprecipitated in these experiments are recognized by the same antibodies that precipitate the lysosomal enzyme activities. Most of the labeled proteins are secreted into the medium along with the lysosomal enzyme activities during axenic growth. During the developmental phase of the life cycle of Dictyostelium, the total amount of the common antigen decreases about 2-fold relative to total cell protein. However, the synthesis of antigenic proteins continues throughout most of development.     

Isolation and characterization of two tryptophan biosynthetic enzymes, indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, from Bacillus subtilis.

Two of the enzymes responsible for tryptophan biosynthesis in Bacillus subtilis have been extensively purified. These proteins are indole-3-glycerol phosphate synthase and N-(5'-phosphoribosyl) anthranilate isomerase. By comparison to the non-differentiating enteric bacteria in which these two enzymes are fused into a single polypeptide, the isolation of the indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase from B. subtilis has demonstrated that the two proteins are separate species in this organism. The two enzymes were clearly separable by anion-exchange chromatography without any significant loss of activity. Molecular weights were determined for both enzymes by gel filtration and sodium dodecyl sulfate-slab gel electrophoresis, and indicated that the indoleglycerol phosphate synthase is the slightly larger of the two proteins. The minimum molecular weight for indoleglycerol phosphate synthase was 23,500, and that for phosphoribosyl anthranilate isomerase was 21,800. Both enzymes have been examined as to conditions necessary to achieve maximal activity of their individual functions and to maintain that activity.     

Human saliva as a source of biochemical genetic markers. II. Genetic interpretations and possible utilization.

Human saliva enzymes are compared to analogous blood enzymes. The genetic interpretations for variants of several saliva proteins are reviewed. The possible use of human saliva enzymes and proteins in population genetic studies and disease diagnosis is discussed.     

Biochemical and genetical approaches to the mechanism of action of penicillin

Since the discovery in 1965 that penicillin inhibits the transpeptidation reaction in peptidoglycan synthesis, a considerable effort has been put into the purification of enzymes that catalyse this reaction. This has resulted in the recognition that bacteria possess multiple forms of these penicillin-sensitive enzymes and has made it difficult to identify the precise target that penicillin inactivates to kill the organism. Recently penicillin-sensitive enzymes have been detected and studies as penicillin-binding proteins on sodium dodecyl sulphate polyacrylamide gels. The availability of this convenient method for identifying penicillin-sensitive enzymes has allowed biochemical and genetical approaches to be used to dissect their roles in the lethal effects of penicillin and other beta-lactam antibiotics. Three penicillin-binding proteins (1 B, 2 and 3) have been identified as killing targets for penicillin in Escherichia coli, whereas four other binding proteins are not implicated in the mechanism of action of the antibiotic. The complex biological effects that beta-lactam antibiotics produce on the growth of E. coli can be explained by their interaction with the three killing targets. Progress in the correlation of penicillin-binding proteins with penicillin-sensitive enzymes and in the development of strains of E. coli that overproduce penicillin-binding proteins is discussed.     

Specific associations of T4 bacteriophage proteins with immobilized deoxycytidylate hydroxymethylase.

Is the enzymatic machinery for DNA precursor biosynthesis linked to the DNA replication apparatus? To identify intermolecular associations among deoxyribonucleotide biosynthetic enzymes and to ask whether these enzymes are linked to replication proteins, we analyzed radiolabeled T4 bacteriophage proteins that bind specifically to a column of immobilized T4 deoxycytidylate hydroxymethylase. More than a dozen T4 proteins and a few Escherichia coli proteins are adsorbed specifically by this column. Several of the T4 proteins were identified by two-dimensional gel electrophoresis and radioautography. These include five enzymes involved in DNA precursor biosynthesis, dCMP hydroxymethylase, thymidylate synthase, dihydrofolate reductase, dCTPase-dUTPase, and ribonucleotide reductase large and small subunits, plus several proteins of DNA metabolism and replication. Analysis of extracts of cells infected with phage amber mutants defective in specific proteins suggested a specific association involving thymidylate synthase and the gene 32 single-strand DNA-binding protein.     

Deubiquitinating enzymes: their diversity and emerging roles

A growing number of important regulatory proteins within cells are modified by conjugation of ubiquitin, a well-conserved 76-amino-acid polypeptide. The ubiquitinated proteins are targeted to proteasome for degradation or alternative metabolic fates, such as triggering of plasma membrane endocytosis and trafficking to vacuoles or lysosomes. Deubiquitination, reversal of this modification, is being recognized as an important regulatory step. Deubiquitinating enzymes are cysteine proteases that specifically cleave off ubiquitin from ubiquitin-conjugated protein substrates as well as from its precursor proteins. Genome sequencing projects have identified more than 90 deubiquitinating enzymes, making them the largest family of enzymes in the ubiquitin system. This review will concentrate on recent important findings as well as new insights into the diversity and emerging roles of deubiquitinating enzymes in the ubiquitin-dependent pathway.     

Enzyme-inhibitor interactions at the plant-pathogen interface

The plant apoplast during plant-pathogen interactions is an ancient battleground that holds an intriguing range of attacking enzymes and counteracting inhibitors. Examples are pathogen xylanases and polygalacturonases that are inhibited by plant proteins like TAXI, XIP, and PGIP; and plant glucanases and proteases, which are targeted by pathogen proteins such as GIP1, EPI1, EPIC2B, and AVR2. These seven well-characterized inhibitors have different modes of action and many probably evolved from inactive enzymes themselves. Detailed studies of the structures, sequence variation, and mutated proteins uncovered molecular struggles between these enzymes and their inhibitors, resulting in positive selection for variant residues at the contact surface, where single residues determine the outcome of the interaction.     

Ubiquitination and deubiquitination: targeting of proteins for degradation by the proteasome

The post-translational modification of proteins by covalent attachment of ubiquitin targets these proteins for degradation by the proteasome. An astounding number of proteins are involved in ubiquitination and deubiquitination of proteins. The pathways are combinatorial, and selectivity of proteolysis will depend strongly on the exact combination of ubiquitinating and deubiquitinating enzymes present at any time. In addition to temporal control, it is likely that these modifications are also regulated spatially. In this review, we discuss the regulation of ubiquitination by enzymes of this pathway and highlight some of the outstanding problems in understanding this regulation.     

Compartmentation of glycolysis in trypanosomes: a potential target for new trypanocidal drugs

In African trypanosomes most enzymes of the glycolytic pathway are found in a microbody-like organelle, called the glycosome. The analysis of their structural and functional properties has shown that these glycosomal enzymes possess some specific features which are absent from the cytosolic proteins of trypanosomes and from the glycolytic enzymes of other organisms, where glycolysis is not compartmentalized within an organelle. The specific properties of the glycosomal enzymes may be responsible for the routing of the proteins from their site of synthesis, the cytosol, into the glycosome, or they may be involved in the proper functioning of the enzymes within the organelle. Whatever the role of the unique features, they are potential targets for compounds that could specifically interfere with glycolysis in trypanosomes. Therefore, a detailed study of the glycolytic enzymes of trypanosomes may lead to the development of therapeutically useful drugs against these harmful parasites.     

Recent developments in dynamic electrochemical studies of adsorbed enzymes and their active sites

This review outlines recent developments in electrochemical investigations of proteins adsorbed on electrodes. The important point about 'protein film voltammetry' is that it addresses the rates of reactions that occur in enzymes - catalysis, inhibition, electron flow - as a function of potential; in other words, it introduces the 'potential dimension' into enzyme kinetics. Some surprisingly subtle, yet significant observations are made, including demonstration of a special role for Mo(V) in the catalytic cycle of Mo enzymes, quantitation of the catalytic bias in multi-centred enzymes such as mitochondrial Complex I, insight into mechanisms of proton transfer in enzymes, and properties of proteins that are covalently attached directly to a gold surface.     

Properties of salt-resistant lipase and lipoprotein lipase purified from human post-heparin plasma.

Lipoprotein lipase and salt-resistant lipase were isolated from human post-heparin plasma. The proteins of human post-plasma lipoprotein lipase and salt-resistant lipase were identified and demonstrated to be immunologically different. Significant differences between the two enzymes in their relative amino acid composition were demonstrated, which indicates that the two enzymes are different proteins. When analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, the enzymes seemed to have monomer molecular weights similar to that of lipoprotein lipase purified from bovine milk.     

Identification and Chromosomal Location of Two Human Genes Encoding Enzymes Potentially Involved in Proteolytic Maturation of Farnesylated Proteins

Two human cDNAs encoding proteins similar to yeast enzymes involved in proteolytic processing of farnesylated proteins like a-factor mating pheromone and Ras2p have been cloned from an ovary cDNA library. These proteins have been tentatively called Face-1 and Face-2 (farnesylated protein-converting enzymes 1 and 2), respectively, and are integral membrane proteins, belonging to distinct families of metalloproteinases. Northern blot analysis of poly(A)+ RNAs isolated from a wide variety of human tissues demonstrated that both genes are expressed in all examined tissues, which suggests that these enzymes play housekeeping roles in normal processes. Fluorescence in situ hybridization experiments showed that the human FACE-1 gene maps to 1p34, whereas FACE-2 is located at 11q13, a region frequently amplified in human carcinomas and lymphomas. On the basis of these results, we suggest that inhibition of Face-1 and/or Face-2 could be part of strategies directed to block the functioning of prenylated proteins activated in oncogenic processes, including Ras proteins.