Comparison of the DING protein from the archaeon Sulfolobus solfataricus with human phosphate-binding protein and Pseudomonas fluorescence DING counterparts

DING proteins represent a new group of 40 kDa-related members, ubiquitous in living organisms. The family also include the DING protein from Sulfolobus solfataricus, functionally related to poly(ADP-ribose) polymerases. Here, the archaeal protein has been compared with the human Phosphate-Binding Protein and the Pseudomonas fluorescence DING enzyme, by enzyme assays and immune cross-reactivity. Surprisingly, as the Sulfolobus enzyme, the Human and Pseudomonas proteins display poly(ADP-ribose) polymerase activity, whereas a phosphatase activity was only present in Sulfolobus and human protein, despite the conserved phosphate-binding site residues in Pseudomonas DING. All proteins were positive to anti-DING antibodies and gave a comparable pattern of anti-poly(ADP-ribose) polymerase immunoreactivity with two bands, at around 40 kDa and roughly at the double of this molecular mass. The latter signal was present in all Sulfolobus enzyme preparations and proved not due to either a contaminant or a precursor protein, but likely being a dimeric form of the 40 kDa polypeptide. The common immunological and partly enzymatic behavior linking human, Pseudomonas and Sulfolobus DING proteins, makes the archaeal protein an important model system to investigate DING protein function and evolution within the cell.

     

Mechanisms of poly(ADP-ribose) polymerase catalysis; mono-ADP-ribosylation of poly(ADP-ribose) polymerase at nanomolar concentrations of NAD

Calf thymus and rat liver poly(ADP-ribose) polymerase enzymes, and the polymerase present in extracts of rat liver nuclei synthesize unstable mono-ADP-ribose protein adducts at 100 nM or lower NAD concentrations. The isolated enzyme-mono-ADP-ribose adduct hydrolyses to ADP-ribose and enzyme protein at pH values slightly above 7.0 indicating a continuous release of ADP-ribose from NAD through this enzyme-bound intermediate under physiological conditions. NH2OH at pH 7.0 hydrolyses the mono-ADP-ribose enzyme adduct. Desamino NAD and some other homologs at nanomolar concentrations act as 'forward' activators of the initiating mono-ADP-ribosylation reaction. These NAD analogs at micromolar concentrations do not affect polymer formation that takes place at micromolar NAD concentrations. Benzamides at nanomolar concentrations also activate mono-ADP-ribosylation of the enzyme, but at higher concentrations inhibit elongation at micromolar NAD as substrate. In nuclei, the enzyme molecule extensively auto-ADP-ribosylates itself, whereas histones are trans-ADP-ribosylated to a much lower extent. The unstable mono-ADP-ribose enzyme adduct represents an initiator intermediate in poly ADP-ribosylation.     

Regulation of poly(ADP-ribose) polymerase. Histone-specific adaptations of reaction products

The post-translational poly ADP-ribosylation of proteins by the nuclear enzyme poly(ADP-ribose) polymerase (EC 2.4.2.30) involves a complex pattern of ADP-ribose polymers. We have determined how this enzyme produces the various polymer size patterns responsible for altered protein function. The results show that histone H1 and core histones are potent regulators of both the numbers and sizes of ADP-ribose polymers. Each histone induced the polymerase to synthesize a specific polymer size pattern. Various other basic and/or DNA binding proteins as well as other known stimulators of poly(ADP-ribose) polymerase (spermine, MgCl2, nicked DNA) were ineffective as polymer size modulators. Testing specific proteolytic fragments of histone H1, the polymer number and polymer size modulating activity could be mapped to specific polypeptide domains. The results suggest that histones specifically regulate the polymer termination reaction of poly(ADP-ribose) polymerase.     

Temporal Patterns of Poly(ADP-Ribose) Polymerase Activation in the Cortex Following Experimental Brain Injury in the Rat

The activation of poly(ADP-ribose) polymerase, a DNA base excision repair enzyme, is indicative of DNA damage. This enzyme also undergoes site-specific proteolysis during apoptosis. Because both DNA fragmentation and apoptosis are known to occur following experimental brain injury, we investigated the effect of lateral fluid percussion brain injury on poly(ADP-ribose) polymerase activity and cleavage. Male Sprague-Dawley rats (n = 52) were anesthetized, subjected to fluid percussion brain injury of moderate severity (2.5-2.8 atm), and killed at 30 min, 2 h, 6 h, 24 h, 3 days, or 7 days postinjury. Genomic DNA from injured cortex at 24 h, but not at 30 min, was both fragmented and able to stimulate exogenous poly(ADP-ribose) polymerase. Endogenous poly(ADP-ribose) polymerase activity, however, was enhanced in the injured cortex at 30 min but subsequently returned to baseline levels. Slight fragmentation of poly(ADP-ribose) polymerase was detected in the injured cortex in the first 3 days following injury, but significant cleavage was detected at 7 days postinjury. Taken together, these data suggest that poly(ADP-ribose) polymerase-mediated DNA repair is initiated in the acute posttraumatic period but that subsequent poly(ADP-ribose) polymerase activation does not occur, possibly owing to delayed apoptosis-associated proteolysis, which may impair the repair of damaged DNA.     

Characterization of human poly(ADP-ribose) polymerase with autoantibodies

The addition of poly(ADP-ribose) chains to nuclear proteins has been reported to affect DNA repair and DNA synthesis in mammalian cells. The enzyme that mediates this reaction, poly(ADP-ribose) polymerase, requires DNA for catalytic activity and is activated by DNA with strand breaks. Because the catalytic activity of poly(ADP-ribose) polymerase does not necessarily reflect enzyme quantity, little is known about the total cellular poly(ADP-ribose) polymerase content and the rate of its synthesis and degradation. In the present experiments, specific human autoantibodies to poly(ADP-ribose) polymerase and a sensitive immunoblotting technique were used to determine the cellular content of poly(ADP-ribose) polymerase in human lymphocytes. Resting peripheral blood lymphocytes contained 0.5 X 10(6) enzyme copies per cell. After stimulation of the cells by phytohemagglutinin, the poly(ADP-ribose) polymerase content increased before DNA synthesis. During balanced growth, the T lymphoblastoid cell line CEM contained approximately 2 X 10(6) poly(ADP-ribose) polymerase molecules per cell. This value did not vary by more than 2-fold during the cell growth cycle. Similarly, mRNA encoding poly(ADP-ribose) polymerase was detectable throughout S phase. Poly(ADP-ribose) polymerase turned over at a rate equivalent to the average of total cellular proteins. Neither the cellular content nor the turnover rate of poly(ADP-ribose) polymerase changed after the introduction of DNA strand breaks by gamma irradiation. These results show that in lymphoblasts poly(ADP-ribose) polymerase is an abundant nuclear protein that turns over relatively slowly and suggest that most of the enzyme may exist in a catalytically inactive state.     

Purification and characterization of an (ADP-ribose)n glycohydrolase from human erythrocytes

An (ADP-ribose)n glycohydrolase from human erythrocytes was purified approximately 13,000-fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59,000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP-ribose)n by this enzyme was exoglycosidic, yielding ADP-ribose as the final product. The Km value for (ADP-ribose)n (average chain length, n = 15) was 5.8 microM and the maximal velocity of its hydrolysis was 21 mumol.min-1.mg protein-1. The optimum pH for enzyme activity was 7.4 KCl was more inhibitory than NaCl. The enzyme activity was inhibited by ADP-ribose and cAMP but not the dibutyryl-derivative (Bt2-cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP-ribose)n glycohydrolase II, but not to those of nuclear (ADP-ribose)n glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986) J. Biol. Chem. 261, 965-969]. Thus, human erythrocytes contain (ADP-ribose)n glycohydrolase II. The kinetics of degradation of poly(ADP-ribose) bound to histone H1 by purified erythrocyte (ADP-ribose)n glycohydrolase was essentially the same as that of the corresponding free poly(ADP-ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP-ribose), much less activity on the corresponding oligo(ADP-ribose) bound to histone H1. The enzyme had more activity on oligo(ADP-ribose) bound to mitochondrial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP-ribose) bound to histone H1. It did not degrade mono(ADP-ribosyl)-stimulatory guanine-nucleotide-binding protein (Gs) and -inhibitory guanine-nucleotide-binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP-ribose)n glycohydrolase II may be involved in extranuclear de(ADP-ribosyl)n-ation, but not in membrane de-mono(ADP-ribosyl)ation.     

Histone shuttling by poly(ADP-ribosylation).

We have found that two nuclear enzymes, i.e. poly(ADP-ribose) polymerase (EC 2.4.2.30) and poly(ADP-ribose) glycohydrolase, may cooperate to function as a histone shuttle mechanism on DNA. The mechanism involves four distinct reaction intermediates that were analyzed in a reconstituted in vitro system. In the first step, the enzyme poly(ADP-ribose) polymerase is activated in the presence of histone-DNA complexes and converts itself into a protein carrying multiple ADP-ribose polymers. These polymers attract histones that dissociate from the DNA as a histone-polymer-polymerase complex. The DNA assumes the electrophoretic mobility of free DNA and becomes susceptible to nuclease digestion (second step). In the third step, poly(ADP-ribose) glycohydrolase degrades ADP-ribose polymers and thereby eliminates the binding sites for histones. In the fourth step, histones reassociate with DNA, and the histone-DNA complexes exhibit the electrophoretic mobilities and nuclease susceptibilities of the original complexes prior to dissociation. Our results are compatible with the view that the poly(ADP-ribosylation) system acts as a catalyst of nucleosomal unfolding of chromatin in DNA excision repair.     

Diadenosine 5', 5"'-P1,P4-tetraphosphate stimulates processing of adp-ribosylated poly(ADP-ribose) polymerase

The effect of diadenosine 5', 5"'-P1,P4-tetraphosphate (Ap4A) on the time course and acceptors of poly(ADP-ribose) synthesis was studied in undamaged and N-methyl-N'-nitro-N-nitrosoguanidine-treated human lymphocytes. Analysis of protein acceptors of poly(ADP-ribose) revealed that treatment with Ap4A stimulated ADP-ribosylation of bands at molecular weights of 96,000, 79,000, and 62,000. Pulse-chase studies showed that these bands were produced as a result of an effect of Ap4A on the processing of ADP-ribosylated proteins rather than on the synthesis of newly ADP-ribosylated proteins. By incubating permeabilized cells in the absence or presence of Ap4A and purified poly(ADP-ribose) polymerase auto-ADP-ribosylated with [32P]NAD+, we showed that the Mr = 96,000, 79,000, and 62,000 bands were derivatives of the prelabeled enzyme. Our results indicate that normal human lymphocytes process auto-ADP-ribosylated poly(ADP-ribose) polymerase to specific lower molecular weight products and that this processing is stimulated by Ap4A.     

HIV-1 Tat protein is poly(ADP-ribosyl)ated in vitro.

Purified recombinant HIV-1 Tat protein stimulated acceptor-dependent reaction of poly(ADP-ribose) polymerase in a dose-dependent manner. Analysis of the reaction products by SDS-polyacrylamide gel electrophoresis followed by immunoblotting with anti-poly(ADP-ribose) antibody revealed that recombinant Tat proteins were covalently modified with poly(ADP-ribose) in the enzyme reaction. Eventhough no significant effect of the modification was detected in the activity of Tat to form a specific complex with TAR (a viral transactivation response element) RNA, the present results raise the possibility that poly(ADP-ribose) polymerase is involved in the regulation of HIV-1 through the modification of a virus-encoded transactivator, Tat protein.     

Changes in mRNA levels of poly(ADP-ribose) polymerase during activation of human lymphocytes

The level of mRNA encoding the nuclear enzyme poly(ADP-ribose) polymerase (ADP-ribosyltransferase, EC 2.4.2.30) was found to be very low in quiescent human lymphocytes and to increase at least 10-fold between 1 and 2 dyas after stimulation with the mitogen phytohaemagglutinin, staying high for several days thereafter. This increase was inhibited by 3-methoxybenzamide (a competitive inhibitor of poly(ADP-ribose) polymerase) but was not affected significantly by aphidicolin. Incubation of activated cells with cycloheximide for 2 h increased the expression slightly. These data demonstrate that, during lymphocyte activation, the level of mRNA of the poly(ADP-ribose) polymerase gene correlates with, and hence is presumably responsible for, the increase in poly(ADP-ribose) polymerase protein detectable by enzyme assay or immunochemistry.     

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.     

PARP-2, A novel mammalian DNA damage-dependent poly(ADP-ribose) polymerase.

Poly(ADP-ribosylation) is a post-translational modification of nuclear proteins in response to DNA damage that activates the base excision repair machinery. Poly(ADP-ribose) polymerase which we will now call PARP-1, has been the only known enzyme of this type for over 30 years. Here, we describe a cDNA encoding a 62-kDa protein that shares considerable homology with the catalytic domain of PARP-1 and also contains a basic DNA-binding domain. We propose to call this enzyme poly(ADP-ribose) polymerase 2 (PARP-2). The PARP-2 gene maps to chromosome 14C1 and 14q11.2 in mouse and human, respectively. Purified recombinant mouse PARP-2 is a damaged DNA-binding protein in vitro and catalyzes the formation of poly(ADP-ribose) polymers in a DNA-dependent manner. PARP-2 displays automodification properties similar to PARP-1. The protein is localized in the nucleus in vivo and may account for the residual poly(ADP-ribose) synthesis observed in PARP-1-deficient cells, treated with alkylating agents or hydrogen peroxide.     

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells. Properties of the purified polymerase.

Poly(ADP-ribose) polymerase from Ehrlich ascites tumor cells, partially purified by chromatography on DNA-agarose, was obtained as a more than 80% homogeneous preparation by isoelectric focusing in a sucrose gradient. The polymerase activity was shown to be associated with the major protein in the preparation. Results obtained by electrophoresis in the presence of sodium dodecyl-sulfate indicated that poly(ADP-ribose) polymerase consists of a polypeptide chain with a molecular weight of 130 000. Ultracentrifugation at non-denaturating conditions indicated that the active enzyme may be an oligomeric form of this polypeptide chain. The isoelectric point of the polymerase was 9.40. The effects of various additions to the assay mixture on the synthesis of poly(ADP-ribose) as well as some kinetic data, are given. It is shown that poly(ADP-ribose) is a highly efficient inhibitor of its own synthesis, and results are presented which suggest that the well-known stimulatory effect of DNA on the synthesis is due to reduction of this inhibitory effect of the product.     

Poly(ADP-ribose) accessibility to poly(ADP-ribose) glycohydrolase activity on poly(ADP-ribosyl)ated nucleosomal proteins

Hydrolysis of protein-bound 32P-labelled poly(ADP-ribose) by poly(ADP-ribose) glycohydrolase shows that there is differential accessibility of poly(ADP-ribosyl)ated proteins in chromatin to poly(ADP-ribose) glycohydrolase. The rapid hydrolysis of hyper(ADP-ribosyl)ated forms of histone H1 indicates the absence of an H1 dimer complex of histone molecules. When the pattern of hydrolysis of poly(ADP-ribosyl)ated histones was analyzed it was found that poly(ADP-ribose) attached to histone H2B is more resistant than the polymer attached to histone H1 or H2A or protein A24. Polymer hydrolysis of the acceptors, which had been labelled at high substrate concentrations (greater than or equal to 10 microM), indicate that the only high molecular weight acceptor protein is poly(ADP-ribose) polymerase and that little processing of the enzyme occurs. Finally, electron microscopic evidence shows that hyper(ADP-ribosyl)ated poly(ADP-ribose) polymerase, which is dissociated from its DNA-enzyme complex, binds again to DNA after poly(ADP-ribose) glycohydrolase action.     

Changes in the level of poly ADP-ribosylation during a cell cycle

In order to analyze the fluctuation of the poly ADP-ribosylation level during the cell cycle of synchronously growing He La S3 cells, we have developed three different assay systems; intact and disrupted nuclear systems, and poly(ADP-ribose) polymerase in vitro system. The optimum conditions for poly ADP-ribosylation in each assay system were similar except the pH optimum. Under the conditions favoring poly ADP-ribosylation, little radioactivity incorporated into poly(ADP-ribose) was lost after termination of the poly ADP-ribosylation by addition of nicotinamide which inhibits the reactions by more than 90% in any system. In the intact nuclear system, the level of poly ADP-ribosylation increased slightly subsequent to late G2 phase with a peak at M phase. The high level of poly ADP-ribosylation in M phase was also confirmed by using selectively collected mitotic cells which were arrested in M phase by Colcemid. The level in mitotic chromosomes was 5.1-fold higher than that in the nuclei from logarithmically growing cells. Colcemid has no effect on the poly ADP-ribosylation. In the disrupted nuclear system, a relatively high level of poly ADP-ribosylation was observed during mid S-G2 phase. When poly(ADP-ribose) polymerase was extracted from the nuclei with a buffer solution containing 0.3 M KCl, more than 90% of the enzyme activity was recovered. The poly(ADP-ribose) polymerase in vitro system was dependent on both DNA and histone—10 μg each. In the enzyme system, enzyme activity was detected throughout the cell cycle and was observed to be highest in G2 phase. The high level at M phase observed in the intact nuclear system was not seen in the other two systems. Under the assay conditions, little influence of poly(ADP-ribose) degrading enzymes was noted on the level of poly ADP-ribosylation in any of the three systems. This was confirmed at various stages during the cell cycle through pulse-labeling and “chasing” by adding nicotinamide.     

Interaction of the ADP-ribosylating enzyme from the hyperthermophilic archaeon S. solfataricus with DNA and ss-oligo deoxy ribonucleotides

The DNA-binding ability of the poly-ADPribose polymerase-like enzyme from the extremely thermophilic archaeon Sulfolobus solfataricus was determined in the presence of genomic DNA or single stranded oligodeoxyribonucleotides. The thermozyme protected homologous DNA against thermal denaturation by lowering the amount of melted DNA and increasing melting temperature. The archaeal protein induced structural changes of the nucleic acid by modifying the dichroic spectra towards a shape typical of condensing DNA. However, enzyme activity was slightly increased by DNA. Competition assays demonstrated that the protein interacted also with heterologous DNA. In order to characterize further the DNA binding properties of the archaeal enzyme, various ss-oligodeoxyribonucleotides of different base composition, lengths (12-mer to 24-mer) and structure (linear and circular) were used for fluorescence titration measurements. Intrinsic fluorescence of the archaeal protein due to tryptophan (excitation at 295 nm) was measured in the presence of each oligomer at 60 degrees C. Changes of tryptophan fluorescence were induced by all compounds in the same range of base number per enzyme molecule, but independently from the structural features of oligonucleotides, although the protein exhibited a slight preference for those adenine-rich and circular. The binding affinities were comparable for all oligomers, with intrinsic association constants of the same order of magnitude (K=10(6) M(-1)) in 0.01 M Na-phosphate buffer, pH 8.0, and accounted for a "non-specific" binding protein. Circular dichroism analysis showed that at 60 degrees C the native protein was better organized in a secondary structure than at 20 degrees C. Upon addition of oligonucleotides, enzyme structure was further stabilized and changed towards a beta-conformation. This effect was more marked with the circular oligomer. The analysed oligodeoxyribonucleotides slightly enhanced enzyme activity with the maximal increase of 50% as compared to the control. No activation was observed with the circular oligomer.     

Inhibition of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, and DNA ligase II by poly(ADP-ribosyl)ation reaction in vitro

Incubation of DNA polymerase alpha, DNA polymerase beta, terminal deoxynucleotidyl transferase, or DNA ligase II in a reconstituted poly(ADP-ribosyl)ating enzyme system markedly suppressed the activity of these enzymes. Components required for poly(ADP-ribose) synthesis including poly(ADP-ribose) polymerase, NAD+, DNA, and Mg2+ were all essential for the observed suppression. Purified poly(ADP-ribose) itself, however, was slightly inhibitory to all of these enzymes. Furthermore, the suppressed activities of DNA polymerase alpha, DNA polymerase beta, and terminal deoxynucleotidyl transferase were largely restored (3 to 4-fold stimulation was observed) by a mild alkaline treatment, a procedure known to hydrolyze alkaline-labile ester linkage between poly(ADP-ribose) and an acceptor protein. All of these results strongly suggest that the four nuclear enzymes were inhibited as a result of poly(ADP-ribosyl)ation of either the enzyme molecule itself or some regulatory proteins of these enzymes.     

Differential assay and biological significance of poly(ADP-ribose) polymerase activity in isolated liver nuclei

Poly(ADP-ribosyl)ation of nuclear proteins is catalyzed by poly(ADP-ribose) polymerase. This enzyme is involved in the regulation of basic cellular functions of DNA metabolism. DNA breaks induced by DNA-damaging agents trigger the activation of poly(ADP-ribose) polymerase increasing its endogenous level. This increase modifies the pattern of poly(ADP-ribosyl)ated chromatin proteins. In this paper we describe a procedure for the isolation of intact nuclei from rat liver to be used for the endogenous activity assay. Artifactual activation of the enzyme was avoided since a very low level of DNA-strand breaks occurs during the isolation of nuclei. We present a series of experiments which prove the ability of this procedure to detect increases in endogenous liver activity without modification of the total level. The application of this technique can be useful for a better understanding of the role of early changes in poly(ADP-ribose) polymerase level in physiological conditions and during exposure to DNA-damaging agents.     

Some properties of the lysozymes in serum and colostrum from cows with high and low lytic power against Micrococcus lysodeikticus

Previous work has uncovered a dominant gene for high bacteriolytic activity of bovine serum against the test bacterium Micrococcus lysodeikticus. This major gene effect is also fully expressed in colostrum. In the present study the lytic power of serum and colostral whey from high and low level cows was subjected to a degree of characterization. It was found that the enzyme activities studied exhibited properties in accordance with those defined for a lysozyme (EC 3.2.1.17), i.e. (1) lysis of a suspension of M. lysodeikticus, (2) basic protein (pI = 10.0 and pI = 10.3 for bovine serum lysozyme (BSL) and bovine colostrum lysozyme (BCL), respectively), (3) molecular weight (MW) approximately 16 000 for both BSL and BCL, (4) liberation of free reducing sugars during action on cell wall peptidoglycan (the kinetics of BSL and BCL differed strongly), and (5) fairly heat stable, especially at acidic pH and relative labile at alkaline pH (BCL was far more sensitive to heating at alkaline pH than was BSL). The dramatic differences in activity between high and low level animals might be due to a major genetic mechanism influencing the amount of, or the activity of, circulating enzyme molecules, rather than a structural gene coding for a certain enzyme with a particular specific activity. This is also supported by the high correlation between the lytic capacity of BSL and BCL in spite of the different properties of these lysozymes (i.e. in respect of pI, enzyme kinetics and heat stability) reported in the present study.