Knowledge and attitudes of Australian veterinarians to animal abuse and human interpersonal violence.

A survey of Australian veterinarians was undertaken to assess their amount of knowledge about, and their attitudes towards animal abuse, human violence and the link between the two. Results from the 185 respondents to a questionnaire that was either mailed out or hand delivered revealed a wide variety of definitions and attitudes towards abuse, with the majority of veterinarians recognising the link between human and animal abuse. The overwhelming majority of veterinarians believed that they should intervene in some way when confronted with either animal or human abuse, although most felt ill-equipped to deal with suspected human abuse. Almost 20% of cases of animal abuse had associated suspected or known human abuse. It is suggested that veterinarians need more resources made available to them to be able to deal more effectively with these situations.

Identification of basic residues involved in substrate binding and catalysis by pyrophosphate-dependent phosphofructokinase from Propionibacterium freudenreichii.

Despite overall low identity of sequence between ATP- and PPi-dependent phosphofructo-1-kinases (PFK), an alignment permits the tentative identification of catalytically important residues in PPi-dependent PFK. Seven basic residues of the PPi-dependent PFK of Propionibacterium freudenreichii, Arg-310, Lys-315, Arg-326, Arg-186, Arg-78, Arg-90, and Lys-148 were chosen on the basis of this alignment for site-directed mutagenesis to neutral residues. Mutants R186A, K315A, and R326A had substantial increases of 43-, 389- and 678-fold, respectively, in Km for fructose 6-phosphate relative to wild-type enzyme and relatively small effects on kcat. The modest kinetic effects of mutations at Arg-78, Arg-90, and Arg-310 were not considered to indicate important roles for these residues. On the other hand, mutant K148M had a Km for PPi that was increased by 132-fold and kcat lowered by a factor of 490. Cho and Cook (Cho, Y.-K., and Cook, P.F. (1988) Biochemistry 28, 4155-4160) concluded on the basis of kinetic and chemical modification data that the enzyme utilizes a proton shuttle mechanism and that two lysines are involved in substrate binding. The present data along with our previous data on mutant D151A support a double proton shuttle mechanism involving Asp-151 and Lys-148 with Lys-148, Arg-326, Lys-315, and perhaps Arg-186 being important for substrate binding.

Identification of active site residues in pyrophosphate-dependent phosphofructo-1-kinase by site-directed mutagenesis.

The primary structure of pyrophosphate-dependent phosphofructokinase (PFK) from Propionibacterium freudenreichii exhibits a low but significant level of sequence identity with Escherichia coli ATP-dependent PFK, permitting the tentative assignment of residues that may be involved in catalysis. Based on these assignments, the roles in catalysis of 2 aspartyl residues (Asp151 and Asp153) and 2 lysyl residues (Lys80 and Lys85) were examined. Mutagenesis of the Asp151 to alanine and serine reduced kcat by a factors of 2 x 10(4) and 4 x 10(5), respectively, while showing no change in Km for either substrate in the forward reaction or for metal ion in the back reaction. The kcat for Asp153 was decreased by a factor of 700 with no change in Km for pyrophosphate and an increase of about 20-fold in Km for fructose 6-P and close to 4-fold for magnesium ion. That these changes in the mutants were not the result of global conformational changes was indicated by their identical behavior during substrate-specific elution chromatography, ion-exchange chromatography, and limited proteolysis by trypsin and subtilisin. Mutations of Lys80 and Lys85 showed no significant changes in kinetic parameters, suggesting no involvement in mechanism or substrate binding. These and other results permit preliminary modeling of the active site of pyrophosphate-dependent PFK.

Identification of critical lysyl residues in the pyrophosphate-dependent phosphofructo-1-kinase of Propionibacterium freudenreichii.

Pyrophosphate-dependent 6-phosphofructo-1-kinase (PPi-PFK) from Propionibacterium freudenreichii was inactivated by low concentrations of the lysine-specific reagent pyridoxal phosphate (PLP) after sodium borohydride reduction. The substrates fructose 6-phosphate and fructose 1,6-bisphosphate protected against inactivation whereas inorganic pyrophosphate had little effect. An HPLC profile of a tryptic digest of PPi-PFK modified at low concentrations of PLP showed a single major peak with only a small number of minor peaks. The major peak peptide was isolated and sequenced to obtain IGAGXTMVQK, where X represents a modified lysine residue, corresponding to Lys-315. Lys-315 was protected from reaction with PLP by fructose 1,6-bisphosphate. As indicated by HPLC maps of PPi-PFK modified with varying concentrations of PLP, a direct correlation was observed between activity loss and the modification of Lys-315. Two of the minor peptide peaks were shown to contain Lys-80 and Lys-85, which were modified in a mutually exclusive manner. Partial protection against modification of these two residues was provided by MgPPi. The data were used to adjust the sequence alignment of the Propionibacterium enzyme with that of ATP-dependent PFK of Escherichia coli to identify homologous residues in the substrate binding site. It is suggested that Lys-315 interacts with the 6-phosphate of fructose 6-phosphate and that Lys-80 and -85 may be located near the pyrophosphate binding site.

Neurite growth modulation associated with astrocyte proteoglycans: influence of activators of inflammation.

Of the three classes of sulphated proteoglycans produced by type 1 astrocytes in vitro and released into conditioned medium, only heparan sulphate (HS) was associated with enhanced neurite growth by sensory neurons following pretreatment of a laminin substratum. Astrocyte-conditioned medium (ACM) produced in the presence of certain inflammatory mediators had reduced titers of neurite-promoting activity. The low activity ACM contained inhibitors of neurite growth. Heparan sulphate proteoglycans may modulate neurite growth when complexed to constituents of the extracellular milieu either directly or by interacting with other growth-promoting or growth-inhibitory factors.

Characterization of glycosaminoglycans produced by primary astrocytes in vitro.

Quantitative biosynthetic studies with cultures highly enriched for glial fibrillary acidic protein (GFAP+) cells of neonatal mammalian brain demonstrated production of four proteoglycans: hyaluronate (HA), heparan sulphate (HS), chondroitin sulphate (CS), and dermatan sulphate (DS). The glycosaminoglycans were present in cell conditioned medium and in the cellular compartment. There were qualitative differences in the subcellular disposition of the various proteoglycans. The ratio of HS to CS/DS in cell extracts was 1:1, while in medium this ratio was 1:6. All of the glycosaminoglycans were associated with core proteins that were integral to the cell membrane and associated with the cell surface by non-covalent interactions involving glycosaminoglycans. Less than 20% of the HS was non-covalently associated with the astrocyte cell surface reflecting in part the proportionately smaller amounts of this proteoglycan released to astrocyte conditioned medium. HS released to medium was undersulphated relative to that associated with cells. The astrocyte can contribute proteoglycans to the extracellular milieu and displays cell surface proteoglycans that have the potential to provide appropriate substrates for neuron adhesion, process extension, and other cell-cell interactions.

Specificity and pH dependence for acylproline cleavage by prolidase.

Catalytic pH dependence for the hydrolytic activity of the enzyme prolidase with a series of dipeptide substrates is found to be generally bell-shaped (kcat/Km) or simple sigmoidal (kcat). An enzymic residue with a pKa value of 6.6 is found to be critically involved in the catalytic mechanism, as is the substrate amino group. Significant catalysis at a pH of 6.6 is also observed for prolidase with (alkylthio)acetylprolines and with haloacetylprolines. A reverse-protonation state mechanism for substrate binding and activation is postulated, involving a chelative interaction of the aminoacylamide portion of substrate with a strongly Lewis-acidic active site metal ion.

Mechanism and inhibition of prolidase.

The pH dependence of Ki for inhibition of prolidase by acetylproline, proline, and trans-1,2-cyclopentanedicarboxylate follows a different pattern in each case, although deprotonation of an enzymic functional group with a pKa value of 6.6 perturbs ligand binding in every instance. Results are most easily explained with prolidase active as a metalloenzyme dimer exhibiting selective cooperative interactions.