Murray R. Grigor

Comparative aspects of milk caseins.

The caseins comprise the major protein component of milk of most mammals and are secreted as micelles that also carry high concentrations of calcium. They are phosphoproteins that represent the products of four genes, equivalent to those that encode the bovine alpha s1, alpha s2, beta, and kappa-caseins. There is considerable variation in the relative proportions of the particular caseins across species. The primary sequences of the alpha s1, alpha s2, and beta-caseins also show considerable species variation consistent with rapidly evolving genes that are proposed to have a common precursor. In contrast, the kappa-caseins exhibit features that demonstrate a separate origin and function where they are proposed to stabilise the micelle structure. This review focuses on comparative aspects of the caseins across a number of species for which information is now available.

The acute-phase protein serum amyloid A3 is expressed in the bovine mammary gland and plays a role in host defence

The serum amyloid A protein is one of the major reactants in the acute-phase response. Using representational difference analysis comparing RNA from normal and involuting quarters of a dairy cow mammary gland, we found an mRNA encoding the SAA3 protein (M-SAA3). The M-SAA3 mRNA was localized to restricted populations of bovine mammary epithelial cells (MECs). It was expressed at a moderate level in late pregnancy, at a low level through lactation, was induced early in milk stasis, and expressed at high levels in most MECs during mid to late involution and inflammation/mastitis. The mature M-SAA3 peptide was expressed in Escherichia coli, antibodies made, and shown to have antibacterial activity against E. coli, Streptococcus uberis and Pseudomonas aeruginosa. These results suggest that the mammary SAA3 may have a role in protection of the mammary gland during remodelling and infection and possibly in the neonate gastrointestinal tract.

The BSP30 salivary proteins from cattle, LUNX/PLUNC and von Ebner's minor salivary gland protein are members of the PSP/LBP superfamily of proteins.

Saliva influences rumen function in cattle, yet the biochemical role for most of the bovine salivary proteins (BSPs) has yet to be established. Two cDNAs (BSP30a and BSP30b) from bovine parotid salivary gland were cloned and sequenced, each coding for alternate forms of a prominent protein in bovine saliva. The BSP30 cDNAs share 96% sequence identity with each other at the DNA level and 83% at the amino acid level, and appear to arise from separate genes. The predicted BSP30a and BSP30b proteins share 26-36% amino acid identity with parotid secretory protein (PSP) from mouse, rat and human. BSP30 and PSP are in turn more distantly related to a wider group of proteins that includes lung-specific X protein, also known as palate, lung, and nasal epithelium clone (LUNX/PLUNC), von Ebners minor salivary gland protein (VEMSGP), bactericidal permeability increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), and the putative olfactory ligand-binding proteins RYA3 and RY2G5. Bovine cDNAs encoding homologs of LUNX/PLUNC and VEMSGP were isolated and sequenced. Northern blot analysis showed that LUNX/PLUNC, BSP30 and VEMSGP are expressed in bovine salivary tissue and airways, and that they have non-identical patterns of expression in these tissues. The expression of both BSP30a and BSP30b is restricted to salivary tissue, but within this tissue they have distinct patterns of expression. The proximity of the human genes coding for the PSP/LBP superfamily on HSA20q11.2, their similar amino acid sequence, and common exon segmentation strongly suggest that these genes evolved from a common ancestral gene. Furthermore, they imply that the BSP30a and BSP30b proteins may have a function in common with other members of this gene family.

Differential expression of the whey acidic protein gene during lactation in the brushtail possum (Trichosurus vulpecula).

The whey acidic protein (WAP) is a whey protein found in the milk of a number of species. We have isolated and characterised a WAP cDNA clone from the brushtail possum (Trichosurus vulpecula) and examined its expression in the mammary gland. The amino acid sequences of WAP from the possum and another marsupial, the tammar wallaby, share 69% identity, however, less sequence identity exists between the marsupial and eutherian WAP sequences (30-37%). The possum and tammar WAP genes consist of three four-disulphide core (4-DSC) domains, with a WAP motif at the beginning of each domain. In contrast, the eutherian WAP sequences consist of two 4-DSC domains with the WAP motif only present in the second domain. This WAP motif is also present in a number of protease inhibitors found in a wide range of species. Phylogenetic analysis of marsupial and eutherian WAP sequences suggests that the ancestral WAP gene has three domains and that one of the domains has been deleted from the eutherian gene. The profile of WAP gene expression in the possum mammary gland changed throughout lactation, with WAP mRNA levels reaching a peak between days 106 and 177 of lactation. The level of WAP mRNA in the mammary gland appeared to be correlated with the level of circulating prolactin in the lactating female and was different to that observed for several other whey protein genes. Overlapping expression of the WAP and early lactation protein genes, both of which are putative protease inhibitors, may provide protection of milk immunoglobulins that are required for the prolonged period of passive immune transfer to the marsupial pouch young.

Subcellular location and differential antibody specificity of arginase in tissue culture and whole animals

Studies in man and other mammals have demonstrated the existence of two forms of arginase, a cytoplasmic form located primarily in liver and a mitochondrial form expressed in lesser amounts in a larger number of organs, but especially kidney. They appear to be encoded in different gene loci. Using a colloidal silica gradient separation technique, we have now located arginase in H4 cells, a rat hepatoma‐derived line, to the cytoplasm and the arginase in human embryonic kidney‐derived line, to the mitochondrion. Antibody prepared against A1 precipitates all the arginase from liver, 50% from kidney and none of the activity from human embryonic kidney (HEK) cells. An antibody prepared against partially purified All, by contrast, precipitates >90% of arginase activity from HEK cells, half from kidney and virtually none from H4 cells or rat liver.

Separate monoacylglycerol and diacylglycerol acyltransferases function in intestinal triacylglycerol synthesis

Abstract The monoacylglycerol and diacylglycerol acyltransferases in microsomes from the rat small intestinal epithelium have been examined in order to: (1) differentiate between the monoacylglycerol acyltransferase and diacylglycerol acyltransferase and (2) establish whether or not there is more than one diacylglycerol acyltransferase involved in the two pathways of triacylglycerol synthesis which operate in these cells. Assays were established for the monoacylglycerol acyltransferase using sn -2-monoolein as substrate and for the diacylglycerol acyltransferase using three different substrates: exogenous sn -2-diolein; diacylglycerol synthesized endogenously from monoolein; or diacylglycerol synthesized endogenously from glycerol phosphate as substrate. Compared to the diacylglycerol acyltransferase, the monoacylglycerol acyltransferase was distributed more widely throughout the intestine and was also relatively insensitive to inhibition by mercuric chloride. In addition, exogenous diolein did not inhibit the monoacylglycerol acyltransferase activity. These observations are consistent with separate monoacylglycerol and diacylglycerol acyltransferase enzymes. The distribution of the diacylglycerol acyltransferase activity assayed either with exogenous diolein or diacylglycerol produced from monoolein was similar in different sections of the intestine and in samples from different rats. Further, the diacylglycerol acyltransferase activity was strongly inhibited by mercuric chloride with all three assays. However, the activity assayed with exogenous substrate was selectively inhibited by dithiothreitol and the non-ionic detergents, Triton X-100 and Tergitol NP-40. This is unlikely to represent the presence of separate enzymes since the same reagents inhibit the activity of the microsomal CDPcholine: diacylglycerol cholinephosphotransferase when assayed with exogenous diolein but not with endogenous diacylglycerol from glycerol phosphate. It is concluded that these reagents interfere with the association of exogenous diolein with the microsomal membrane and that there is only one diacylglycerol acyltransferase active in the small intestinal epithelium.

Phylogenetic analysis of three lipocalin-like proteins present in the milk of Trichosurus vulpecula (Phalangeridae, Marsupialia).

Abstract. Three proteins have been identified in the milk of the common brush tail possum, Trichosurus vulpecula that from sequence analysis are members of the lipocalin family. They include β-lactoglobulin, which appears to have two forms; a homologue to the late-lactation protein found in tammar, Macropus eugenii; milk; and a novel protein termed trichosurin. Whereas β-lactoglobulin and trichosurin are both expressed throughout lactation, the late-lactation protein is not detected in samples taken before days 100–110 of lactation. The cDNAs encoding each of these proteins have been isolated from cDNA libraries prepared using possum mammary mRNA and sequenced. Phylogenetic analysis showed that the T. vulpeculaβ-lactoglobulin, along with two other macropod β-lactoglobulins, forms a subclass of β-lactoglobulins distinct from those for eutherian mammals; both marsupial late-lactation proteins appear to have similarities to a family of odorant-binding proteins, whereas trichosurin has similarities to the major urinary proteins of rodents.

Lysozyme and alpha-lactalbumin from the milk of a marsupial, the common brush-tailed possum (Trichosurus vulpecula).

Lysozyme and alpha-lactalbumin have been identified using N-terminal sequence analysis of whey proteins from the common brush-tailed possum, Trichosurus vulpecula after separation by two-dimensional denaturing electrophoresis. Both proteins were purified from pooled possum milk using ion exchange chromatography and gave mass values of 14,896 and 13,985 Da respectively by MALDI-TOF mass spectrometry. Clones containing the full coding sequences of the genes for both proteins were isolated from a possum mammary cDNA library and the DNA sequence of the coding region determined. The inferred protein sequences were used in phylogenetic analysis of both protein classes. These showed that the T. vulpecula alpha-lactalbumin, along with other marsupial alpha-lactalbumins, formed a family distinct from the eutherian alpha-lactalbumins and the alpha-lactalbumin of a monotreme, the platypus, consistent with the separate evolution of the marsupials. By contrast the T. vulpecula lysozyme was shown to be similar to the ruminant stomach lysozymes and primate lysozymes and quite distinct from the Ca2+-binding lysozymes found in the milk of the echidna and horse.

Multiple molecular forms of rat mammary glucose-6-phosphate dehydrogenase: Proposed role in turnover of the enzyme

Polyacrylamide gel electrophoresis shows that more than 80% of the total glucose-6-phosphate dehydrogenase activity of rat mammary homogenates exists as three dimeric forms. During lactation all three forms are present in equivalent amounts, but 24 h after the removal of the pups to stop lactation, there is a shift to the fastest migrating form (band I). During this period the ratio of enzyme activity to tissue glutathione concentration decreased. Using partially purified preparations of the enzyme it was shown that dithiothreitol could convert all the activity to the slowest migrating form (band III) and that diamide could reverse this change giving rise to band I only. Reduced glutathione could partially convert the enzyme to the band III form. Experiments using iodoacetic acid, iodoacetamide, p-chloromercuribenzoate, and mercuric chloride are also described. Based on these observations it is proposed that band I and band III represent fully oxidized and fully reduced forms of the enzyme, respectively, and band II a partially oxidized form. The oxidized and reduced forms are catalytically equivalent. However, the oxidized form is the most rapidly inactivated by chymotrypsin or mammary microsomes. Mammary microsomes can also catalyze the oxidation of the enzyme. The oxidation precedes the inactivation by microsomes and also occurs at lower microsome concentrations. It is proposed that the microsomal oxidation of the enzyme is the initial step in the turnover of the enzyme.

Differences in mitogenic responses to angiotensin II, calf serum and phorbol ester in vascular smooth muscle cells from two strains of genetically hypertensive rat

Vascular smooth muscle cells cultured from spontaneously hypertensive (SHR), New Zealand genetically hypertensive (GH) rat strains and their control strains (Wistar-Kyoto (WKY) and normal Wistar (N) rats) were compared for mitogenic responses [( 3H]-thymidine incorporation) to angiotensin II (AII), fetal calf serum (FCS), and the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA). SHR cells showed enhanced basal [3H]-thymidine incorporation and increased responses to all three factors. In contrast, basal and FCS-stimulated [3H]-thymidine incorporation was the same or less in GH than in N cells. However, DNA synthesis was greater in GH cells in response to AII, AII + TPA or FCS + TPA. These results suggest that vascular smooth muscle cells from both hypertensive rat strains display enhanced mitogenesis, but the enhancement occurs via different intracellular signalling pathways.