Action of a cell wall proteinase from Lactococcus lactis subsp. cremoris SK11 on bovine alpha s1-casein.

The cell wall-associated proteinase from Lactococcus lactis subsp. cremoris SK11 was partially purified and incubated with alpha s1-casein for various times up to 48 h. Sixteen trifluoroacetic acid-soluble oligopeptide hydrolysis products were identified by determination of the amino acid sequence. Eleven of these oligopeptides originated from the 78-residue sequence comprising the C-terminal region of alpha s1-casein and were present among the products after the first 60 min of digestion. Three oligopeptides from the N-terminal region and two others from the central region of the alpha s1-casein sequence were also present among the early digestion products although in smaller amounts than most of the oligopeptides from the C-terminal region. No clear consensus sequence of amino acid residues surrounding the cleavage sites could be identified.

Identification of a catalytically essential nucleophilic residue in sheep liver cytoplasmic aldehyde dehydrogenase.

Sheep liver cytoplasmic aldehyde dehydrogenase was labelled by reaction with the substrate p-nitrophenyl di[14C]methylcarbamate. After tryptic digestion and peptide fractionation the labelled residue was identified as Cys-302. This is the first unequivocal identification of the essential enzymic nucleophile in the esterase activity of aldehyde dehydrogenase. By implication, Cys-302 is probably also the residue that is acylated by aldehyde substrates and the first residue that is modified by disulfiram.

Bovine ceroid-lipofuscinosis (Batten's disease): the major component stored is the DCCD-reactive proteolipid, subunit C, of mitochondrial ATP synthase.

The ceroid-lipofuscinoses (Batten's disease) are a group of recessively inherited lysosomal storage diseases of children and animals in which there is intracellular accumulation of a fluorescent lipopigment in a wide variety of cells. Lipopigment bodies isolated from pancreas, liver, kidney and brain tissue from a heifer affected with ceroid-lipofuscinosis contained between 55 and 62% protein. A dominant component comigrated on LDS-PAGE with the major low molecular weight protein stored in ovine ceroid-lipofuscinosis. It was identified by amino acid sequence and mass spectroscopy as the full subunit c of mitochondrial ATP synthase, normally found only in the inner mitochondrial membrane, where it is estimated to account for 2-4% of the membrane protein. In pancreatic lipopigment it accounted for at least 40% of the total lipopigment mass and this storage was considered specific to the disease. No other mitochondrial proteins were found in storage bodies. These results are similar to those found in studies on the ovine and the late infantile and juvenile human forms of the disease. It is concluded that bovine ceroid-lipofuscinosis is also a proteolipid proteinosis in which subunit c of mitochondrial ATP synthase is specifically stored in lysosome derived organelles.

Evidence for reactivity of serine-74 with trans-4-(N,N-dimethylamino)cinnamaldehyde during oxidation by the cytoplasmic aldehyde dehydrogenase from sheep liver.

A nucleophilic group in the active site of aldehyde dehydrogenase, which covalently binds the aldehyde moiety during the enzyme-catalyzed oxidation of aldehydes to acids, was acylated with the chromophoric aldehyde trans-4-(N,N-dimethylamino)cinnamaldehyde (DACA). Acyl-enzyme trapped by precipitation with perchloric acid was digested with trypsin, and the peptide associated with the chromophoric group was isolated and shown to be Gln-Ala-Phe-Gln-Ile-Gly-Ser-Pro-Trp-Arg. After redigestion with thermolysin, the chromophore was associated with the C-terminal hexaresidue part. If the chromophore is attached to this peptide, serine would be expected to bind the aldehyde and lead to the required acylated derivative. Differential labeling experiments were performed in which all free thiol groups on the acylated enzyme were blocked by carboxymethylation. The acyl chromophore was then removed by controlled hydrolysis and the protein reacted with [14C]iodoacetamide. No 14C-labeled tryptic peptides were isolated, suggesting that the sulfur of a cysteine cannot be the acylated residue in the precipitated acyl-enzyme.

Ovine ceroid lipofuscinosis. The major lipopigment protein and the lipid-binding subunit of mitochondrial ATP synthase have the same NH2-terminal sequence.

Previous studies on lipopigment isolated from sheep affected with ceroid lipofuscinosis (Batten's disease) showed that the disease is a lysosomal proteinosis, involving specific storage of peptide(s) that migrate in dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 3500. This band is the dominant contributor to the lipopigment mass. When purified total lipopigment proteins were loaded onto a protein sequencer, a dominant sequence was found, identical to the NH2 terminus of the lipid-binding subunit of protein translocating mitochondrial ATP synthase. This sequence was determined to 40 residues and a minimum estimate of 40% made for its contribution to the lipopigment protein mass. The full lipid-binding subunit has physical and chemical properties similar to those of the specifically stored low Mr peptide, which may be the full protein or a large NH2-terminal fragment of it. Lipopigments in the human ceroid lipofuscinoses also contain a major component with similar physical and chemical properties. These and previous results indicate that the genetic lesion in ovine ceroid lipofuscinosis causes an abnormal accumulation of this peptide in lysosomes, i.e. the disease is a proteolipid proteinosis, specifically a lysosomal mitochondrial ATP synthase lipid-binding subunit proteinosis. The analogous human diseases are likely to reflect storage of the same or similar peptides.

A mass-spectrometric sequence study of the enzyme ribitol dehydrogenase from Klebsiella aerogenes.

The first detailed results of the application of a low-resolution mixture analysis approach to the sequence analysis of an enzyme, ribitol dehydrogenase, are given. Examples of the interpretation of the spectra of peptide mixtures derived from this protein are described. Evidence for new fragmentation patterns observed is reported, together with an explanation of the generation of ambiguous sequences by use of a low-specificity enzyme, thermolysin. The overall sequencing strategy evolved is assessed.