Manfred Pavlík

Primary structure of cathepsin D inhibitor from potatoes and its structure relationship to soybean trypsin inhibitor family.

A novel effective procedure for the purification of cathepsin D inhibitor from potatoes (PDI) was developed. The amino acid sequence of PDI was determined by analysis of the cyanogen bromide digest and of the limited tryptic and chymotryptic digests of the protein. The inhibitor is a single polypeptide chain protein consisting of 188 residues with a simple sugar moiety attached to Asn‐19. The tentative disulfide pairings are also suggested. The sequence data clearly indicate that PDI is homologous with the soybean trypsin inhibitor (STI) (Kunitz) family. The active center of PDI for trypsin inhibition was identified as Pro‐Val‐Arg‐Phe in analogy to STI.

Comparison between prochymosin and pepsinogen from lamb and calf

1. Prochymosin (EC 3.4.23.4) and pepsinogen A (EC 3.4.23.1) from Mongolian lamb (Ovis platyurea) were purified to homogeneity by salt precipitation, gel filtration and ion-exchange chromatography. 2. Immunoelectrophoresis shows partial immunochemical identity between chymosins and pepsins from lamb and cattle, respectively. 2. Activity determinations, N-terminal amino acid sequences and amino acid compositions also show a close relationship between the proteinases from lamb and cattle. 4. Lamb prochymosin and pepsinogen are both glycosylated.

Identification of 17–18 kDa zona pellucida binding proteins from boar spermatozoa

Zona pellucida (ZP) binding proteins from boar spermatozoa were compared with antigens recognized by ACR.2 and ACR.3 monoclonal antibodies. The ZP binding proteins of 55, 53,45 and 38 kDa are identical with various forms of boar acrosin immunologically detected by ACR.2 antibody. The ZP binding proteins of 17 and 18 kDa are recognized by ACR.3 antibody. The N‐tenninal amino acid sequence of the 17 kDa protein revealed that it is not derived from an acrosin molecule.

The characterization of two new low molecular weight proteins (LMPs) from Streptococcus pyogenes.

Two novel extracellular mitogenic substances were isolated from Streptococcus pyogenes strain NY-5 and characterized. The purification steps involved an initial enrichment of the proteins from culture supernatant by silica gel adsorption, followed by ion exchange chromatography and gel filtration. The purified materials were homogeneous in SDS-PAGE, showed estimated molecular weights of 12 kD and isoelectric points of 4.7 and 4.3, respectively. Both proteins (LMP-12k-4.3pI and LMP-12k-4.7pI) demonstrated lymphocyte transformation activity at a concentration of 0.1 microgram/ml. The LMP-12k-4.7pI showed a 69.2% homology of the amino acid sequence with that of a phosphocarrier protein of Staphylococcus aureus and with a total identity in the active centre. The same protein was also isolated from streptococcal group C strain H46A with an N-terminal amino acid sequence being identical. The LMP-12k-4.7pI demonstrated biochemical properties identical with those of the earlier described streptococcal pyrogenic exotoxin type D. The LMP-12k-4.3pI did not show such a clear relation to other functional proteins.

Protein chemical characterization of Mucor pusillus aspartic proteinase. Amino acid sequence homology with the other aspartic proteinases, disulfide bond arrangement and site of carbohydrate attachment.

The amino acid sequence of Mucor pusillus aspartic protenaise was determined by analysis of fragments obtained from cleavage of the enzyme by CNBr and limited tryptic digestion. The protenaise is a single polypeptide chain protein containing 361 amino acid residues, cross‐linked by two disulfide bonds. A sugar moiety composed of two GlcNAc residues and four neutral sugar residues is asparagine‐linked to the chain. The sequence of M. pusillus proteinase is highly homologous with the M. miehei protonaise (83% identity). The homology with other aspartic proteinases is low (22–24%) and indicates that the Mucor proteinases diverged at an early evolutionary phase. The most conservative regions of the molecule are those involved in catalysis and forming the binding cleft and the core region of the molecule.

Type 1 M protein of Streptococcus pyogenes: N-terminal sequence and peptic fragments

Limited proteolysis of the surface of type 1 Streptococcus pyogenes by pepsin gives rise to fragment Pep M1 of M r 20270 as the main product which covers the N‐terminal part of the M protein. The amino acid sequence was determined of the N‐terminal region of the M protein representing the most exposed part of the molecule on the surface fibrils of streptococcal cells, which seems to be very important for the differentiation of the individual serological types. The sequence differs from the homologous N‐terminal sequences of types 5, 6 and 24, and shows a homology with sequences repeating in the chain of type 24. Fragment Pep M1 binds to fibrinogen; the absence of its 30 N‐terminal amino acid residues, however, abolishes this interaction which is believed to play a role in the virulence of S. pyogenes.

Modification of serine residues during sequential degradation of proteins and peptides by the phenylisothiocyanate method

A novel method of serine determination during Edman degradation of proteins and peptides which is based on the reaction of serine degradation products with alkyl thiols is described. The physicochemical characteristics and the structures of the reaction products, i.e., of 5-[alkyl-thio)methyl)-3-phenyl-2-thioxo-4-imidazolinones, were determined. The procedure described permits the products of serine degradation to be obtained in a 70-80% yield.