Amino acid sequences of mammalian kazal-type proteinase inhibitors from salivary glands.

1. The amino acid sequences of bikazins (the double-headed Kazal-type proteinase inhibitors from submandibular glands) isolated from the snow leopard (Unica unica), the European mink (Mustela lutreola), and the European pine marten (Martes martes) were determined. 2. N-terminal domains of bikazins are characterized by a cysteine residue spacing that differs from that of C-terminal domains of bikazins and other Kazal-type proteinase inhibitor domains. 3. N-terminal sequences of bikazins seem to be specific for, and highly conserved within, each Carnivora family.

Visualization of lectin-like proteins in human placenta by means of anti-plant lectin antibodies.

Proteins antigenically cross-reactive with lectins were sought in the placenta by immunohistochemistry using polyclonal antibodies raised in rabbit against four well-known lectins: Concanavalin A, Wheat germ agglutinin, Ulex europaeus agglutinin, and Phaseolus vulgaris leukoagglutinin (PHA-L), as well as one antibody raised in goat against PHA-L. Even at high dilutions of the primary antibody, strong staining was obtained after short incubations, in patterns generally resembling those obtained for placental lectins by other means, such as those based on binding capacity for glycosylated probes. One of the immunohistochemical patterns distinguishes with great clarity between the trophoblast cell layers, thus relating to developmental and functional parameters; another localises PHA-L-immunoreactivity to the syncytiotrophoblast. These results underline the validity of the immunohistochemical screening as an approach in its own right. Both positive and negative controls were applied to the immunohistochemical methodology. These controls showed that the staining patterns obtained relate to the specificities of the primary antibodies employed; i.e. to lectins. The PHA-L-like cross-reactivity was analysed immunochemically. In electrophoretically separated and Western-blotted placental extracts there were found anti-PHA-L-binding fractions of apparent molecular weights 30 kDa, 58 kDa and 67 kDa. Control studies of the PHA-L antigen showed anti-PHA-L-binding fractions of approximate molecular weights 32 kDa and 60 kDa. The 30 kDa fraction from placenta and the 32 kDa fraction from PHA-L antigen bound lactosylated BSA but not fucosylated BSA.(ABSTRACT TRUNCATED AT 250 WORDS)

Separation of peptides on Empore thin-layer chromatography sheets and blotting onto polyvinylidene difluoride membranes with subsequent gas phase sequencing.

Methods for the separation of peptides on a new type of thin-layer chromatography (TLC) sheet and blotting onto polyvinylidene difluoride (PVDF) membranes with subsequent gas phase sequencing are described. For validation, the A and B chain of insulin were chromatographed on Empore TLC sheets and either extracted or blotted onto PVDF membranes. The advantages and disadvantages of thin-layer chromatography on Empore sheets versus high performance liquid chromatography (HPLC) are discussed, along with the possibility of combining the two methods. In addition, TLC was combined with electrophoresis (fingerprinting) for the separation of complex peptide mixtures. Blotting from TLC sheets onto PVDF membranes was performed in two ways: contact diffusion and electrophoretic transfer. In our experiments electroblotting was more effective. Amino acid sequence determination of the B chain of insulin was possible both after extraction from the TLC sheet and after blotting onto PVDF membranes. In the former case, liquid phase sequencing and, in the latter case, gas phase sequencing was performed. The possibility to blot from TLC sheets onto membranes, e.g. PVDF, may prove useful in many fields, for example in biochemistry, and in molecular and cell biology.

Purkinje cell antibodies in a patient with cerebellar disorder: detection of responsible antigenic proteins.

A 34-year-old male patient developed a neurological disorder and signs of cerebellar degeneration, with antibodies against Purkinje cells in the serum, a syndrome previously described as "paraneoplastic cerebellar atrophy". Antibody reaction of the patient's serum was demonstrated by immunohistochemistry on sections through the rat and human cerebellum. Purkinje cells demonstrated granular staining of cytoplasmic proteins and proximal dendrites with nuclear sparing. In an immunoblot, the antibodies from the patient's serum reacted with proteins from an extract of rat cerebellum. Only a few distinct proteins from the complex mixture of cerebellar proteins were found to bind with the serum antibodies when using a combination of affinity chromatography and sodium dodecyl sulphate (SDS) gel electrophoresis. The molecular masses of the proteins differed significantly from those identified in patients reported in the literature. Protein denaturation by SDS and 2-mercaptoethanol resulted in a decrease of antibody binding capacity. After immunosuppressive therapy and plasmapheresis, the reaction of the patient's serum with Purkinje cells was greatly diminished; however, only slight clinical improvement was observed. No sign of neoplasm could be found with repeated examinations. The immunological aspects of this case suggest that cerebellar degeneration may be linked to a previously unreported autoimmune response.

[Diagnosis of alcoholism based on detection of a transferrin variant by polyacrylamide gel electrophoresis and immunoblotting]. / Die Diagnose von Alkoholismus auf der Grundlage des Nachweises einer Transferrinvariante durch Polyacrylamid-Gelelektrophorese und Immunoblotting.

A sensitive method is discribed for the diagnosis of alcoholism. The method is based on the detection of a transferrin variant (carbohydrate deficient transferrin = CDT) in plasma of alcoholics. The determination of CDT, the presence of which is characteristic for chronic alcoholism, is performed in three steps: The plasma proteins are separated by polyacrylamide gel electrophoresis and then transferred electrophoretically onto a nitrocellulose sheet; finally, the transferrin types on the nitrocellulose sheet are specifically detected by an antibody reaction. With the exception of certain cases (genetic variants, rare diseases) CDT is found only during chronic alcohol consumption. In comparison to other markers for chronic alcoholism an advantage of CDT is its higher specificity. A further advantage of the method is that CDT can be identified with high sensitivity by the use of a relatively small amount of technical equipment.

Direct immunoblotting from histological sections of brain onto nitrocellulose: evaluation with the protein neurophysin.

A method for the isolation and localization of proteins and peptides from histological sections of rat and human brain by immunoblotting is described. For validation, the well-characterized protein neurophysin was electrophoretically transferred from formaldehyde-fixed or fresh tissue sections onto a nitrocellulose membrane. Neurophysin on the nitrocellulose membrane was detected by a specific antibody reaction. The antibody against neurophysin was visualized either by using secondary antibodies, conjugated with peroxidase or by protein A gold, followed by enhancement with silver. With this simple and fast method, neurophysin (or other proteins and peptides) can be identified on nitrocellulose membranes in areas that correspond to anatomically defined regions. Since the procedure combines the advantages of precise regional localization of polypeptides with the specificity of antibody-antigen reactions, the method may prove useful for rapid screening of the distribution of peptides or proteins in (brain) tissue.

The amino-acid sequence of the double-headed proteinase inhibitor from badger (Meles meles) submandibular glands.

Badger submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of fox, dog, lion and cat in both domains. As in fox and dog inhibitor, the trypsin-inhibiting domain of badger inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of lion and cat. Domains I and II of badger inhibitor are structurally related both to the sequenced inhibitors of fox, dog, lion and cat and to the sequenced monovalent secretory pancreatic trypsin inhibitors. The sequence of the badger inhibitor is N-terminally extended by four amino acids in comparison to fox and dog inhibitors and extended by eight amino acids in comparison to lion and cat inhibitors. Furthermore, the badger inhibitor is C-terminally extended by two amino acids in comparison to the lion inhibitor and by three amino acids in comparison to all other sequenced inhibitors.

The diagnosis of CSF fistulae on the basis of detection of beta 2-transferrin by polyacrylamide gel electrophoresis and immunoblotting.

A sensitive method is described for the detection of beta 2-transferrin, a transferrin-variant found only in cerebrospinal fluid (CSF). The determination of beta 2-transferrin, whose presence is characteristic of CSF-admixtures in secretions, is performed in three steps. The proteins of the secretion are separated by polyacrylamide gel electrophoresis and then transferred electrophoretically onto a nitrocellulose sheet. Finally, the transferrins on the nitrocellulose sheet are specifically detected by an antibody reaction. The bands are visualized either by using antibodies conjugated with peroxidase or by protein A gold. With the exception of certain cases (Ritchie, R. F. & Smith, R. (1976) Clin. Chem. 22, 497-499; Görg, A. et al. (1983) Human Genetics 64, 222-226) beta 2-transferrin is found only in cerebrospinal fluid, and not in other body fluids. Therefore the detection of beta 2-transferrin can be used for the diagnosis of rhino- and otoliquorrhea. The advantage of this method is that beta 2-transferrin can be unequivocally identified by the use of a relatively small amount of technical equipment. CSF can therefore be clearly identified in secretions. An additional advantage of the method is its high sensitivity.

[Protease inhibitors as tumor-associated growth factors]. / Proteaseninhibitoren als tumorassoziierte Wachstumsfaktoren.

Two apparent endothelial cell growth factors were isolated and characterized from serum free cell culture medium of hepatoma cells by McKeehan et al. The factors were identified as proteinase inhibitors with known primary structure. They are the pancreatic secretory trypsin inhibitor (HPSTI) and the double headed inhibitor HI-30, the inhibitory active part of the inter-alpha-trypsin inhibitor complex. We were able to isolate acid resistant inhibitory active material from serum free culture medium of 4 out of 11 tumor cell lines, which we have analyzed. The cell lines were not derived from liver cells. The inhibitory active material was identified as the inhibitor HI-30 by N-terminal amino acid sequence analysis. The results indicate that HI-30 is a real growth factor, since it is expressed as well in tumor cells which are not derived from liver cells.

The amino-acid sequence of the double-headed proteinase inhibitor from fox (Vulpes vulpes) submandibular glands.

Fox submandibular glands contain a double-headed secretory proteinase inhibitor. Its amino acid sequence was determined. Extensive homologies were found between this inhibitor and the corresponding inhibitors of cat, lion and dog in both domains. As in dog inhibitor the trypsin-inhibiting domain of fox inhibitor contains an Arg residue in the reactive site in contrast to a Lys residue in the inhibitors of cat and lion. Domains I and II of fox inhibitor are structurally related both to the sequenced inhibitors of cat, lion and dog and to the sequenced monovalent secretory pancreatic trypsin inhibitors. In comparison to cat and lion inhibitors the N-terminally extended sequences of fox and dog inhibitors seem to be characteristic for the inhibitor of Canidae.

cDNA cloning of human inter-alpha-trypsin inhibitor discloses three different proteins.

Inter-alpha-trypsin inhibitor (ITI) is a serum protein of unknown function. Part of the molecule (formerly called HI30) is closely related to a tumor-derived protein acting as a growth factor for endothelial cells. We screened a human liver cDNA expression library with antibodies raised against human ITI and isolated several clones which could be divided into three groups according to their DNA sequences. The cDNA of the first group codes for a protein composed of alpha 1-microglobulin (alpha 1M) and urinary trypsin inhibitor (UTI) and is identical to that encoded by a clone originally found by screening a human liver cDNA library with oligonucleotides derived from amino-acid sequences of the two Kunitz-type domains of UTI. The proteins derived from the cDNA of the second and the third group of clones are distantly related to each other, but unrelated to the protein derived from group 1 clones. Partial amino-acid sequencing of ITI isolated from serum allowed the verification of large parts of the cDNA-derived amino-acid sequences. The results favour the view that ITI is not a single chain protein, but rather a very tight complex of several components or a mixture of such complexes.

The amino-acid sequence of the trypsin-released inhibitor from sheep inter-alpha-trypsin inhibitor.

The amino-acid sequence of the inhibitory part of the sheep serum inter-alpha-trypsin inhibitor (ITI) was determined. The inhibitor is composed of two covalently linked Kunitz-type domains. The reactive site of the C-terminal antitryptic domain contains arginine in position 71 (P1) and glycine in position 73 (P'2), whereas ITI derived inhibitors hitherto investigated contain phenylalanine in these positions. The reactive site of the N-terminal elastase inhibiting domain contains leucine in position 15 (P1) and methionine in position 17 (P'2), as in ITI-derived inhibitors of pig and horse.

The amino-acid sequences of the double-headed proteinase inhibitors from cat, lion and dog submandibular glands.

Cat and lion submandibular glands each contain a double-headed secretory proteinase inhibitor. Their amino-acid sequences were determined, and the amino-acid sequence of the inhibitor of dog submandibular glands was revised. Extensive homologies were found between these inhibitors in both domains. The trypsin-inhibiting domains of cat and lion inhibitors, however, contain a Lys residue in the reactive site in contrast to an Arg residue in the dog inhibitor. Domains I and II of cat, lion, and dog inhibitors are structurally related both to each other and to the sequenced monovalent secretory pancreatic trypsin inhibitors, Notable differences in inhibitory properties of canine and feline inhibitors are discussed with respect to sequence differences.

[New methods of diagnosing cerebrospinal fluid fistulas using beta 2-transferrin or prealbumin--principles and methodology]. / Neue Methoden zur Diagnostik von Liquorfisteln mit Hilfe von beta 2-Transferrin oder Prälbumin--Grundlagen und Methodik.

Two new methods were developed to identify pure CSF and CSF in nasal secretion. One method is based on measurement of beta 2-transferrin, a transferrin variant found only in CSF, whereas the other one is based on determination of the albumin-prealbumin ratio. The determination of beta 2-transferrin is made in two steps. Firstly, the protein variants beta 1- and beta 2-transferrin are isolated selectively from the complex protein mixture by an affinity procedure. The transferrin variants are then separated by a highly resolving polyacrylamide gelelectrophoresis and identified by staining. Advantages of the method are that blood or nasal secretion does not disturb the test and that beta 2-transferrin can be identified with high accuracy. Therefore, CSF can be differentiated unambiguously from other secretions. The determination of the ratios of concentration of the proteins albumin and prealbumin by immunoelectrophoresis is a further possibility to identify pure or almost pure CSF. This sensitive method, which is fast and easy to perform, is very helpful in corroborating the diagnosis of a CSF fistula. On the basis of this method one cannot get a false positive result, but a false negative one by high contamination of CSF with blood or nasal secretion. Therefore, the method of beta 2-transferrin determination must be carried out if there is any doubt.