Robert J. DeLange

Human bone morphogenetic protein (hBMP)

Abstract Human bone morphogenetic protein (hBMP) was chemically extracted from demineralized gelatinized cortical bone matrix by means of a CaCl2·urea inorganic-organic solvent mixture, differential precipitation in guanidine hydrochloride, and preparative gel electrophoresis. hBMP is isolated in quantities of 1 mg/kg of wet weight of fresh bone, and has the amino-acid composition of an acidic polypeptide. The mol wt is 17 to 18 k-Da (kilodaltons). Implants of the isolated 17-kDa protein are very rapidly adsorbed and produce a smaller volume of bone than protein fractions consisting of 24-, 17-, and 14-kDa proteins. Since the isolated 24- and 14-kDA components lack hBMP activity, the kinetics of the bone morphogenetic processes including the function of other proteins as carrier molecules, await investigation.

A Bovine Low Molecular Weight Bone Morphogenetic Protein (BMP) Fraction

A low MW bone morphogenetic protein fraction (BMP) is quantitatively extracted from bovine bone matrix by an inorganic-organic CaCl2-urea solvent mixture and fractionated by ion exchange and gel chromatography. The BMP fraction induces differentiation of perivascular mesenchymal type cells into cartilage and bone inside the mouses thigh, outside of double walled diffusion chamber, in muscle pouches in the rabbit anterior abdominal wall, and in 0.8 cm trephine defects in the rats skull. Bovine BMP may consist of electrophoretic components ranging from 12 K to 30 K in MW. The main components correspond to a MW of 23 K, 18 K and 12 K when they are compared with the mobilities of standard proteins. Because it was invariably present in all of the fractions with osteoinductive activity, circumstantial evidence leads to a 17 to 18 K component for a BMP. The possibility of a diameter monomer system for BMP activity also warrants consideration. The polypeptide portion constitutes only about 80% to 85% of the dry weight of the mixture of the three electrophoretic components, and suggests that the BMP fraction contains glycoproteins. Characteristically, glycoproteins migrate anomalously on SDS gels and create doubt about whether the major bands represent true MW. Nevertheless, the data clearly point to the low MW protein fractions for the direction of future work on BMP.

3 Leucine Aminopeptidase and Other N-Terminal Exopeptidases

Publisher Summary This chapter focuses on leucine aminopeptidase and other N-terminal exopeptidases. The classic N-terminal exopeptidase—leucine aminopeptidase—was first observed in the extracts of swine intestinal mucosa. Although several substrates could be used to assay leucine aminopeptidase, L-leucinamide is most often employed because it is commercially available or is easily prepared. It is rapidly hydrolyzed by leucine aminopeptidase, but it is not hydrolyzed by dipeptidases or by many other proteolytic enzymes that may be present at the various stages of purification of the enzyme. For the assay of leucine aminopeptidase during purification procedures, the titrimetric procedure as described by Hill et al . has been widely used because many samples may be conveniently assayed at one time. The highly purified preparations of leucine aminopeptidase were found to contain traces of contaminating endopeptidases, which could be inactivated by treating the preparations with diisopropyl fluorophosphate (DFP) and iodoacetate (SO). The inactivation of the contaminating enzymes is unnecessary when leucine aminopeptidase is used for complete hydrolysis of peptides.

[28] Preparation and bioassay of bone morphogenetic protein and polypeptide Fragments

Publisher Summary Among the cellular and extracellular components of the organic matrix of dentin, bone, and various tumors is a bone morphogenetic protein (BMP). The identity of BMP is based upon its capacity to induce the differentiation of mesenchymal-type perivascular connective tissue cells into the bone. This chapter discusses a standard method of preparing BMP from either bovine or human bone. There are also some methods of cleavage of the molecular structure of BMP to produce the smallest unit of protein structure with osteoinductive activity, designated as bone morphogenetic polypeptide (BMP-p). The most available source of BMP is demineralized bovine or human cortical bone matrix. The most direct method for the preparation of BMP consists of the chemical extraction of HCl-demineralized bone in a 4 M solution of GuHCl. This extract consists of a complex mixture of intra- and extracellular proteins that is extremely difficult to fractionate. A slightly more manageable extract with a high level of BMP activity is obtainable from insoluble gelatinized bone matrix prepared by sequentially extracting the relatively soluble noncollagenous proteins. This removes about 20% of the non-BMP relatively soluble proteins from the system. The BMP is tightly bound to insoluble protein aggregates within the densely packed cross-linked structure of cortical bone matrix.

Histone-like protein in the Archaebacterium Sulfolobus acidocaldarius

The Archaebacterium Thermoplasma acidophilum contains a basic chromosomal protein remarkably similar to the histones of eukaryotes. Therefore, it was of interest to examine a different Archaebacterium for similar proteins. We chose to examine Sulfolobus acidocaldarius because it is thermophilic, like T. acidophilum, but nevertheless the two organisms are not particularly closely related. Two major chromosomal proteins were found in S. acidocaldarius. The smaller of these was soluble in 0.2 M H2SO4 and had a molecular weight of 14500. The larger was acid-insoluble and had a molecular weight of about 36000. Together, the proteins protected about 5% of the DNA against nuclease digestion and stabilized about 50% against thermal denaturation. Overall, the properties of these proteins were intermediate between those of the Escherichia coli protein HU and T. acidophilum protein HTa.

Estrogen and progesterone receptor sites in malignancies of the uterine cervix, vagina, and vulva.

Cytoplasmic receptors for 17 beta-estradiol (ER) and progesterone (PR) were measured in uterine cervical, vaginal, and vulvar carcinomas by the dextran-coated charcoal (DCC) technique. Tissues from 30 patients with cervical carcinoma were examined. Thirteen percent (2 of 16) of well-differentiated squamous carcinomas had positive ER, and 19% (3 of 19) had positive PR. None of the three patients with moderately well-differentiated disease have positive ER or PR, while two of five patients with poorly differentiated lesions contained measurable ER and PR. In contrast, all four of the well-differentiated adenocarcinomas of the cervix had detectable ER, and three of four for PR. Neither of the two patients with poorly differentiated adenocarcinoma had either ER or PR. None of the five vulvar and seven vaginal epidermoid carcinomas studied had ER or PR activity. Hormonal therapies may be useful in the treatment of adenocarcinoma of the cervix.

Calf thymus histone III: Sequences of the amino- and carboxyl-terminal regions and of the regions containing lysyl residues modified by acetylation and methylation

Abstract Sequence studies on the tryptic peptides from maleylated calf thymus histone III showed one site of e-N-methylation in the sequence Arg-Lys (CH 3 ) 0–2 -Ala-Ser-Pro-Ala-Thr-Gly-Gly-Val-Lys-Lys-Pro-His-Arg, and two sites of e-N-acetylation in the sequences Arg-Lys-Ser-Thr-Gly-Gly-Lys(Ac)-Ala-Pro-Arg and Arg-Lys-Gin-Leu Ala-Thr-Lys(Ac)-Ala-Ala-Arg. The NH 2 -terminal sequence of histone III is probably Ala-Arg,and the sequence of 20 residues at the CDOR-terminus has been established. There is little or no similarity when these regions of calf thymus histones III and IV are compared.

Thermoplasma-Acidophilum Histone-Like Protein - Partial Amino-Acid-Sequence Suggestive of Homology to Eukaryotic Histones

Thermoplasma acidophilum is a freeliving mycoplasma-like organism that has a small basic protein tightly bound to its DNA. The N-terminal sequence of this protein has been determined. It has a distanct but statistically significant homology to eukaryotic histones H2A, H3, and to Escherichia coli protein HU.

Determination of estrogen and androgen receptors in Trichomonas vaginalis and the effects of antihormones

Trichomonas vaginalis, a common genital pathogen, was found to possess both specific estrogen and specific androgen receptors. These 4.3 S macromolecules were proteinaceous in nature. Both metronidazole-resistant and metronidazole-sensitive strains possessed both types of sex hormone-binding proteins. The estrogen receptor binding was competitively inhibited by the antiestrogen tamoxifen citrate, and the androgen binding was competitively inhibited by the antiandrogen cyoctol. The presence of these specific receptors may allow the use of hormonal and antihormonal manipulation in the treatment of infections caused by these organisms.

Estrogen and progesterone receptors in ovarian neoplasms

The cytoplasmic receptors for 17 beta-estradiol (ER) and progesterone (PR) were measured in 39 malignant and 15 benign ovarian neoplasms. All eight endometroid carcinomas had positive ER sites, one-half contained PR. The number of ER binding sites decreased as tumor grade increased. Conversely, none of the 11 mucinous tumors contained either ER or PR receptors. One-half of the well-differentiated serous tumors had ER (57 +/- 23 fmole/mg protein) while none of the poorly differentiated tumors had measurable binding. In serous carcinomas, PR was only detected in well-differentiated lesions (447 +/- 240 fmole/mg protein). Only one of 15 benign neoplasms contained ER and PR receptors. Correlation of tumor grade and type may help to plan hormonal therapies in advanced ovarian malignancies.